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Methanogens - Science topic

Methanogens are microorganisms that produce methane from single and two carbon compounds as a metabolic byproduct in anoxic conditions. They are classified as archaea, a group quite distinct from bacteria. They are common in wetlands, where they are responsible for marsh gas, and in the guts of animals such as ruminants and humans, where they are responsible for the methane content of belching in ruminants and flatulence in humans. To date only 133 methanogens have been isolated throughout the world.
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We are performing an experiment to estimate the change in the microbial profile in the gut of dairy cattle to different diets. The experiment's main aim is to see if there is a reduction in methane emissions and a change in the microbial profile of the gut. We are planning to perform a microbiome analysis to get a complete estimate of the change in the microbial profile.
The question here is;
1. Is microbiome testing the best method to go about estimating the reduction in methane emissions? (as it can be used to estimate the methanogenic archaea in the gut). Is the result really reliable?
2. Is In-VItro testing to estimate methane testing a good way to measure methane emissions, is this testing reliable?
Thank you.
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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.
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Commonly, for determining the activity of acetoclastic methanogens from anaerobic seed sludge (with low remaining carbon source), I use sodium acetate as main carbon source and analyse the max slope for methane production to get the value of its activity. However, I have problem to determine the activity of acetoclastic methanogens from the microbial sample with high remaining carbon source (such as starch and fibers). The remaining carbon source from this sample can also be used by microorganisms to produce acids then methane. The separation (centrifugation) of starch and fiber from microbial sludge is also difficult, since they have similar density. The control test shows similar methane production with experiment test (+sodium acetate). Would you like to give me some suggestions for this test?
Thank you very much
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Victor S Garcia-Rea. Thank you very much
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Hi everyone! I'm trying to identify methanogens from wastewater using flow cytometry . Any idea on how to go about it? which stains or flurochromes we have to use? how do we go about isolation of methanogens after that.
Will be grateful for your inputs...:-)
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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
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The SMA test is a metabolic assay to evaluate the ability of the anaerobic sludge to convert organic substrate into methane. its advantages include:
Helping to determine the maximum organic loading to be applied to a reactor, determination of minimum sludge mass to retain in reactors during desludging, gives indication of any inhibition or toxicity in the reactor etc.
Several studies on SMA tests with different substrates only give the SMA values attained, I am yet to come across an article which specifically mentioned which SMA range is best for anaerobic systems, and above or below which thresholds indicate a reduction in sludge activity when using sodium acetate and glucose as substrates.
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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
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I recently conducted DNA-SIP experiment where substrate was 13C labelled carboxyl group (99%) model compound (e.g. Decanoic acid-1-13C, benzoic acid). The sequencing data indicates that 75% of the reads were Methylotrophs (Methylobacillus). Remaining 25% are Pseudomonas, Sediminibacter, etc. I have previously observed methanogens in similar system so I believe that methane production is likely. However, I am not sure why Methylotrophs would be that much high? I was expecting some other bugs to be high who might be responsible in degradation of these model compounds but it didn't come along. But I am also considering that the Methylobacillus might have been feeding on the products of other compounds? In that case, what degradation pathways one can expect with such high abundance of methylotrophs.
Can you please provide some insights or I am thinking in a wrong direction?
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here is the final story i generated.
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Hello,
I have quantified both methanogens (mcrA target gene) and methanotrophs (pmoA target gene) within stream sediment samples and we have measured potential methanogenesis and methane oxidation from the source streams.
The mcrA gene is a single copy gene and so I do not need to worry about this when interpreting the qPCR data and calculating cell specific methanogenesis. However, for the pmoA gene, I have read in an old paper from 1995, there can be duplicate copies present in some MOB (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC176995/pdf/1773071.pdf). I also see in this more recent paper (within the SI), that when calculating cell specific methane oxidation, the authors divided their value by 1.62, stating this was the mean copy number for pmoA (https://www.nature.com/articles/s43705-021-00026-y#citeas). There however, no reference to back this up.
Does anyone have any insight and/or references to support how many copy numbers of the particulate methane monooxygenasegene (pmoA) gene are present within a cell?
Thanks in advance,
Kate
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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
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Hello,
I am currently working on my Master thesis and wanted to know that if we are producing biohydrogen (I-Stage) and biomethane (II-Stage) in a two-stage process from microalgae simultaneously and if we wanted to recirculate the digestate back again, which actually would contain methanogens, how can we separate it so it won't hinder the hydrogen-producing bacteria in the first reactor.
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You have to disable methanogen for biohydrogen production. so you can use the acidic and thermal pretreatment.
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In the literature about extremophiles, it is common to find the range of temperature, hydrostatic pressure, pH, salinity and water activity over which living organisms are able to thrive (i.e., survive and reproduce).
In contrast, it is very rare to find this information regarding the environmental redox potential (Eh). Recently, I found a paper that reported the range between -450 mV (for methanogens) and +850 mV (for iron oxidizers) (Beech et al., 2000).
Does anyone know another reference reporting the Eh range for life?
And does anyone know for which organisms there is experimental evidence of thriving at the highest and lowest Eh of such range?
---
Beech et al. (2000) Simple methods for the investigation of the role of biofilms in corrosion. https://efcweb.org/efcweb2019_media/Documents/WP_TF/WP10/MICbook.pdf
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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.
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I used a two staged anaerobic digesters in plant design dealing with rice industry waste such as rice husks, rice straws, rice bran, and etc. The confusing part is do i need to feed in microbiomes such as acetogens and methanogens into the bioreactors? If not, the inoculum should consists the microbiomes before entering the bioreactors? Please advice
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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.
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With a catalytic pretreatment of the CO2 and H2 mixture to activate the reaction, then a second biological process using methanogenic bacteria, could the efficiency of the process be improved and the production of methane increased?
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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.
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Could you suggest me a solution for generating methanogenic bacteria during the anaerobic digestion process? Shall I add cow manure to produce them? or addition is not necessary?
Thanks.
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I know that faeces contains such microb, but addition of cow dung will facilitate the production of methane.
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I am trying to look at the population of methanogens in my sample of food waste and biosolids. I fix my sample(0.1ml) with 9.9ml Foramide. Then I take 0.1ml of that stain with acridine orange and filter through polycarbonate filters. However when I look through the microscope, the methanogens are in colony and also there is large amount of biomass. So I do not think my results would be acurate. I want to know is there any method by which I can separate the biomass and the microbes at the same time.
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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
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I have a question about the electron donor and acceptor of methanogens
for Hydrogenotrophic methanogens i think the donor is hydrogen and acceptor is carbon dioxide
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It is helpful
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During anaerobic digestion, what are the operation conditions or chemicals added to inhibit the activity of methanogens and allow the digestor to operate in the acidogenesis phase for VFAs production?
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High organic load and/or low HRT will be the efficiency to produce VFA.
Yann
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Hi, can anyone suggest me media for culturing methanogens from sludge samples collected from anaerobic bioreactors
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Hi friends, I am working on mcrA gene. To do this, I need a database, reference taxonomy, and the relevant stuff to draw a taxonomy bar plot. SILVA and Greengenes either have data for bacteria and archaea, but I was said it is required to construct my own classifier by different database for mcrA gene. Could you please help me how to begin and generate a special database/ reference to a functional gene?
By the way, is there another way to provide a taxonomy bar plot without QIIME2 software pakages? Thanks
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Hi if you want to make your database just use this script! It will be fast and easy!
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hello everyone , i want to check the diversity of Methanogen of a particular soil , please suggest me any idea or pipeline for this analysis, and also suggest me a particular primer for the amplification of methanogen.
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16S primers for Archaea or mcrA primers.
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I have read somewhere that if we have to measure cod reduction then we should first adjust cod upto 1000 ppm
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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
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I want to know the selection of optical wavelength and other points to note in the process ,thanks.
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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.
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Does anyone know the range of pressure that 120ml anaerobic serum bottles can tolerate (10-100 psi)?
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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
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I am looking for the protein expression of Methanosarcina mazeii that can get expressed in the presence of acetate. I am not much aware of steps. I was planning to do cell-lysis and then SDS-PAGE to check the protein expression. What other steps I should take to do this? And can we link the protein expression with ant metabolic pathway? Any suggestion would be really helpful.
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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.
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Through the soft drink wastewater treatment and using fermenter, how can I stop fermentation at the highest concentration of VFA and don't let the methanogens drive the process to the biogass production?
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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.
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I would like to know the methanogen species that produces the most methane if anyone knows or knows of a paper please.I want to grow a pure culture methanogen and measure the amount of methane it produces as part of my project.
Susie
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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?
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The discovery of the ability of some cyanobacteria (CB) to form methane may have great impact, first of all, on the terrestrial phototrophic biotachnologies. It is fantastic that CB are able to convert CO2 directly into such eco-friendly fuel as methane! Of course, it is not so simple now to separate O2 and CH4 produced by CB in same photoreactor but can be defenitely resolved sooner or later. Additionally, this finding sounds promising for the development of extraterrestrial biological in situ resources utilization (BioISRU). About 12 years ago I proposed a closed BioISRU loop to support Space Exploration Please see the attchment). It was well known then that CH4 might be convenient fuel for this goal. Therefore, I propose to convert CB biomass into methane with the help of anaerobic methanogens. However, the discovery of Bizic-Ionescu et al may simplify proposed cycle and make it more affordable for Space Exploration. Moreover, I would recommend to the authors to work on a proposal for ESA. Congratulations! Igor Brown, PhD
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Kenneth M Towe
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
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Hello, everyone. The production of methlymercury (MeHg) was primarily conducted by anaerobic microorganisms including sulfate- and iron-reducing bacteria, methanogens, and other syntrophs. Thus, the amount of inorganic mercury (Hg(II)) that is bioavailable to these organisms is an important factor for controlling Hg(II) methylation. Due to the complexity of Hg speciation in sediments, quantification of the bioavailable fraction remains a great challenge. So, which method would be potential strategies ? the concentrations of Hg(II) in pore water ? the contents of low-molecular-weight (LMW) thiols (e.g. cysteine, thioglycolic acid) which could affect its bioavailability and thus MeHg production? passive sampling like diffusive gradients in thin films (DGT)? a selective model organism using to stimulate the Hg uptake process into methylating microbes?
I don't know the performance difference between those methods. So if anyone can share your opinion or scientific story, I'd be most grateful.
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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
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Hello professional colleagues, please, i have results from DNA Sequencing (16 S) of samples from my anaerobic digesters. The sequencing results have further been run via Qiime pipeline. However, I am not an expert in microbiology or molecular biology. Please, can anyone advise me on how to do the phylogenetic tree, separate the methanogenic archaea, or the different microbial groups, plots, etc. I can view the results in on Qiime 2 view, but that appears to be all I know for now. Any advise will be highly appreciated. Thanks.
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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.
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What is the simplest, quickest, and/or cheapest way of finding out whether a molecule is involved in a novel quorum sensing / cell-cell signaling / signal transduction pathway?
Most literature explains assays that build on systems that have already been established, involving AHLs, AI-2, DKPs, DSFs, HAQs, etc., but our suspected signal molecule is not similar to those. It might be involved in cessation of cell growth, and we sort of have growth curves at different concentrations of the compound.
Outside of forward and reverse genetics, which would be especially difficult for our organism, an Archaeal methanogen, I’ve seen the most plausible options in this paper:
Affinity chromatography and photo-affinity labeling don’t seem simple or quick or cheap, though.
I’d appreciate any suggestions.
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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.
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I need to add ampicillin and vancomycin in media for isolating mathanogens. What concentration of stock should I prepare and at what concentration I have to add it. What will be the working concentration specific for methanogen isolation.
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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
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Have a nice day fellow scientists,
Since me and my supervisor weren´t able to determine what is going on with my media, I am seeking help among more experienced and skilled. I will be thankful for every kind of help, which may give me a bit of insight on where I was doing mistakes.
I will have various questions with photos which may help with description of my problems. I am really sorry for my grammar as my language skills are not as advanced as they should be.
I am having this problems for about the month and half, since the winter begun. We are maintaining 21 degrees of Celsius in laboratory, so this may not play role. Whenever I am working with my cultures, I am working in the laminar box or outside, always with the burner working nearby.
  • I am preparing this medium as described in the link up to 1 L of volume to the sterilized Schott bottle while gassing the liquid with N2/CO2 whole time. After the first process, I added the reducing agents - L-cysteine and H2S, with pH maintained around pH = 7,05 and redox potential of -300 mV. The medium was able to sustain this condition for whole month as part of the test. The resazurin solution was used as anoxic indicator.
  • After the preparation I dispense medium into 150 mL serum bottles, also sterilized prior to the use. The volume of the media inside is approximately 20 mL and all actions are accompanied with gassing by N2/CO2. The atmosphere in the serum vial is then flushed and exchanged for H2/CO2 (5:95), closed with the butyl rubber stopper and sealed with aluminium caps. In this condition I store them in the dark at laboratory temperature.
  • After a day I observed that almost 60% of my media turned whitish or pinkish with white haze and white spongy-like precipitation. Others seemed ok. The pH dropped to 5,8 - 6,2 and ε renged from -30 mV to 20 mV. Also a distinct acidic smell appeared when I opened the vials. (Picture 1 and 2)
I´ve never witnessed this condition before, since I am preparing the medium, so I don´t know what may be going on.
Also, after inoculating the vials with my cultures, the medium turned white after 15 minutes, with precipitation containing what I believe are lysed cells, since I was unable to see any living cells on the native preparate, nor on the Gram. (I was working with DSMZ cultures of Methanobrevibacter smithii and Methanospirillum hungatei, inoculating 1 mL via pregassed syringes.)
This weekend I was working on the medium again, but we had different (smaller Volume) gas bomb containing the H2/CO2 (5:95). The number of failed media went bellow 20% but there are some with the whitish haze.
So my hypothesis is, that since I can´t afford the oxygen scrubber, it might be contaminated by the oxygen or something, since the problem introduced after the bomb was low and started running out. But I am not sure, because I´ve never had such problem before, working with anaerobic media.
Another topic is cultivation of the anaerobes themselves.
I wanted to ask what are the most frequent anaerobic or facultative anaerobic contaminants during the work with anaerobes. Since I am unable to isolate and sequenate the samples at the moment due to the finances, the information would come in handy.
I was working with rather-pure cultures with possible minor contaminations. I processed the cultures with antibiotics (Vancomycin+Clindamycin) and ended with what u can see in the picture no. 3.
The picture is 1 mL filtrate stained with DAPI seem to contain the Methanospirillum itself with something representing round cells, sometimes forming pairs. I am unable to recognize the source, or the contaminant itself which may be some kind of methanogen as well.
Are there any handy and fast techniques to prevent it, or to identify the methanogens (I assume I may use the fluorescence microscopy by Doddema and Voegels 1978) which I may use in the laboratory.
Thank you very much for your help and time, I will be grateful for anything.
Martin Černý
Bc. student at the University of Masaryk
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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.
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All media available for the enumeration and growth of methanogens seems to be based on formulations to desired composition.
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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.
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Kindly give me a simply protocol
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Dr. Martin Blaser,
Thank you very much for your valuable suggestion....
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Which factors or parameters must I change to stop the methanogenic activity in my UASB reactor?
The objective is to have a UASB reactor with only acid-producing bacteria.
My UASB reactor is pilot scale and is working with municipal wastewater, the current temperature is 25 C and it is producing methane.
I would like to stop it producing methane but keeping the acid producing bacteria.
Should I try to lower the pH to 5.0? (Current pH=6.7)
Should I try to lower the temp? (Current temp=25C)
Should I try to acidify the influent wastewater?
Suggestions?
Thanks in advance
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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.
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The different biofilm carriers show a key role to enrich methanogens as well as to prevent the microorganisms from being washed out in the effluent. Therefore, biofilm carriers in anaerobic digestion process is able to improve biogas yield and methane content. 
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Hello dear 
I attached an article and a link for you. I hope be helpful
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The hydrolysis phase of the biogas production process includes the degradation steps of macromolecules to the individual components and finally to fatty acids, CO 2 and H 2 , but also various intermediates. In these degradation processes, a variety of microorganisms is involved or the degradation takes many steps or reactions.
The products of the hydrolysis phase are a substrate for the methane formation by methanogenic bacteria or archaea. For scientific investigations of a double-stage biogas process, which focused on methanation stage, is it possible to prepare a substrate in the lab instead of operating a hydrolysis stage? 
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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.
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I am trying to Isolate methanogens from a methane producing consortia. 
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Thank you Denny!!
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Hello,
I am currently testing various methanogenic mediums for growth of methanogenic archaea, but the MM medium keeps failing to create a blackish sediment of Na2S in the solution after the preparations. Instead it creates milk-like haze in the solution with white precipitation on the base.
I am preparing medium in the flask under anoxic condition of CO2/H2 (g) atmosphere with the N2 (g) atmosphere of the other solution. 
I have checked the temperature of the solution prior to adition of Na2S if it was cool enough, I have tackled the pH with 0,1 M NaOH solution, or I left it unchanged and I´ve regulated it after completing it.
I´ve been working on the MM medium for some time and I´ve never been adjusting pH or watched the temperature of the solution. I also made a new solution of Na2S and managed to get a new, pure substance, but nothing has changed.
Dis somebody have this issue with MM medium preparation? If yes, please, help me with this step, thus I can continue in use of this medium for cultivation.
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are you using ultrapure (milli Q) water?
Also check if your Na2S 9H2O stock solutiuon is well prepared
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Most biological and chemical anaerobic oxidation and methanogenic reactions which take place during the AD process are characterized by a low Gibbs energy exchange (ΔG~0), mainly due to the absence of strong external electron acceptors such as oxygen. This low-energy exchange makes some key reactions during AD near to thermodynamic equilibrium. The thermodynamic equilibrium model shows there is a possibility that the bioprocesses occur near the equilibrium and it should include the development of biokinetic models of AD process, but not yet.
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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.
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I want to isolate methanogens from rumen liquor in small ruminants. Can anyone recommend the simplest and economical method for above mentioned activity?
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Hello, we offer an anaerobic gassing station for Hungate tubes. You can download details from our website qcal.de.
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Thermal hydrolysis is a pretreatment before fermentation. The tracer then has to be nontoxic to methanogens, but also able to induce a clear response. Thanks
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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.
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I plan to set up anaerobic chemostats and want to feed one of the cultures DSMZ recommended media with H2/CO2 gas, we have applikon chemostat systems in our lab for reference. I'm wondering about the practicalities of feeding in a gaseous substrate and would appreciate any diagrams/advice that could help.
Best wishes,
Ciara
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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. 
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AS I treated digested sludge for 30min at 90C but CH4 production is still noticed.
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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.
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I am doing experiments with indigenous species isolated from black shale. Surprisingly the consortium containing only bacterial species (without methanogens)  produces methane with native as well as with sterilized shale.  I am going to repeat the experiment with more accurately sterilized samples (t=100 deg, on 3 following days) but if methane production still persists - either the isolates are not pure but co-cultures with some aero- and/ or thermotolerant Methanosarcinas (more likely) or there is another mechanism for methane production? Many thanks for any suggestions!
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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
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I am working on phylogenetic analysis of the mcrA genes from methanogens and I only have basic knowledge of genetics. First, I aligned all the sequences with the default option from Geneious v.7.1, then I trimmed both ends. Next, I used MOTHUR to create OTUs at different taxonomical levels. After that, I used the blastn function from the Geneious to find the closest relatives of each of the OTU and cut them to the similar length and created neighbor-joining trees. Each node of the trees has the bootstrap value between 8%-27%, ridiculously low. If someone could point out what I've done wrong that would be great. If anyone is willing to analyze the data since this is way over my head, my mentor and I will gladly offer co-authorship.
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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.
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In the scientific literature there is information about the use of various organic and inorganic additives to improve the efficiency of the process of methane production. I'm interested, in what the culture of microorganisms (biological means) are used for the intensification of this process?
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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
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Some say adding Fe(III)Cl to digester not only diminishes H2S ppms but also improves the anaerobic digestion efficiency.
Before I thought Fe(III)Cl works only with H2S and does not interact with methanogens. Does it? If yes, how? 
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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.
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I am investigating the wastewater treatment process of a wastewater with COD of 7000 mg/L. Wastewater enters into a tank with HRT of 14 days and anaerobic process takes place in this reactor. According to Metcalf & Eddy's instruction, 2600 mg/L of CaCO3 is added to the tank. But pH of outlet stream is about 4.8. why? and how can I fix this problem? in other words, I want to set pH at 7 to activate methanogenic bacteria to treat the wastewater?  
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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
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The experts who have researched about biogas or anaerobic bacteria  
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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.
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It is assumed that to be a two stage system, the HRT should be greater in the acidogenic reactor because the hydrolysis step is which required most time.
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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. 
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CSTR using anaerobic mixed culture with coal as the substrate. HRT=SRT. Started at 40 day, dropped to 30 day, and ended on 20 day RT. Looking for other examples where increased growth rate improved the apparent yield in a chemostat or CSTR.
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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
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need any information and images about this organism and how this organism produce methane genetically?
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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 .
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I am wondering whether PAM is possibly present in anaerobic digestion system and might effect the overall production of biogas in the digesters
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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.
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Does anybody isolate and identify the organism that produce methane in rumen?
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Those are all arch bacteria. does not need to go rumen for search but you can source it out from cattle dung.  
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I'm studying anaerobic digestion, and during the experiments we saw a big changes in time in level of ammonium nitrogen in fermented biomass. It has correlation with biogas production yield.  We want to corelate it with methanogenesis intensivity, and activity of different groups of microorganisms, but what biochemic impact can have level of ammonium nitrogen?
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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
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Landfill leachates are reported to have high concentration of ammonia. If treated using anaerobic technologies will it cause any inhibition to the methanogens? If it cause some inhibition what is the best way to solve the problem.
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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.
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If possible, we prefer services provided by out-source companies. Looking at the recent literature, it seems qPCR is still the dominant technique for studying mcrA gene. While there are qPCR services, I would like to know if there are other options like next-gen sequencing. Thank you for your help.
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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 
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Methane Pluming has been characterized on the Mars surface by NASA scientists? May that evidence be related to the presence of methanogens under the soil?
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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.
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In particular,would the complete retention of slow-growing methanogenic organisms improve the applicability of anaerobic processes to more waste streams? How
would AnMBRs compare to existing high-rate anaerobic processes, for example,
the upflow anaerobic sludge blanket (UASB) reactor? What waste streams
would be most suitable to treat with AnMBRs? What design and operational
challenges exist?
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Thank you very much Pravin.
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There is very little or no growth while I am using the liquid portion of fungal pretreated bagasse as sole carbon source.
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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. 
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I am doing experiment waste water methane production and oxidation. I used acetate as a carbon source in the Synthetic Waste Water. I want to detect Methanogens and Methanotrophs in my systems and in my lab, we can do DGGE analysis. Therefore I request relative researcher to help me to prepare the gene specific primer for DGGE analysis that possible cover most of the Methanogens and Methanotrophs. Any references in this regard will be highly appreciated.
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Thanks for your kind cooperation and the information @ Mustafa Vohra & Halina E Tegetmeyer.
Hope I can follow your referece
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The requirement for methanogens  are 20% CO2 and 80% N2. How do we obtain this mixture?
we use an anaerobic chamber that allow to use 2 gasses but we cannot control the amounts of gases. 
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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!!!
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I need to isolate these strains from ruminal fluid to use them for further investigations, but most of works published about methanogens use molecular methods. We have an anaerobic chamber in the laboratory and I found general requirement of methanogens but need more specific features of these species.
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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
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I am trying to build a two (or more) stage AD reactor and thus we need to inhibit the methanogens in the 1st reactor. The classical approach is to lower the pH with acid solution but it turns impracticable for medium to high sized reactors. Does anybody any idea how to achieve this objective? Thanks in advance for your collaboration input.
Servio Tulio Cassini  Brazil
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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.
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I want to study cover crop biomass application to the methanogenesis in rice paddy soil
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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
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The use of enzymatic pretreatment to enhance the hydrolysis phase in the anaerobic digestion reveals the accumulation of LCFA in the reactor; these can coat the methanogens and inhibit methane formation.
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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.
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I need some standard protocol for FISH analysis of soil samples, I want to check methanogenic archaeal population and diversity by using Fluorescent in situ Hybridization (FISH)?
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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.
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What is the best composition of microorganisms (acidogenic and methanogenic) in a biodigester?
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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.
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In production of gas in farms or any other place to aproach solid organic waste.
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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.
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Methanogenic Archea are small microorganisms involved in the decomposition of organic matter in soil, by using H2 or some times CO2 as a substrate and eventually produce methane which is the 2nd highest green house gase after after CO2.
Archea identification, activity, diversity and community structure can determine the production and oxidation rate of methane.
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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. 
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Granular sludge is the key factor for an efficient operation of an upflow anaerobic sludge blanket (UASB) reactor. However in some cases in an UASB reactor we lost granules for many reasons.
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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.
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To target specific methanogens and acido, acetogens identification, V3 and V4 metagenomics study is sufficient to show the microbial profile ? 
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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.
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The majority of methane producer is not acetoclastic methanogen but hydrogenotropic methanogen. Does it mean something is going wrong during the anaerobic digestion? 
How many anaerobic digestion models are nowadays being recognized beside ADM1? 
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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.
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Tetrakis Hydroxymethyl Phosphonium Sulphate and methanogenic bacteria
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I think you can try some strains of Bacillus subtilis. 
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Media for methanogens.
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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
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Does anyone know of a practical case where the knowledge gained through the analysis of a metagenome is used to improve a production process?
I'm particularly interested in examples in the anaerobic digestion field. For example, if through metagenomics or similar approaches a lack of hydrolytic bacteria is detected, an addition of these into the actual process resulted in an increase in the biogas production. 
A follow up to this would be. Are the results obtained with metagenomics really useful for the improvement of processes such as the anaerobic digestion, or they are only interesting for the microbiolgical point of view?
Thanks. 
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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".
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I'm trying to figure out how to isolate methanobacterium to create starter fluid for(inoculum) the biogas digester without using animal manure. Preferably using household products or organic waste. The nature of my project is such that this must be done by a person with little of no education and it must not be expensive, so  no labs and I cant buy the methanogens.  
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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. 
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I am interested in understanding how I can detect production of methane gas from hydrocarbon degrading bacteria.
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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.