Questions related to Microbial Ecology
Evolution runs in perpetuity for billions of years, with innumerable remarkable
innovations inspired and catalyzed by both cooperation and conflict. Bacteria in nature are categorized into beneficial and harmful ones. They have evolved the capacity to communicate chemically to coordinate attacks on others, and a willingness to commit suicide for the greater good of the community. At the very early stage of biological evolution Mother Nature conducted a great experiment: Bacteria and Archaea came together in a fusion event to
synthesize a whole new domain of life, the Eukarya. Over the past 600 million years the Bacteria, Archaea and microbial Eukarya have continued to evolve into brand new niches. In the process thet have created new substances for bacteria to exploit and new environments to inhabit. What conditions detemine the nature of interactions? Do such interactions offer some clues? How such interactions could be exploited to solve the environmental problems humanity is facing today?
I am currently doing a PCA on microbial data. After running a Parallel Analysis to determine the number of factors to retain from the PCA, the answer is 12. Since my idea is to save the factor scores and use them as independent variables for a GLM together with other variables, I was wondering:
- Should I definitely save the factor scores of all 12 factors (which would become too many variables) or I can save only a few of them (e.g., the first 3 which together explain a 50% of the variance) for the GLM?
- If I can save a lower number, should I re-run the PCA retaining only that lower number (e.g. 3) or just use the factor scores already obtained when retaining the 12 ones?
Thank you all for your time and help!
Hi, can anyone recommend any papers exploring the relationship between microbial biomass and microbial diversity? Some papers I have looked at just say there’s a moderately positive correlation in their introductions but it’s not the focus of the study or the studies cited.
I also would love if anyone could share papers expounding on the Fungal:Bacteria and G+:G- bacterial ratios and their link to ecosystem functioning/soil health. I have tried searching for studies that go deeper into why these ratios are the objects of many experiments‘ focii, but I still feel I am missing some gaps in their connection with soil health (and soil biodiversity/biomass).Thanks!
I am working on an iron inducible promoter screen and would like to perform it on solid media. However, using agar may contaminate my control media with iron and I would like to use some method to deplete the usable iron to the bacteria. I have seen a few papers that use 2,2'-dipyridyl to do so, but I am skeptical because I have never heard of this method and am having trouble finding literature pertaining to this topic. If anyone has experience or can steer me to an article discussing the use of the substance in this way I would appreciate it.
Dear Friends and Colleagues,
To date very few journals are available to accept video articles for submission/publication in our field, although these types of articles are of paramount importance in microbial ecology in order to share new techniques and approaches.
For this reason, I decided to open a Collection titled “Multi-targeted approaches to evaluate microbial interactions and ecosystem assemblages in diverse environments” on the Journal of Visualized Experiments (JoVE): https://www.jove.com/methods-collections/919.
The purpose of the Collection is to offer a comprehensive overview of wet- and dry-labs perspectives in the field of microbial ecology.
The most recent Impact Factor for JoVE (ISSN 1940-087X) is 1.163, according to the 2019 Journal Citation Reports released by Clarivate Analytics in 2020, and the Journal is currently indexed in the major databases, including PubMed, EMBASE, Scopus and Web of Science.
After submission, each manuscript will be editorially and peer reviewed, which is typically a 1-2 month process. Once a text article passes review, a script will be generated. Generally, a filming date will be scheduled within 4-8 weeks after acceptance. A videographer will be sent to the authors’ site to film the procedure.
If you are interested, either message me or submit an abstract here:(https://www.jove.com/methods-collections/submit-an-abstract?collection_id=919).
Prof. Elaine CP De Martinis, Ph.D – Full Professor, University of São Paulo, Brazil
Otávio GG Almeida, B.Sc. – Ph.D. candidate, University of São Paulo, Brazil
I am looking for future collaborators in India working in the field of microbiome and metagenomics, with focus on antimicrobial resistance. Any suggestions?
The content obtained for the C & N in the soil samples of the forest floors is increased this time and in 2020 is 5 times more than that of the C & N obtained in 2008, simultaneously the same forest stands in four districts is increased by areas observed in the India State of Forest Report 2019, not only that the forest canopy is much lush green in comparison to 2008, is this change be considered as the evidence of the climate change, though the time span of only 12 years is very short for interpretation of the evidence of the climate change.
Gut microbiota play significant roles in human health. With the help of Next-gen sequencing, we know much about the compositions of bacteria in the gut but little is known about their activities. What is the default state of bacteria in the gut? Are most bacteria living in the gut at near stationary phase with basal metabolic activity? Do they become more active after a meal? Or they are always active? For example, what will a probiotic species do in the gut after they are taken by people? How many and how long will they colonize and grow in the gut?
1. There are several database containing nifH sequences like fungene (19500 seq), arb database (32,954 seq), zehr (72000 seq) and farnelid (5,00,000 seq). what is the difference between these databases ?
2. Can fungene pipeline be used for taxonomic classification of nifH functional gene ?
3. Any other pipeline/tool available for taxonomic classification of nifH gene ?
I would like to find a tool (if it exist ...), to predict the porportion of r vs K strategist in samples from a metabarcoding study.
For example, the tool I need (R package or equivalent) could work similarly to functional predictive tools such as Tax4Fun, FAPROTAX or PICRUST, but instead of predicting functions, it investigate ecological strategy such as r vs K as explained by the "r and K selection theory".
Does the material of the 96-well plate (PVC or Polystyrene) make a difference on the attachment of the biofilm to the surface? I am able to grow a mature Mycobacterium biofilm on the air-media interface, just need a better way of quantifying, as most of the bacteria is washed away when rinsed with saline. Plate counts works fine, but need a more high throughput method. Also, what stain will work the best to use for mycobacterial EPS?
I am processing a 16s RNA next gen sequencing data set and trying to compare between my samples the effects on organisms involved in the nitrogen cycle. I am just wondering if there is some sort of database or even a good paper that goes over all of the known organisms in the nitrogen cycle. The more detail the better but i would settle for just a simple list
I am working on a project aimed to determine the influence of long-term fertilization on soil microbial communities. I am sampling both the rhizosphere and the bulk soil and hope to use the current best choice of primers for targeting bacterial and archaeal 16S rRNA genes. Initially I planned to use the primer pair 341F/785R, which targets the V3-V4 region of 16S and is reported to have good domain coverage for both bacteria and archaea. However, I now also have the option to use separate, archaea (956F/1401R)- or bacteria (969F/1406R) -specific primers, which target the V6-V8 region of 16S. The benefits of the separate primers are better coverage for archaea, and less eukaryotic sequence contamination, but the V3-V4 primers are the standard tool typically used in similar research. I am confused with which set should I proceed with or if there are any other primer sets I should be considering?
I have two treatment groups of sample plus a group control. Each treatment and control have own three replicates. I am using Qiime2 for data analysis. I am in dilemma to the following ways below. My question is here:
For normalization prior to statistical analysis, do I need to get an arrange value of three replicates then normalize my data or without calculation the average?
I have atmospheric microbiome data and want to know how I should be analyzing alpha diversity. I used unweighted unifrac for beta diversity and thought that I should use Fath's PD but don't really understand the difference between Faith and Chao1.
I don't think Shannon is going to be good because my data has the presence of many low abundant taxa and is zero inflated.
Any help is greatly appreciated!
Has such a question been answered in a paper? Or does anyone have experience with that?
Is there a possibility that bacteria show low solubilization index on PVK agar medium but show quite a high solubilization efficiency in PVK broth when quantified? If so, Kindly share some literature.
I am currently investigating Archaeal and Fungal populations in a micro biome dataset, and while digging in the literature I came across the linked paper below in which they utilized Permanovas to dissect their data. Can someone (in laymen's terms) explain what this test does and why it is useful in this situation?
In a fermentor reactor, I have detected several "Uncultured bacterial clone (Prevotella)" with >95% of identity which were dominant and appeared to inhibit the fermentation process under certain operational conditions where the main fatty acids were acetate and butyrate. However, they were also present in the microbiome under other operational conditions where the reactor performance was different.
I have read that some Prevotella species can produce bacteriocins but I am wondering if this has only been determined for a few well known species or is it a more general feature for the genus Prevotella?
And what (pH, substrate availability, redox) might trigger bacteriocin production in this or other genera?
Hey! Dear colleagues,
I am designing my experiment. I want to know which nuclease can cut linear dsDNA but don't cut circular dsDNA?
To be more specific. I am looking for a nuclease that cut the extracted genomic DNA but don't cut the plasmid DNA.
I'm quite confused about using DESeq2 to find the differential abundant taxa in microbiome studies, especially when there are more than two groups of the factor. I know DESeq2 was initially used for RNA-seq to detect the regulation of gene expressions. It's easy to understand when there are only two groups, e.g. treated vs. untreated. We can easily say which taxa was up-regulated by looking at the log2fold change (positive or negative).
BUT what about when there are three groups, control vs. treat1 vs. treat2? The DESeq2 can still handle this situation, but then I have no idea how to interpret the log2fold change. If we detected some taxa that were significantly different, how can we know in which group these taxa were up-regulated?
Although some people suggest to do the pairwise comparison, it's still unclear to me how to do it and how to interpret it. Does anyone have very good recommendation or idea about this?
Thanks in advance.
For a dark root that doesn't clear with 10% KOH in autoclave for 1 hour, 24 and also 48 hours at room temperature, would it be possible and time effective to directly clear it with 0.5%NH4OH+0.5% H2O2?
Hi fellow researchers,
I have recently begun a PhD in an interdisciplinary team that involves some microbial ecology in field experiments on my part.
Correspondingly I am looking for a way to determine especially prokaryote diversity from soil and water samples (types of species and relative abundance basically) in a mobile setup.
It should be relatively quick and especially not too expensive to buy up-front.
I am not sure about FISH as the technique seems to be more about looking at the occurence of specific strains that you already know before-hand (and thus know the complementary DNA).
PCR techniques use quite a lot of rather costly equipment and is pretty lab-bound to my experience.
The abundance itself could be achieved by DAPI/Hoechst staining and fluorescent microscopy.
Any way to get a sense of types of prokaryotes involved / general diversity in the field though?
May alternatively look at fungi in the field aswell so if that would work in a similar fashion that would also be great.
Any ideas would be much appreciated.
I always have some problems with the formula while doing PERMANOVA. For example, adonis(distance.bray ~ x+y, sample.data, permutations = 999) and adonis(distance.bray ~ y+x, sample.data, permutations = 999) gave me the different results of the R square values for factor x and y. So what are the difference between these two formula? And which one should I trust?
In parallel with a project I am currently working on ("BiodiverCité", the inventory and characterization of the wetlands of the urban area of Bordeaux in France), partners will have to opportunity to collect samples of water over a few dozen streams carefully selected. While they are interested in the hydrological and chemical characteristics of these rivers and their associated fauna, I had the idea to take advantage of this sampling campaign to focus on the microflora and, later, possibily establish a link between the different data produced.
I am interested in two types of microflora : on the one hand, bacteria, archaea and other unicellular organisms (but mainly bacteria I must admit) and, on the other hand, the viral microflora.
Concerning the bacterial flora, I will unfortunately exclude any pasteurian approach, because of lack of time and lack of means.
I started with the idea of centrifuging the collected water samples and carrying out a DNA extraction which would join a DNA library to be analyzed once the financial means have been collected.
For the viral flora, I thought to carry out a precipitation of 1M NaCl, PEG6000 10% on the supernatant to concentrate the viruses in a volume easily handled and storable to have a phagolibrary in which to possibly find phages directed against bacterial species of interest (eg F. psychrophilum or other pathogenic species in aquaculture) and secondly, carry out a phage nucleic acid extraction to constitute a phagoDNAlibrary exploitable in the longer term.
My questions are:
1) What volume of sample should be collected to have enough bacterial DNA to make metabarcoding ? For the soil, the specialists of Genosol (INRA, France) urge me to extract from at least 500 mg of soil for bacterial and 1 g for fungal. But for water ?
2) Is the NaCl / PEG6000 precipitation suitable for a long-term phago-library ? I fear that this step is too brutal for some phages and that even if I concentrate theoretically in number of viral particles, I lose in infectivity (due to damage to viral fibrils). If this technique is suitable, is the use of PEG8000 better recommended to ensure a wider enrichment spectrum (for podoviridae for example, more difficult to precipitate if I understand my literature) ?
3) I know there are publications on viromics but I have not looked into detail: what minimum sample volume (before precipitation) is desirable to have enough DNA for metagenomics (or just qPCR detection of well known virus) ? 200 ml ? 1L ? 10L ??? (2 ml :) ?)
Thank you for your attention.
While checking the effect of inhibitors on protease enzyme it was found that iodoacetate instead of inhibiting the protease activity it has enhanced the protease activity. I searched alot but could't find the possible reason for this. Can anyone explain what could be the possible reason for the same. Thank you.
If you want to do taxonomic profiling of your ngs data, mainly comprising the variable regions of 16S (v3-v4), which database you will prefer and why ?
not only in all known to me textbooks, but also in my academic environment, plants are only discussed as carbon sinks and therefore planting forests is said to be one of the main strategies to reverse or reduce some effects of climate change.
But: About 10 years ago a german team from MPI published - and that seemed to be a shock for some climate researchers - that plants (especially forests) are able to produce tremendous amounts of methane under "normal" aerobic conditions autonomously (1). There were even attempts to link the ending phase of the ice ages with forest growth in the way that besides volcanic or solar activity forests were responsible for the warm up (and not vice versa).
Still in any lectures at my institution only common methane sources (microbes, anaerobic environments as bogs and swamps, cattles, humans,...) are discussed with students while - as said - plants are only considered as carbon sinks.
How so? Do you know something about the latest discoveries in this field (maybe i missed some relevant publications)? Are plants excluded as methane sources because of new investigations? Or is it an open (and not well known) problem that is not recognized broadly by environmental scientists?
(1) Keppler F, Hamilton JTG, Brass M et al (2006) Methane emissions from terrestrial plants under aerobic conditions. Nature 439, 187-191.
We are running 1.5% agarose gels (using 1x TBE as a buffer), for 8 hours at 120 watts.
Gels have continuously been very smeary and bands are in the shape of an upside down 'u'. Due to the strange nature of the bands, we have run the same samples multiple times and the number of bands and band lengths also vary every time (The two attached pictures represent different samples, though).
In addition the current when running the gel seems to be uneven.
We have already tried altering the amount of sample that we load into the gel, and altering PCR conditions.
Has anyone ever seen this issue before and know what could be the cause?
I want to compare bacterial communities using 16s rRNA sequences. I used Illumina Miseq and I has big differences in the number of OTUs and reads in all the samples. I use Primer-6 software to get diversity indexes and comparative analysis (ie. MDS, CAP). The question is: Is it necessary to normalize the total number of reads for these analyses and comparisons? If it is true, Which methodology is recommended?
I have to prepare biological samples (natural fibrous material like luffa sponge) for SEM analysis to understand the colonization of Methanogenic Archea in biofilms. after fixation in glutaraldehyde followed by dehydration in graded series of ethanol solutions it require to be completely dried by Critical point drying or lyophilization. as i have no facility of these two, is there any alternative or shall i simple dry it in drying oven?
I am analysing microbial communities from different sources regarding cheesemaking (milk, starter culture, cheeses) and I would like to know which is the best way to correlate these microbial communities with environmental samples such as latitude, longitude, altitude, etc.
I am currently using Qiime and phyloseq, but I want to give a try with Vegan. Anyone could help me with some functions from this software regarding my main question?
Thanks a lot!
DNA isoalation of microbial community for DGGE. I m looking for an explanation and recent papers in support.
How many samples should be included for the authenticity of 16S metagenomic data for human gut microbiota?
I'm looking for some papers on using selective media to identify and enumerate certain soil based probiotics. May you know any papers, please advise. Thanks
Does anybody know which is the best way of calculating alpha and beta diversity from molecular fingerprint techniques? I have done DGGE with environmental samples in order to determine the diversity of microalgae, I have the band patterns with the number of species in each sample, but I'm not sure how to calculate diversity, taking into account that I don't have abundance data, only number of species.
Hi, I am approaching to the identification of nano and microplankton using an inverted microscope with epifluorescence.
I would like to differentiate the organisms in 7 classes:
Autotrophic dinoflagellates, autotrophic flagellates, cryptophytes, diatoms, prymnesiophytes, heterotrophic dinoflagellates and
This type of classification has been used by Landry et al. 2014 (http://dx.doi.org/10.1016/j.dsr2.2014.02.006).
Using the epifluorescence I can distinguish heterotrophs from autotrophs, but I do not now how to identify the organisms for each class.
Do you know how to do it?
Thank you, any help will be more than welcome,
We are looking for the stool standard with known bacterial abundance which we can use as a control to find if any differences between two stool DNA extraction method.
I am analyzing my 18sRNA Soil samples for Eukaryota classification with Mother pipeline. I used Silva.nr_v123.align database and silva.nr_v123.tax in alignment. In result, as i see in taxonomy file there are classification up-to class level of species but not classification up-to species name.
Please let me know how to get classification upto species names.
OTU Size Taxonomy
Otu000001 27283 Eukaryota(100);Opisthokonta(100);Nucletmycea(100);Fungi(100);Zygomycota(100);Mucoromycotina(100);
Otu000002 24765 Eukaryota(100);SAR(100);Alveolata(100);Ciliophora(100);Intramacronucleata(100);Spirotrichea(100);
Otu000003 18535 Eukaryota(100);Opisthokonta(100);Holozoa(100);Metazoa_(Animalia)(100);Eumetazoa(100);Bilateria(100);
Otu000004 15052 Eukaryota(100);Opisthokonta(100);Holozoa(100);Metazoa_(Animalia)(100);Eumetazoa(100);Bilateria(100);
Horizontal gene transfer is the primary mechanism for the spread of antibiotic resistance in bacteria. How to detect when there is HGT between bacteria and their hosts?
I am analysing microbial assemblages attached to marine microplastics using 16S metagenomics, and I have a query about maximising the quantity of DNA I can extract from the substrate?
I have been using the DNeasy Kit, Qiagen (Formerly PowerSoil, MoBio) to remove the attached assemblage from the plastic (recommended by Harrison et al, 2014), but I am struggling to extract any DNA - according to PCR amplification and 0.8% agarose gel electrophoresis. I am aware that the associated biomass, ergo DNA yield, is typically very low.
One paper (Debeljak et al, 2017) suggests that I should incorporate an additional lysis step using ReadyLyse solution (Epicentre) to maximise cell lysis. However, I was wondering if there was a cheaper alternative? For example, I was thinking of creating a Lysozyme:TE Buffer solution to the desired molarity, although I'm aware that the enzymes are derived from different sources and have different efficiencies.
If anyone has any feedback/recommendations, that would be very useful!
Thanks in advance.
I have some river DNA samples (i.e., biofilm, water, sediment) for which I would like to know about the amounts (or relative proportion) of eukaryotic and prokaryotic DNAs before metagenomic sequencing.
Any comments or insights would be greatly appreciated!
I have recently come across some papers that suggest 16S RNA sequencing as a technique to know the active microbial population in metagenome and require some views about it. Also if this experiment is effective, I would like to know how to specifically isolate rRNA as most kits offer specific isolations of mRNA only.
I isolate bacteria from jhum soil and get may Burkholderia species, can you suggest any reason for their abundance and an article for reference in this respect.
I would like to know if there is a detailed biological classification of autotrophic or facultative autotrophic microorganisms.
How to increase the production of certain molecules from certain bacteria in communities of microbiome without changing these community profiles?
Can fastidious or even unculturable marine bacteria be grown in situ? If so, what are the current methodologies for doing so?
I have two groups of fecal samples and want to compare α-diversity between the groups. I already know the Shannon index for the microbial community of each sample, and I know I can compare one sample with another sample in terms of α-diversity, but I would like to know a way to compare groups of samples, not one sample to another. I am thinking it more like a comparison of a group of ecosystems against a different group of ecosystems, always in terms of α-diversity. The attached paper from Nature seems to have calculated something like a mean relative inverse Simpson's index (Figure 1a).
Thank you in advance.
Some possible species used are E. coli and S. aureus
Municipal and industrial wastewater often contains a cocktail of a multitude of heavy metals and nutrients. Bacteria present in such wastewater may develop multiple heavy metals resistance to cope with such heavy metal stress as an adaptive strategy. These bacteria with multimetal resistance property have the potential for remediating the wastewater or soil contaminated with multiple heavy metals. I expect some enlightening and enriching inputs from RG friends and researchers.
I want to isolate microbes from fermented fish but confused which type of microorganisms should be targeted and what media can be used to isolate them.
I want to screen soil samples for azotobacter and azospirillium, so is there any plate or chemical assay to quickly screen samples, after that when I identify it as any one above them, then please suggest any molecular or other method to identify the strain of the same..
Hi, I am looking for sequencing my soil samples. My objective to study bacterial diversity in the response of nitrogen fixation. Could you suggest me the good target gene and primers. I am wondering to go with MiSeq (2*250) technique. Thanks..
I want to analyse the biomass, abundance and functional diversity of different flora and fauna in my experimental samples. Please suggests me precise and standard methods for this purpose.
Hello , everyone. I measured the rhizospheric fungal community by ITS2 sequencing and found greater within site dissimilarity in rhizospheric compartment than thoes in bulk soil. I wonder of priority effects could be operating in rhizospheric compartment? How can I directly test the priority effects? Can I take the bulk soil as the initial state and the rhizospheric as the final state and then compare the difference between these two state? Thank you for your consideration!
I'm working on degradation studies. The sample I have, is a consortia of unknown microorganisms. I want to study diversity of microorganisms on gene basis particularly functional gene community, which are taking part in degradation. Can anyone help me out in this.
Can you suggest me a method to create a heat map based on actual data and not the relative percentages? I need it because I have different treatments with increasing the overall population! For example, in one treatment, I have a species A with number 30, and species B with number 70. On the other hand, in second treatment, I have species A with number 300 and species B as 700. So, the heat created on relative percentages will say that there is no difference however there is a major difference in the expression of each species population. I have such a dataset for which I want a descriptive illustration. Can anyone help me to figure out a way for such a problem. Thanks in advance!
Does anyone know of a consolidated, easy-to-access resource for identification/growth conditions of culturable bacteria?
According to metagenomics, DNA is extracted from a mixture of microbial community in soil, cloned into a vector and later on a metagenomic library is constructed. the ability of the community to produce a specific protein or antibiotic is also tested. However my doubt is that how the specific organism of interest can be isolated from the metagenome library which has hundreds of microbial community. there also exists a possibility that we may not know the primer sequence for a novel microorganism not yet studied which may be producing the specific antibiotic.Can somebody make this point clear to me
I am currently working on microcystis which is a very toxic algae. We are near to having a monoculture and would like to get a whole genome sequencing done when that has been achieved. What are some reliable companies that do genome sequencing and how much does it usually cost? Or is this something that you would have to purchase instruments for?
I am trying to grow cultures of either Microcystis or Anabaena but I am having trouble getting them to take off. Right now I am using a modified version of COMBO with limited to no results. Any help or advice would be appreciated! Thank you!