It is somewhat less than precise method, given the difference in nature and complexity of proteins and complex carbohydrates. However, it is a commonly used and generally accepted method. When you publish your findings, readers in your field will understand the points of references (and their limitations). 

Dr. Alam is right: EPS is pretty complex and may easily vary in composition from one sludge (source) to another. However it is well established that the bulk of EPS is polysaccharide and proteins so quantifying those two may give an idea of how much EPS is present. I am not for adding up the two though-- I don't really think it is necessary. See how EPS is "quantified" in this paper: http://pubs.acs.org/doi/abs/10.1021/es5033426

http://pubs.acs.org/doi/abs/10.1021/es5033426

EPS can be obtained by determining total protein and poli sacaride in  sludge of the centrifuged. EPS = Protein +Polisacaride.

Simply put, EPS comprises of dead bacterial cells in sludge, which is nothing but the waste sludge, that is not used in an Activated Sludge Process (ASP). In normal biological treatment processes, 40% of the sludge ends up as waste sludge. Therefore if the total concentration of sludge in the process is 10 g/l, then EPS could be around 4g/l. I hope this is a simple, and correct practical answer.

Thank you very much for all the useful advice and suggestions. But, I am still thinking about the sum of proteins and polysaccharides. The unit of proteins is (mg/gMLVSS as BSA) and the unit of polysaccharides is (mg/gMLVSS as glucose). Can we calculate something with different units? If possible, what is the exactly unit of EPS: mg/gMLVSS as BSA or glucose or something else?

This is a good question, and it's not really well defined or discussed in the literature.  Most researchers tend to discuss them separately (protein vs polysaccharides), and others have simply added them together to get a total EPS value.  The units are really an equivalent mass based on that specific standard (BSA or glucose), so you really won't be able to resolve those units to be the same.  Most readers interested in the topic recognize this, and understand the units, so adding them together to get a total EPS is acceptable and has been used in the literature before.

One thing to consider is that there's some artifacts in the Bradford method that you're using.  For example, we've found if you use different standards such as BSA or some another protein like ovalabumin, the standard curves are very different (slopes differ by a factor of 2) which then gives very different values for the protein content of your samples. The Lowry method is more consistent in it's response to different proteins and gives more reliable values.  There's a nice paper that looks at some of these issues which is linked below.

Good luck.

http://www.sciencedirect.com/science/article/pii/0043135494902615#

I don't know if it will help, but some references are made in the paper noted below to methods we have used for quantifying EPSs following guide lines used in Japan.  For units, I have always just used:  % of dried sludge with respect to the individual components (i.e., % protein, % carbs, % N-acids, etc.)

Bhattii et al., "Characterizatin of Denitrifiying Granular Sludge ..." J. of Bioscience and Bioengineering, Vol. 91, No. 4, 373-377. 2001.

EPS can be measured by determining total protein, polysaccharide and carbohydrates and summary as DOC. But: you have to extract the eps from the sludge by adding ion exchange resin to the sludge, followed by gently stirring the mixture (sludge and resin). Then you can separate by centrifugation the sludge/resind from the liquid phase followed by filtration of the liquid phase by syringe filter units (0,45 µm; optimised filter with repsect to the protein content). Now you can measure with specific methods each single parameter of EPS in supernatant. I recommend literature review. In case you still have problems after this, don't hesitate to contact me.

PS: using BSA standard to measure proteins didn't work in our case, we used lowry method.

The extraction of SMP and EPS are based on a physical­­­-chemical extraction (sodium hydroxide-formaldehyde) method. SMP and EPS (or free EPS and bound EPS) are normalized as the sum of polysaccharide and protein, measured colorimetrically by the methods of Dubois et al.  and Lowery et al. respectively. Bovine serum albumin (BSA) is used as a protein standard, and glucose is used as a polysaccharide standard. Concentration of soluble microbial products (SMP) and extracellular polymeric substances (EPS)  are analyzed in the samples .

If you want to know the actual concentration of EPS: Extract it, lyophilize a certain volume of the EPS solution and weigh out the final solid after freeze-drying. This can give you the actual mass of "total EPS" per volume of culture or number of cells etc. For this method to work though you have to make sure all the cells are removed, as well as salts from media and small molecular weight metabolite (e.g. via dialysis). From previous studies we also understand that the method you use for extracting EPS affects its composition. If you are only interested in soluble EPS fraction then a simple centrifugation method may work.

Best wishes.

Me to want to know the calculating the EPS production What formula i have to follow.............. please send me the exact formula for calculating the EPS production using phenol-sulfuric acid method and proteins by Bradford method. 

Or you can measure the sugar concentration in EPS

Hi,

Firstly, talking about EPS, we all know that its composition is extremely complex and it contains not only proteins and polysacharides but also carbohydrate, humic acid, uronic acid, DNA and etc... so I personally don't think that it's correct to indirectly calculate EPS production simply by the sum of proteins and polysaccharides. If you take a look in the paper in the attached review paper, they said sometimes humic acid percentage can take up to 20%. So isn't that in some cases (which you don't know if it happens to your sample or not), you underestimate your EPS production too much?

Secondly, take a closer look to the principle of phenol-H2SO4 and Bradford method, they normally  use glucose and BSA as the standard, and we surely know those are not the main composition of EPS's polysaccharides and proteins.

Hence, I think it's not recommend to calculate EPS = proteins + polysaccharide, and I think you should consider Adeyemi S. Adeleye's suggestion. We usually do dialysis the EPS suspension against DDW and then precipitate EPS by cold EtOH overnight and then lyophilize to calculate the total EPS.

Hope it can help.

http://www.sciencedirect.com/science/article/pii/S0734975010001035

We are trying to quantification EPS in sourdough. We follow the bibliography protocols: We centifugate the sample, then the clear supernatant was collected, and the EPS was precipitated by adding ethanol overnight. The precipitate was recovered by centrifugation and dissolved in distilled water. At this point we have the problem that the solution is not dissolved well and it is turbid. How we can obtain a clear dissolution with out pellet?

you sould read my articles

Isabel: Consider adjusting pH to alkaline (e.g. 8). This, with prolonged mixing should help with dissolution.

Article A critical review of extracellular polymeric substances (EPS...

According to my two researches:

Article Assessment of nanoclay-sludge ratios in sequencing batch mem...

Assessment of nanoclay-sludge ratios in sequencing batch membrane bioreactors: Azo dye removal and fouling alleviation

and

Article Comparison of high-concentration azo dye removal by long HRT...

Comparison of high-concentration azo dye removal by long HRT in MSBRs’ bioaugmented with GAC and sponge media

Most part of it (if it is sludge) is humic and protein (especially humic), and the concentration of carbohydrate is low in comparison with sum of humic and protein. Therefore, if there is any error, it will be reduce.

In addition, the numbers (ABS or absorption) on the spectrophotometer for proteins (and also humic) are low, for high concentrations of them. So there is no big different between BSA and other standards (the error is high for all of the standards).