Cintia C. Lobo(1)*, Nora C. Bertola(1), Edgardo M. Contreras(2), Noemí E. Zaritzky (1,3)

MONITORING AND MODELING 4-CHLOROPHENOL BIODEGRADATION KINETICS BY PHENOL-ACCLIMATED ACTIVATED SLUDGE BY USING OPEN RESPIROMETRY

Cintia C. Lobo(1)*, Nora C. Bertola(1), Edgardo M. Contreras(2), Noemí E. Zaritzky (1,3)

Supplementary Data

Item SD1.In the following paragraphs, the fitting procedure isdescribed. Model #1 is discussedas an example. However, the procedure is valid for the other tested models.

The aerobic degradation of 4CP by phenol-acclimated activated sludge was represented by the following reactions:

(SD1)

(SD2)

where R4CP and R4CC are the oxidation rates (expressed in mM/h) of 4CP and 4CC, respectively.

In the simplest model (Model #1) it was assumed a Monod-type expression for R4CP and R4CC (see Table 2 in the manuscript):

(I) / (II)

where qm4CP and qm4CC represent the specific consumption rates (mmol/gTSS/h) of 4CP and 4CC, respectively, K4CP and K4CC are the semisaturation constants (mM), X (gTSS/L) is the biomass concentration, and [4CP] and [4CC] are the concentrations of 4CP and 4CC, respectively.

According to reactions (SD1) and (SD2), the change of 4CP, 4CC and P as a function of time in a batch system is:

(SD3)

(SD4)

(SD5)

Reaction (SD1) indicates that one mol of oxygen is consumed during the oxidation of one mol of 4CP. According to Eq.(SD2), one mol of oxygen is consumed during the oxidation of one mol of 4CC. To take into account that the exogenous oxygen uptake rate (OURex) after the pulse of 4CP was a little higher than the initial one, it was assumed that this increment is proportional to [P]. Based on these considerations:

(SD6)

Where qP is a proportionality constant. Thus, Model #1 is comprised by eqs.(SD3) to (SD6) along with eqs.(I) and (II).

The next step is to translate these equations to GEPASI format. The main screen of GEPASI is:

The following reactions were used to represent the model in the GEPASI window called “Reactions”

The third reaction was included to take into account the start-up process during the respirometric assays, which includes several factors such as the finite time response of the dissolved oxygen sensor and transport phenomena. The term x represents the active biomass, which is formed from xi, the resting biomass. We use a similar approach in previous works (Contreras et al., 2008a,b; Ferro Orozco et al., 2010; Contreras et al., 2011).

The first reaction represents the oxidation of 4CP to 4CC. Because x appears in both sides of the reaction, its concentration is not affected by this reaction. Similarly, the second reaction is the oxidation of 4CC. The last reaction was included to represent that the exogenous oxygen uptake rate (OURex) after the pulse of 4CP was a little higher than the initial one (see the next paragraphs)

Using the window called “Kinetics”, kinetics corresponding to each reaction can be defined:

Note that the window “Kinetics” is a key window that allows the investigation of the different tested models. That is, changing the “kinetic type” one can change the kinetic equation (e.g., the model). Besides the predefined kinetic equations, the user can also define own models using the button “Kinetic Types” of the main screen:

The user can add a desired equation using the button “Add” of the screen “user-defined kinetic types”. Within this screen, Model #5 was implemented.

In the field “Constants” of the window “Kinetics”, initial values for the parameters must be introduced. These values are only the seed for the fitting procedure, not the fitted ones. Using the button “Help on Kinetics”, one can see corresponding the equation. For example, the kinetic expression corresponding to the first reaction is:

To represent the exogenous oxygen uptake rate (OURex) (Eq.(SD6) in the GEPASI language, we use the button “Add” of the window “Functions” in the main screen of the program:

In this case, the function named OURex was composed by three fluxes (e.g., reaction rates), which represent the three terms of Eq.(SD6).

The initial concentrations of the species can be introduced in the window “Metabolites” of the main screen:

Once the model and the initial conditions are defined, one can fit the model to the experimental data using the screen “Fitting” of the main screen:

In this screen the user must define the Data File, Data Format, and fitting method. Also, the user can define which parameters will be fitted and their boundaries. Then, pressing the “Run” button, the fitting procedure starts. The obtained fitting results corresponding to the tested models are depicted in Table 2.

Item SD2. Figure 2b shows a linear increase of the soluble COD consumed (ΔCODS) as a function of the 4CP degraded (Δ4CP). The slope of this line depends on the compounds that are in solution. In the present work, the initial soluble COD (CODS0) depended on the initial 4CP concentration ([4CP]0) and on the inert COD (CODi; e.g., microbial detritus):

(SD7)

where ThOD4CP = 208 gCOD/mol4CP is the theoretical oxygen demand of 4CP. Then, as long the reaction proceeds, several intermediates such as 4CC, 3-chloromuconate (3CM), or 5-chloro-2-hydroxymuconic semialdehyde (5C2HMS), may appear. If all these intermediates are within the cells (e.g., intracellular intermediates) or are adsorbed onto the biomass, CODS at any time only depends on the concentration of 4CP:

(SD8)

In this case, the soluble COD consumed (ΔCODS) as a function of the 4CP consumed (Δ4CP) is

(SD9)

Conversely, if a given intermediate (P) remain in solution, CODS depends on 4CP and P concentrations:

(SD10)

Thus,

(SD11)

According to the literature (Gao et al., 2009), for all the reported intermediates (P) of the aerobic biodegradation of 4CP, one mol of P is produced per mol of 4CP oxidized. However, the literature is not clear regarding if these compounds are within the cells (e.g., intracellular intermediates), in solution, or are adsorbed onto the biomass. At this point, it must be considered that 4CP removal experiments performed in this work were performed using biomass concentrations similar to those observed in actual activated sludge systems. These high biomass concentrations favored the adsorption of the produced intermediates. If this is the case, only a fraction of P could be in aqueous phase. If the solution is initially devoid of any intermediate, the actual concentration of P in solution is

(SD12)

wherefP (molP/mol4CP) is the amount of P in solution per mol of 4CP degraded. Thus, combining eqs.(SD11) and (SD12):

(SD13)

The term between parenthesis in Eq.(SD13) is the slope of the plot of the soluble COD consumed (ΔCODS) as a function of the 4CP consumed (Δ4CP) (Fig. 2b). Because ThOD4CP = 208 gCOD/mol4CP is known, from the regression line depicted in Fig. 2b, a value for ThODPfP = 45 ± 5 gCOD/mol4CP was obtained.

Item SD3. To calculate the change of pH due to the oxidation of 4CP, it was assumed that phosphates (15 mM) are the only buffer system in the culture medium. Because other buffers may be present also (e.g., carbonates), this assumption leads to an overestimation of the drop of pH caused by the release of chlorides and protons during the metabolism of 4CP. From the standpoint of pH, the degradation of one mol of 4CP produces one mol of a given (or a mixture of) organic acid and the release of one mol of chloride and protons. Depending on the pKa of the produced organic acids, these acids may be fully protonated, in which case no effect on pH can be observed, or completely dissociated. Thus, during the first steps in the aerobic degradation of one mol of 4CP, 1 to 2 mol of H+ are released, one from HCl and the other from the organic acids produced. These considerations were also adopted by Farrel and Quilty (1999) to interpret the changes of pH during the aerobic biodegradation of mono-chlorophenols by a mixed microbial community.

To calculate the change of pH due to the degradation of 4CP, firstly a buffer phosphate pH 7.5 (15 mM) was simulated using the software CHEAQS Pro version (P2011.1). Then, using the Titration Tool of the software, this buffer was theoretically titrated with HCl or H2SO4 to simulate the release of one or two protons per mol of 4CP degraded. Figure SD1 shows that calculations using the software CHEAQS demonstrated that the observed decrease in pH values were compatible with the release of only one mol of H+ per mol of 4CP degraded.

Figure SD1. pH changes due to the aerobic biodegradation of 4CP. Dots: experimental data. Lines: calculated pH values using the software CHEAQS assuming 1 and 2 H+ released per 4CP degraded

Item SD4. Equations corresponding to the tested models

Model #1

Model #2

Model #3

Model #4

Model #5

References

Contreras, E.M., Ruiz, F., Bertola, N.C. 2008a. “Kinetic modelling of inhibition of ammonia oxidation by nitrite under low dissolved oxygen conditions”. Journal of Environmental Engineering 134(3), 184-190 ISSN: 0301-4797

Contreras, E.M., Albertario, M.A., Bertola, N.C., Zaritzky, N.E. 2008b. “Modelling phenol biodegradation by activated sludges evaluated through respirometric techniques”. Journal of Hazardous Materials 158(2-3), 366-374 ISSN: 0304-3894

Contreras, E.M., Ferro Orozco, A.M., Zaritzky, N.E. (2011) “Biological Cr(VI) removal coupled with biomass growth, biomass decay, and multiple substrate limitation”. Water Research 45, 3034-3046 ISSN: 0043-1354

Farrell A, Quilty B (1999) Degradation of mono-chlorophenols by a mixed microbial community via a meta- cleavage pathway. Biodegradation 10:353-362 doi:10.1023/a:1008323811433

Ferro Orozco, A.M., Contreras, E.M., Zaritzky, N.E. 2010 “Dynamic response of combined activated sludge-powdered activated carbon batch systems” Chemical Engineering Journal 157, 331-338 ISSN: 1385-8947

Gao J, Ellis LB, Wackett LP (2009) The University of Minnesota biocatalysis/biodegradation database: improving public access Nucleic acids research:gkp771

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