Characterization of the protein fraction of the extracellular polymeric substances of three anaerobic granular sludges

Extracellular polymeric substances (EPS) play major roles in the efficacy of biofilms such as anaerobic granules, ranging from structural stability to more specific functions. The EPS of three granular anaerobic sludges of different origins were studied and compared. Particularly, the peptides from the protein fraction were identified by mass spectrometry. Desulfoglaeba and Treponema bacterial genera and Methanosaeta and Methanobacterium archaeal genera were prominent in all three sludges. Methanosaeta concilii proteins were the most represented in EPS of all three sludges studied. Principally, four proteins found in the three sludges, the S-layer protein, the CO-methylating acetyl-CoA synthase, an ABC transporter substrate-binding protein and the methyl-coenzyme M reductase, were expressed by Methanosaeta concilii. Mainly catabolic enzymes were found from the 45 proteins identified in the protein fraction of EPS. This suggests that EPS may have a role in allowing extracellular catabolic reactions. Electronic supplementary material The online version of this article (10.1186/s13568-019-0746-0) contains supplementary material, which is available to authorized users.


Introduction
In 1995, Frølund's team proposed a modified method of Lowry's procedure to determine proteins and humic substances (HS) (Frølund et al. 1995). As the Folin-Ciocalteu's phenol reagent (FCPR) is used for colorimetric determination of both proteins and HS (Box 1983;Lowry et al. 1951), they estimated which part of the color development was due to protein versus HS in samples that contain both. With CuSO 4 , the total absorbance is equal to the sum of the absorbances from proteins and HS. Without CuSO 4 , the color development (blind absorbance) is due to HS, in addition to chromogenic amino acids. They observed that HS were responsible for a decrease of the color developed by proteins to 20% but there was no decrease for humic acids, in the absence of CuSO 4 . The mutual interference of proteins and humic compounds was addressed as following: A protein = 1.25 (A total -A blind ) A humic = A blind -1/5 A protein where A total is the total absorbance at 750 nm (A 750 ) with CuSO 4 , A blind is the total A 750 without CuSO 4 , A humic is the A 750 due to humic compounds, and A protein is the A 750 due to proteins.
In the particular case of exopolymeric substances (EPS) extracted from mixed anaerobic biofilms, those equations yielded results particularly high in HS and low in proteins, compared to the literature. Following further investigation, we propose here to update the Frølund's method for the determination of proteins and humic substances when they are both present in the same sample.

Material and methods
Bovine serum albumin (BSA) (Sigma-Aldrich #A7030, MilliporeSigma Canada Co., Oakville, Ontario) and humic acid (HA) (Sigma-Aldrich #53680) were used as the standards for measurement of proteins and humic compounds, respectively. A dilution series of calibration standards in water were prepared, either with HA, or BSA, to give concentrations of 0 to 1 mg/mL. A few drops of concentrated NaOH were used to dissolve HA in water. Solution A is freshly made daily with 50 mL of solution B and 1 mL of solution C. Solution B contains 2% Na 2 CO 3 in 0.1M NaOH. Solution C contains 1% sodium tartrate dihydrate with or without 0.5% CuSO 4 . A few drops of concentrated H 2 SO 4 were used to dissolve CuSO 4 . Solution D contains FCPR (purchased from Sigma-Aldrich, #47641) diluted with an equal volume of HCl 2M. Each mL of sample is mixed with 5 mL of solution A. Thereafter within maximum 10 minutes, 0.5 mL of solution D is added and immediately mixed. Absorbance is read at 750 nm after between minimum 30 minutes and maximum 1 hour using a spectrophotometer (DR3900, Hach, London, Ontario). Total absorbance (A total ) was measured in presence of CuSO 4 while blind absorbance (A blind ) was measured with CuSO 4 omitted, using the series of standards with known BSA or HA concentrations (C BSA or C HA , in mg/mL), to yield the calibration factors from the calibration plot slope, either F BSA (i.e. ∆C BSA /∆A total ) or F HA (i.e. ∆C HA /∆A total ).

Results
Mixed solutions of HA and BSA were prepared with various combined concentrations of HA and BSA from 0 to 1 mg/mL for each. Total absorbance (A total ) and blind absorbance (A blind ) were measured on all samples, and the concentrations of proteins and humic substances were estimated using the formulas (4) and (5) below, to be confronted with their nominal values (concentrations added) in order to test the adequacy of the Frølund's equations (2) and (3) above.
C protein = A protein F BSA (4) C humic = A humic F HA (5) where C protein and C humic are the concentrations (mg/mL) in proteins and humic substances, respectively, and A protein and A humic are given by the equations (2) and (3), respectively.
Figure 1-A diplays the plots of the protein concentration measured using the Frølund's equations as above described, as a function of the nominal or added concentration of proteins, with no HA added and with different concentrations of HA added. That is, each dot corresponds to the average measured protein concentration of several samples, all with the same protein concentration added, while humic acid concentrations ranged from 0 to 1 mg/mL. We observed that the discrepancy between the measured BSA concentration as estimated according to Frølund and the expected concentrations is inversely proportional the concentration of the HA that the sample contains (not shown). The presence of HA in the assay clearly lowers the detection of BSA. In presence of 1 mg/mL of HA, the BSA concentration is underestimated by almost half. Even in absence of HA, BSA concentrations remain underestimated by 30% when calculated with equations (4) and (2).
Similarly, Figure 2-A diplays the plots of the HA concentration measured using the Frølund's equations as above described, as a function of the nominal or added concentration of HA when proteins are absent and with different protein concentrations. That is, each dot corresponds to the average measured HA concentration of several samples, all with the same HA concentration added, while BSA concentrations ranged from 0 to 1 mg/mL. As with the results obtained for protein measurements, the change for the HA estimates using equations (5) and (3) is directly related to the added BSA concentration (not shown). When there is no BSA, the HA estimated value coincides with the expected value. In contrast, the addition of 1 mg/mL of BSA in the samples overestimates the estimated values by 0.1 mg/mL, for expected HA concentrations near 1 mg/mL, and by 0.3 mg/mL, when HA concentration tends to zero.
This means that chromogenic amino acids represent a higher color development in the absence of CuSO 4 than previously described. We estimated that this color development was higher by 40% instead of 20% (equation (8) instead of equation (3)). A ration R (equation 6) is also introduced in equation (2) to offset the underestimation of protein fraction when HA fraction tends to be high ( Figure 1-A), and the coefficient of the total absorbance in the same equation is reduced accordingly (equation 7). The use of equations (6), (7) and (8)

R = A blind / A total (6)
A protein = 1.15 A total -R A blind (7) A humic = A blind -0.4 A protein (8) Fig. S1. Measured protein concentration (A, using the Frølund's equation (2); B, using the newly developed equation (7)), as a function of the nominal (added) protein concentration. Each dot corresponds to the average measured protein concentration of several samples, all with the same protein concentration added, while humic acid concentrations were varying from 0 to 1 mg/mL. Phase-contrast microscopy (Laborlux S, Leitz, Germany; magnification 1000X). Cells after 8 minutes of sonication, dilution 1/100 in buffer.