In this paper (doi: 10.1016/j.csbj.2021.11.043) we used an EPS extraction protocol, but the bacterial cells were killed in the process. How important is it for you to have the cells be alive?

I have sonicated cells before in order to break up biofilms and the cells were still alive afterwards (10% amplitude with pulse of 5s on ice), so you can try.

Hope that helps :)

Hey thanks. Cool paper. I tried using 5% amp sonication, but did not seem to work. I could try 10. We use EPS as an inhibitory molecule in immunologic studies, so the purest EPS (free of bacterial components) is ideal. right now I’m doing sequential spins, followed by filtration , then looking for CFU and amount EPS recovered. we typically use enzymes, DNase, RNase, prot K.

we are using the MsGG media on a B sub strain that has an IPTG inducible EPS promoter. we grow in LB to OD 0.6 then subculture into Msgg at OD 0.1, then let that go 10-12 hours. We use the ethanol ppt method but have not tried the phenol extraction. I was also thinking of omitting the iron form the msg recipe. we just can’t seem to pellet out bactera grown in Msgg

Thanks for taking a look at the paper. If your approach is not working, you can try the EPS extraction method described in the paper. 10-12 hrs seems on the low side to get lots of EPS production, we always grew at least 3 days for mature biofilms. Are you growing macrocolonies on agar plates or pellicle formation in liquid MsGG?

Hi Tatyana,

so the strain of bacillus subtilis we’re using has the WT EPS locus cloned in under an IPTG inducible promoter. The EPS is suppose to be secreted instead of being attached to the bacteria. I grow in LB (10mL) to OD 0.6-0.8. Wash with PBS and resuspend in msg. I subculture that into msgg (250 mL) at an OD of 0.1 Let that go 12 hours and isolate EPS using centrifugation and EtOh precipitation, then quantitate using concentrated sulfuric acid Method then read the absorbance at 315nm. So we haven’t done the pellicle formation. we are avoiding agar plates because when we try structural analysis we have what we think contaminating mannose form the LB-agar plates.

what we are thinkng is that there is so much carbohydrate (usually get 1 - 1.4 mg/mL) that we can not properly pellet bacteria because it is getting stuck in the EPS matrix.

Just to be clear you have the EPS locus inserted into the chromosomal DNA? Its not on a plasmid? I think that sonication should work to separate the bacteria from the EPS and maybe test longer sonication steps at 10% amplitude, so like up to 10 times 5 second pulses and see if that works. I also found this paper, that compares different EPS extraction methods from Aerobic Granular Sludge, but I think would still be relevant to you as well (. 2016 Sep 26;(115):54534. doi: 10.3791/54534J Vis Exp). Good luck :)

Yes, it’s inserted into the genome. thanks. That’s exactly what I’m doing now as far as Sonication. Appreciate your help! I’ll update you if something works

Before resorting to sonication, consider milder methods like vortexing, gentle shaking, or enzymatic treatment (using enzymes that degrade specific components of the EPS matrix). These might be sufficient to release the bacteria without the risks associated with sonication. You might also consider acidification or temperature lowering. If you need to completely disrupt the biofilm regardless of bacterial viability, sonication or a combination of methods (e.g., temperature lowering followed by mild sonication) might be necessary.

Carlos Guadarrama-Perez Hi Carlos,

Thank you for your input. We use the purified EPS in immunological studies, specifically as an inhibitory molecule. With that, I have considered EPS matrix inhibitors as that is a great idea, but have not tried any yet.

I have tried sonication, changing the pH of the culture, as well as filtration. All of which do not greatly affect bacterial numbers by looking at CFU. But, we lose majority of our EPS.

To induce biofilm formation in Bacillus subtilis, I've found two effective methods:

I use 15k rpm for 30 mins to clarify the supernatant. Are you using the right speed?

Carlos Guadarrama-Perez

1. For sonicaton, I have tried 5, 10, 15, 20, and 25% amplitude. I then centrifuged samples down and compared CFU of that to the original stock. It made no difference in clarifying the supernatant.

2. For pH, I changed the pH to 8, 9, 10, and 11 for there last hour of the culture and then again compared CFU after spinning.

3. Our strain of B. sub is a laboratory strained with a WT EPS locus under the control of IPTG promoter. It is a task mutant which secretes the EPS rather than having it be attached to the cells. So for this strain we moved away from growing in LB or agar plates because we believe when we do composition or structural analysis we have a lot of contaminating mannose. So what we do is grow in LB to od 0.6 - 0.8. spin down, wash with PBS. We then resuspend in PBS in inoculate to a starting OD of 0.1-0.2 in MSGG ( a minimal media specifically made for biofilm formation in B sub. At the end of the process we get 30-40 mg of EPS out. We have have that when we do not induce the strain with IPTG we can clarify the sup. We think two things 1) other the EPS is still attached to bacteria because there is so much of it or 2) the molecule is so big that we are spinning that down with out bugs.

4. Our first spin is 5000 rpm for 30 min, then pour off sup and spin again at 14000 rpm for another 30 min. We have tried longer spins too and that does not work, We have ultracentrifuged the sups at 50,000 x g and that clarifies the sup but again lose more than 50% of our EPS

I would attempt acidification to disrupt the charges in the colloidal suspension. This could potentially neutralize charges on the EPS and bacteria, potentially leading to their aggregation and precipitation. Sonication might not effectively separate bacteria from their EPS, as they are often tightly bound through strong chemical interactions.

If no precipitation is observed after acidification, it could indicate several possibilities:

Weak interactions: The interactions between the bacteria and EPS might be weak or reversible, preventing significant aggregation.

Steric hindrance: The EPS matrix itself might prevent close contact between particles even after charge neutralization.

Insufficient conditions: The specific conditions used for acidification (pH, time, temperature) might not be optimal for inducing significant changes in the suspension.

Read:

Srikanth, R., Reddy, C. H. S. S. S., Siddartha, G., Ramaiah, M. J., & Uppuluri, K. B. (2015). Review on production, characterization and applications of microbial levan. Carbohydrate Polymers, 120, 102-114. https://doi.org/10.1016/j.carbpol.2014.12.003

S. Comte, G. Guibaud, M. Baudu, Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods, Enzyme and Microbial Technology, Volume 38, Issues 1–2, 2006, 237-245, https://doi.org/10.1016/j.enzmictec.2005.06.016.