16 Billion Bacteria in 1 Gram of Vermicompost? Piloting Quantitative Sequencing

Almost a year ago now I sequenced a few samples as a part of Southern SARE vermitea research grant for Nicky Schauder at Permaculture Gardens. This 1 vermicompost and 2 vermitea (coming soon) samples were the first samples we performed "quantitative" sequencing. So far, all of our sequencing results have been relative abundance (ex: 3% percent Bacillus). Now we are able to estimate the number of cells in a sample or for each genus (ex: 20 million cells of Bacillus per gram). Surprisingly, this technique is not commonly done in the microbiome world. However, it seems pretty useful in regards to quantifying total beneficial bacteria or pathogens per gram of compost or milliliter of tea. It also allows us to better compare vermi- products to other better characterized biological inoculants.

Vermicompost Bacterial Quantification

As you can see in the report below, I originally reported a whopping 80 billion bacteria cells per gram of vermicompost. In all honesty, this estimate is a bit high because we are actually quantifying is 16S genes (the gene used to identify bacteria) and not cells. This is a problem because each species of bacteria can have a different number of copies of the 16S gene. In short, a better estimate is probably around 16 billion bacterial cells per gram of vermicompost.

 If you want to know more about how this process works and the biases involved I'll post the nerdy details in a section at the bottom.

Aggrego Data Quantitative Microbiome Report

I want to start showing where the Aggrego Data Microbiome report is at right now so you can see the direction we are going. The organism summary of the current Aggrego Data Quantitative Bacterial Microbiome Report is below.

How does 16 billion cells per gram compare to soils and compost?

80 or 16 billion is a huge number, so lets try to put those numbers into perspective. Context always helps understand these enormous numbers better... so how many bacteria have been found in soil or other composts?

Soil Comparison

A paper published in 2014 using 3d fluorescent microscopy to quantify cells says that in general bulk soils contain about 100 million bacterial cells but can contain up to 10 billion cells per gram of soil.

It doesn't seem too surprising to me that vermicompost could have over 100x bacteria than average soils and1.5x-2x more bacteria per gram of the most biologically rich soil. We should consider soils tend to have a lot of heavy minerals and much less organic matter than vermicompost, however vermicompost does have a lot of water weight. Either way, it looks like we are in the right ball park here.

A Note on Testing Bias

It's important to remember that every every kind of test has some sort of bias. When thinking about all the ways we quantify bacteria, most have a pretty significant bias especially in diverse environment samples. We can mitigate that bias by testing everything the same way and then comparing results between samples. While the individual results might not be a perfect reflection of reality, a collection of data points can usually tell the answer we are after.

We will quantitatively sequence some soil samples soon and see what kind of bacterial estimate we get to better compare soils to vermicompost.

Thermophilic compost comparison

Surprisingly, I couldn't easily find good references for the number of bacteria per gram of compost or vermicompost. I found one paper that estimates 1 billion colony forming units (CFU) per gram of thermophilic compost.

A CF What?

A colony forming unit (cfu)is basically the same thing as a cell. A CFU is specifically a "viable" bacterial cell (it has to be alive to start growing) from a sample that grows on an agar plate and replicates to form colony big enough to count. One bias of molecular methods, such as DNA sequencing, is it possible to sequence the DNA of dead cells. This bias is likely not a big deal in compost because it is very biologically active and dead cells would be consumed rapidly.

Counting CFU's is done by plating out a dilution of the sample on agar plates (image below). You can use different media types to select for what types of bacterial species you are looking for. It is most commonly done for pathogen detection in water samples.

This study is likely under estimating the true number of cells in compost

Because they are using a "culture dependent" method (only ~ 1% of bacteria can be cultured). However, we are still in the billions of cells range and because of the methodology used in this study we would expect it to be less than the 16 billion estimated in this vermicompost.

Final Remarks

It should be interesting to see the variation in bacterial density as we test more tea and compost samples. At a glace it's looking like vermicompost is incredibly bacterially dense. We'll take a look at the number of bacteria in a milliliter of vermitea from Nicky's samples very soon too.

Please comment below if you have any questions or general comments. I'd love to hear if anyone has heard of other estimates of bacterial densities in composts.

Nerdy Details and Biases of Quantitative Sequencing

Alright, if you're still reading and want to know how we are actually quantifying bacteria, it's fairly straight forward. We use a bacterial control of 2 known species, one gram positive and one gram negative.

Note on Gram + and - Bacteria

Most bacteria can be classified as either Gram + or - based on their cell wall. The test is called a "Gram" stain after the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884. They are called G + or G- because of the way they stain with dyes under a microscope.

G+ bacteria stain purple and have a thicker cell wall primarily composed of a polymer called peptidoglycan. This structure traps the dye and also makes the cells harder to break open (lyse) to get the DNA out. We use a species of each Gram type to make sure we are evenly quantifying both types of bacteria.

Cellular Concentration Estimate

Each G+ and G- control has a known concentration of cells and 16S copy number. We add a certain number of cells into each sample right before extracting the DNA. The DNA from the control cells is extracted along with the sample DNA and sequenced. The two control species are identified and filtered out of the data during analysis. We can then take the percentage of the control species in the sample with the known number of cells we added to calculate the total 16S gene copies in each sample. The gene copy number can then be converted to cells based on a gene copy number.

So why isn't this a perfect way to quantify organisms?

The main bias has to do with the fact that the gene we are using to identify the organisms (16S), has a different number of copies in each bacterial cell depending on the species. Some organisms only have a single copy, but the maximum that I have seen is as high as 21!

Because we don't know the copy of number of every species and we don't have species level resolution with this type of sequencing, we have to estimate the copy number. For example, if instead we assumed the average 16S copy number per cell was 2 rather than 1, then we would estimate half the number of total cells.

There is a very incomplete database of 16S copy numbers in each species that we could try to reference against to get a better copy estimate for each species. This practice is rarely done in microbiome research and the bias is just accepted. However it is something we may try to do in the future to get a more exact cell count of specific organisms.

Hope that was kind of clear. Any questions please comment down below if I can clarify anything!