Bioprocess and Biosystems Engineering

Influence of linear alkylbenzene sulfonate and ethanol on

the degradation kinetics of domestic sewage in co-digestion
with commercial laundry wastewater

Journal: Bioprocess and Biosystems Engineering

Manuscript ID BPBSE-19-0018.R1

Type of Manuscript: Research Paper

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Date Submitted by the

11-Mar-2019
Author:
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Complete List of Authors: Macedo, thais; Universidade de Sao Paulo Escola de Engenharia de Sao
Carlos, Hydraulic and Sanitation
Silva, Edson Luiz; Federal University of São Carlos, Chemical Engineering
Zaiat, Marcelo; Universidade de Sao Paulo Escola de Engenharia de Sao
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Carlos, Hidráulica e Saneamento;

sakamoto, Isabel; Universidade de Sao Paulo Escola de Engenharia de
Sao Carlos, Hydraulic and Sanitation
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Varesche, Maria Bernadete; USP, SHS; USP, SHS

Keywords: sand, DGGE, substrate inhibition, co-substrate, microbial diversity

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Page 1 of 40 Bioprocess and Biosystems Engineering

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Influence of linear alkylbenzene sulfonate and ethanol on the degradation kinetics
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7 of domestic sewage in co-digestion with commercial laundry wastewater
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10 Thaís Zaninetti Macedo1*, Edson Luiz Silva2, Isabel Kimiko Sakamoto1, Marcelo
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12 Zaiat1 and Maria Bernadete A Varesche1
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15 Corresponding author e-mail: thazmacedo@gmail.com
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18 Thaís Zaninetti Macedo - Laboratory of Biological Processes, Department of Hydraulics and
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Sanitation, Engineering School of São Carlos – University of São Paulo (EESC – USP) Campus
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23 II, São Carlos, SP CEP 13563-120, Brazil, +55 (16) 3373-8356, thazmacedo@gmail.com.
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Edson Luiz Silva - Department of Chemical Engineering, Federal Univ. of São Carlos, Rod.
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28 Washington Luiz, Km 235, SP 310, São Carlos 13565-905, São Paulo, Brazil, +55(16) 3351-
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30 8042, edsilva@ufscar.br
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33 Isabel Kimiko Sakamoto - Laboratory of Biological Processes, Department of Hydraulics and
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35 Sanitation, Engineering School of São Carlos – University of São Paulo (EESC – USP) Campus
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37 II, São Carlos, SP CEP 13563-120, Brazil, +55 (16) 3373-8357, varesche@sc.usp.br.
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40 Marcelo Zaiat - Laboratory of Biological Processes, Department of Hydraulics and Sanitation,
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42 Engineering School of São Carlos – University of São Paulo (EESC – USP) Campus II, São
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44 Carlos, SP CEP 13563-120, Brazil, Tel: +55 (16) 3373-8360, zaiat@sc.usp.br.
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47 Maria Bernadete A Varesche - Laboratory of Biological Processes, Department of Hydraulics
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49 and Sanitation, Engineering School of São Carlos – University of São Paulo (EESC – USP)
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51 Campus II, São Carlos, SP CEP 13563-120, Brazil, +55 (16) 3373-8356, varesche@sc.usp.br.
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 Bioprocess and Biosystems Engineering Page 2 of 40

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Influence of linear alkylbenzene sulfonate and ethanol on the degradation kinetics
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7 of domestic sewage in co-digestion with commercial laundry wastewater
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10 Abstract
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13 The influence of ethanol on the degradation kinetics of linear alkyl benzene sulfonate (LAS) and
14 organic matter was investigated using batch experiments with different initial LAS
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16 concentrations (8.3 mg L-1 to 66.9 mg L-1) and biomass immobilized on sand. The data were
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fitted with a substrate inhibition model. Concentrations of 2.4 mg LAS L-1 and 18.9 mg LAS L-1
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19 (without and with ethanol) provided the maximum LAS utilization rate by the biomass (Sbm).
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21 For LAS degradation, ethanol addition favored a lower decrease in the specific substrate
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22 utilization rate (robs), even at the LAS concentration usually reported as inhibitory (>14.4 mg L-
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1). For organic matter degradation, robs was higher with ethanol. Higher biomass differentiation
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25 was observed at higher LAS concentrations. With ethanol, microbial selection occurred at LAS
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27 concentrations near Sbm. At higher LAS concentrations, the dominance and diversity values did
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not change significantly with ethanol, whereas without ethanol, their behaviors were irregular.
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32 Keywords: sand; DGGE; substrate inhibition; co-substrate; microbial diversity

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35 Introduction
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38 The presence of linear alkyl benzene sulfonates (LAS) in frequently used
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40 products, such as personal care and household products and specifically in laundry
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detergents, results in considerable concentrations of LAS in wastewater. LAS is present
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45 in domestic sewage in concentrations ranging from 1 mg LAS L−1 to 18 mg LAS L−1 [1]
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47 and in higher amounts in commercial laundry wastewater (12 to 1024 mg LAS L−1 [2]).
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49 Surfactants are necessary and irreplaceable compounds in this application due to
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52 their excellent cleaning, emulsification, and foaming properties, as well as their low cost
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54 [3]. In general, wastewater treatment plants (WWTP) are not designed to remove
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56 surfactants. Furthermore, the presence of surfactants may result in damage to the
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performance and operation of WWTP due to foam formation and/or biological toxicity
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Page 3 of 40 Bioprocess and Biosystems Engineering

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[4]. Competitive inhibition in activated sludge biomass was recently demonstrated by
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7 [5]. In addition, the presence of LAS decreases the substrate affinity for biomass and
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9 decreases the maximum growth rate [6]. High LAS concentrations (100 mg L-1) in
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11 activated sludge systems increase inhibition in the system degradation capacity,
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14 resulting in elevated effluent residual chemical oxygen demand (COD) [7].
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16 Although several investigations regarding aerobic and anaerobic biological LAS
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18 degradation have been reported, there is still much information, mainly concerning the
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actual wastewater, to explore to improve such studies. Among bench-scale biological
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23 reactors, the expanded granular sludge bed (EGSB) reactor and the fluidized bed reactor
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25 (FBR) have been used with commercial standard LAS and with laundry wastewater
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containing LAS treatment, showing removal efficiencies greater than 90% [8,9,10]. The
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30 EGSB reactor was applied in the co-digestion of commercial laundry wastewater with
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32 domestic sewage on the pilot and bench scales [11,12, respectively]. In all cases, the
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34 removal efficiencies of LAS were lower than 60% for LAS concentrations above 20 mg
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37 L-1.
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39 A great challenge in LAS biological treatment is to avoid a reduction in the

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41 removal efficiency at high concentrations. In a FBR, the LAS removal efficiency
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decreased from 75% to 65% when the LAS influent concentration increased from 10 mg
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46 L-1 to 23 mg L-1 in diluted laundry wastewater [13]. In an EGSB reactor, the LAS
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48 removal decreased from 93% to 59% when the LAS concentration in diluted laundry
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wastewater increased from 12 mg L-1 to 29 mg L-1 [14]. Motteran et al. [15] also
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53 correlated high LAS concentrations from laundry wastewater to microbial community
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55 inhibition involved in methane production in bath reactors.
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A high LAS concentration may result in toxicity to microorganisms. Garcia-
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7 Morales et al. [16] observed 50% acidogenic activity inhibition (EC50) with a LAS
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9 concentration of 18.9 mg L-1, while methanogenic microbial were more LAS sensitive
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11 (EC50 of 6.3 mg L-1). LAS inhibition in biological reactors was observed for
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14 concentrations above 20 mg L-1 by Macedo et al. [10], and the addition of co-substrate
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16 may influence the biological performance. Macedo et al. [17] observed an increase in
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18 the surfactant removal efficiency (51% to 73% of the LAS removal efficiency for ~20
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mg L-1 of LAS influent) when ethanol was added as a co-substrate in a FBR used to
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23 treat laundry wastewater compared to the addition of sucrose and ethanol. These authors
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25 concluded that the microbial community capable of degrading LAS was favored in the
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presence of only ethanol. Andrade et al. [18] evaluated the optimal conditions of LAS
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30 degradation in denitrifying assay tests and found that ethanol (co-substrate) and nitrate
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32 (electron acceptor) were statistically significant factors (p < 0.05) in surfactant removal,
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34 except for at high LAS concentrations (60 mg L-1 LAS).
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37 The co-metabolism depends on the specific substrate as well as on the microbial
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39 species. Some studies reported that the initial LAS concentration and the addition of
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41 ethanol as a co-substrate interfered with the LAS removal efficiency. Estimation of the
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kinetic parameters can provide a better understanding of the degradation processes.
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46 However, few studies have reported data for the behavior of specific kinetic parameters.
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48 Moreover, although the LAS inhibition kinetics have been suggested in the literature, no
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study has identified or modeled the LAS inhibition kinetics in the presence of ethanol.
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53 Thus, evaluating the LAS degradation kinetics of specific wastewater compositions
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55 could be useful to optimize its subsequent biological treatment.
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Page 5 of 40 Bioprocess and Biosystems Engineering

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In this context, this work intends to estimate the kinetic parameters of LAS
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7 degradation and organic matter uptake using ethanol as a metabolic co-substrate.
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9 Denaturing gradient gel electrophoresis (DGGE) was employed to verify differentiation
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11 in the microbial composition of the biomass adhered to the support material according
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14 to changes in the LAS concentration or substrate composition.
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16 2. Materials and methods
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18 2.1. Support material colonization and biomass adaptation
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21 Before performing the assay tests, a FBR was operated for 45 days in 3 different stages
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24 for support material colonization and activation of the biomass enzymatic apparatus in
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26 the presence of LAS with laundry wastewater and domestic sewage (Table 1). The
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28 reactor was made of PVC and acrylic with a working volume of 19.8 L (2.52 m high;
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31 0.1 m diameter) and operated with a hydraulic retention time (HRT) of 18 h. The
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33 upflow velocity was constant with effluent recirculation (24 m h-1). Sand (0.84-1.19 mm
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diameter) was used as the support material [8].

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2.2. Inoculum
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41 The FBR was inoculated in a closed circuit with inoculum from the activated sludge
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43 system applied to the domestic sewage treatment system of the Volkswagen Company
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45 (São Carlos, SP, Brazil) [19]. The sludge had a total volatile solid (TVS) concentration
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of 3.9±0.08 g TVS L-1. A period of 38 days was enough to reach a reactional steady
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50 state. For 7 days, under the same operational conditions, the reactor was fed in an open
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52 circuit to wash out excess biomass (Table 1). In stage I of inoculation, the reactor was
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fed with a synthetic substrate consisting of yeast extract (200 mg L-1), ethanol (50 mg L-
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1), salt solution (50 g L-1 NaCl, 1.4 g L-1 MgCl2, and 0.9 g L-1 CaCl2), and sodium
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59 bicarbonate [17] in addition to tap water. In stages II and III, the reactor was fed
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domestic sewage (191±6 mg COD L-1) plus laundry wastewater (658±8 mg COD L-1)
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7 (~20 mg LAS L-1) and 200 mg L-1 sodium bicarbonate (Table 1).
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9 2.3. Collection and characterization of laundry wastewater and domestic
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11 sewage
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The laundry wastewater was collected from a commercial facility located in São
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17 Carlos, SP, Brazil and stored in 5 and 10 L high-density polyurethane bottles at 4°C.
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19 Domestic sewage was collected directly through a wastewater interceptor from the
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21 neighborhood near Campus 2 of the University of São Paulo (São Carlos, Brazil). The
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24 commercial laundry wastewater and domestic sewage were characterized previously
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26 (Table 2).
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28 2.4. Experimental design of assays
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The reactors were composed of 2 L bottles with a reaction volume of 1 L, which
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34 contained a LAS mixture of laundry wastewater and domestic sewage in volumetric
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36 proportions, which resulted in different initial LAS concentrations between 8.3 mg L-1
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38 and 66.9 mg L-1 (Table 3). All tests were performed in duplicate in the presence (assay
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41 I) and in the absence of ethanol (assay II) as a metabolic co-substrate (50 mg L-1;
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43 Macedo et al. 2015). Sodium bicarbonate was added as a buffering agent (200 mg L-1).
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45 Batch reactors were inoculated with biomass attached to sand obtained from FBR
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operation, with 24.4 mg SSV L-1. The reactors were agitated at 120 rpm at a constant
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50 temperature of 30°C for 144 h. In R5 and R5’, the wastewater composition was
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52 supplemented with commercial standard LAS to obtain higher LAS concentrations.
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2.5. Physico-chemical analysis
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Page 7 of 40 Bioprocess and Biosystems Engineering

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To characterize the wastewater, analyses of COD, pH, and volatile suspended
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7 solids (VSS) were performed according to Standard Methods for Examination of Water
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9 and Wastewater [20]. The alkalinity was quantified by titration according Ripley et al.
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11 [21]. Volatile fatty acids (VFAs) were quantified by HPLC according Penteado et al.
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14 [22]. The concentration of LAS was obtained using online column-switching solid-
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16 phase extraction (SPE) coupled with liquid chromatography/tandem mass spectrometry
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18 (LC-MS/MS) [23]. For extracting adsorbed LAS, the sand with attached biomass was
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collected at the end of the operation and washed three times with methanol according to
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23 Duarte et al. [24]. The LAS mass balance included the surfactant added (influent),
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25 recovered in the effluent (liquid phase) and adsorbed on the biomass and support
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material in the reactor [25].
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30 2.6. Degradation kinetics parameters of LAS and COD
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32 Profiles of LAS and COD concentration in the bulk liquid against time were
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34 obtained for each reactor in distinct 144-h experiments. The filtered COD and LAS
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37 analyses of samples were taken at the initial time and after 2 h, 4 h, 6 h, 8 h, 12 h, 24 h,
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39 36 h, 48 h, 60 h, 72 h, 96 h, and 144 h.
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41 At the end of the experiments, the sand was washed with caustic soda and
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distilled water to remove the attached biomass. Then, the VSS mass was determined
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46 according to Chen and Chen [26]. The biomass concentration (X) was obtained by
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48 taking the mass of VSS measured divided by the total liquid volume of the system.
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The initial observed specific substrate utilization rates (robs) were calculated as a
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53 function of COD and LAS (Sb) for each initial substrate concentration by adjusting the
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55 COD and LAS profiles using polynomial regression analysis according Zaiat et al. [27]
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57 (equation 1):
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7 (1)
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11 The behavior of robs as a function of LAS concentration was analyzed to verify
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13 whether inhibition occurred through an adaptation of the “Rapid method to assess
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16 substrate inhibition in anaerobic fixed-bed reactors for wastewater treatment” described
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18 by Zaiat et al. [27]. A substrate inhibition expression presented by Andrews [28],
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20 proposed originally for enzymatic kinetics [29], was adjusted to the fit the experimental
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data through the rearranged second-order polynomial expression described Zaiat et al.
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[27] to estimate the apparent kinetic parameters (equation 2).
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30 (2)
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where robs is the initial observed specific substrate utilization rate, is the apparent
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38 maximum specific reaction rate that would be achieved if no inhibition occurred, is
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the initial substrate concentration is the apparent constant of substrate saturation
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43 and is the apparent constant of substrate inhibition.
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46 Data of /robs were plotted as a function of LAS ( ), and the rearranged
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48 inhibition expression [27] was fitted to the experimental data, thus permitting the
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estimation of the apparent kinetic parameters.
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53 The substrate concentration at which the biomass activity is inhibited was
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estimated from equation 2 for =0. In such conditions, the resulting equation 3 is:
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5 (3)
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where is the LAS concentration that provides the maximum substrate (LAS/COD-
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13 LAS) utilization rate by the biomass.
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15 2.7. DNA extraction/PCR–DGGE
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18 At the end of the experiments, samples of attached biomass were collected for
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20 analysis by PCR–DGGE in the Bacteria domain [25]. Total DNA extraction was
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22 performed according to Griffiths et al. [30]. Primers 968F (with a clamp GC) and
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1392R [31] were used. The DGGE banding patterns were analyzed using BioNumerics
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27 V3.5 software. The similarity coefficients were determined according to the Pearson
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29 coefficient, and the dendrogram was determined by an unweighted pair group method
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with an arithmetic average (UPGMA) algorithm using BioNumerics V3.5. The Alpha
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34 diversity (Chao1, Shannon, Simpson and Dominance) was quantified using Past
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36 software.
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3. Results and Discussion

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41 3.1. LAS kinetic degradation
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44 The initial observed specific substrate utilization rates (robs) obtained as a
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46 function of LAS concentration in experiments with and without ethanol are presented in
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48 Table 4. The values of robs were calculated for a 24.4 mg VSS L-1 biomass concentration
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51 (X).
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53 The robs values increased with the LAS concentration from R1 to R3 (8.7 mg
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55 LAS L-1 to 24.0 mg LAS L-1) and from R1’ to R3’ (8.3 mg LAS L-1 to 24.2 mg LAS L-
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57 1).
58 Similarly, the values of robs from the assays were similar in R1 and R1’, R2 and R2’,
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and R3 and R3’ (Table 1). A decrease in robs was observed for higher LAS initial
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7 concentrations (R4, R5, R4’ and R5’). Such decrease was less noticeable in the presence
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9 of ethanol (assay II), with robs values almost two times higher in assay II (with ethanol)
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11 for R4’ and R5’ than in assay I (without ethanol) for R4 and R5.
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14 Therefore, the maximum robs was reached at intermediary substrate
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16 concentrations, revealing the occurrence of inhibition. In this way, the substrate
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18 inhibition expression presented by Andrews [28], originally proposed for enzymatic
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kinetics [29], was adjusted to the data through a rearranged second-order polynomial
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23 expression according Zaiat et al. [27] (Figure 1).
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25 Data of S0/robs were plotted as a function of LAS without (assay I) or with

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ethanol (assay II), and the rearranged inhibition expression [27] was fitted to the
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30 experimental data (Figure 1(a)), thus permitting estimation of the apparent kinetic
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32 parameters (Table 5).

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34 Maximum overall rates (kapp) of 0.069 mg LAS mg-1 SSV h-1 and 0.0510 mg
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37 LAS mg-1 SSV h-1 were observed in assays I and II, respectively, and the maximum
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39 substrate utilization rate was reached when the substrate inhibition (r*) was 0.051 mg
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41 LAS mg-1 SSV h-1 and 0.033 mg LAS mg-1 SSV h-1 in assays I and II, respectively
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(Figure 1(b)).
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46 Although the values of kapp and r* were higher in assay I, the obtained values for
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48 Ksapp and Kiapp were higher in assay II (with ethanol). Furthermore, the LAS
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concentration that provides the maximum substrate utilization rate by the biomass (Sbm)
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53 was found to be 2.39 mg LAS L-1 in the absence of ethanol (assay I) and much higher,
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55 18.98 mg LAS L-1, in the presence of ethanol (assay II) (Table 2). These values are in an
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57 LAS concentration range that can be found in some industrial and urban residual waters.
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Mungray and Kumar [1] reported concentrations of 1-18 mg L-1 in domestic sewage.
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7 Oviedo et al. [7] reported that surfactant can reach concentrations up to 20 mg L-1 in
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9 sewage treatment plants (STP). Therefore, according to the results presented herein,
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11 ethanol addition could facilitate the biological treatment of high LAS concentrations.
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14 However, co-substrate addition may be unnecessary to treat wastewater with low
15
16 concentrations of LAS.
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18 Although the specific substrate utilization rates (robs) are supposed to be lower
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for LAS concentrations above Sbm, a slight decrease was observed for a large range of
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23 substrate concentrations in assay II, as presented in Figure 1 (b).
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25 The robs values were higher in assay I (without ethanol) than in assay II (with
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ethanol) for LAS concentrations above 14.4 mg LAS L-1 (graph intersect in Figure
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30 1(b)). After that, in the absence of ethanol, the specific substrate utilization rate
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32 decreases to approximately half of the maximum value. The specific substrate

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34 utilization rate (robs) slightly decreased in assay II (with ethanol) compared with that in
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37 assay I. Furthermore, ethanol addition favored higher values of specific substrate
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39 utilization at higher LAS concentrations. Therefore, ethanol addition is unnecessary for

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41 LAS concentrations below 14.4 mg LAS L-1, which leads to cost savings in the
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treatment of wastewater with low amounts of LAS. However, alcohol addition could be
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46 helpful in improving the degradation rates of LAS when higher surfactant
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48 concentrations are present.
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Interestingly, most studies on LAS inhibition report a LAS concentration close
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53 to that observed at the intersection point (Figure 1(b)). In most anaerobic biological
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55 reactor studies, for influent concentrations exceeding 15 mg L-1, in general, reactors
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57 present instability in COD and LAS removal [9,32,33,13,14;12,11]. Mösche e Meyer
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[34] detected an immediate 50% inhibition of acetate degradation at concentrations
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7 above 14 mg LAS L-1. According Garcia et al. [35], taking into account the homologue
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9 distribution of commercial LAS in the liquid phase of the anaerobic digesters of a
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11 WWTP, an EC50 of 14 mg L-1 can be considered for LAS toxicity on anaerobic
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14 microorganisms.
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16 Therefore, the addition of 50 mg L-1 ethanol favored higher specific substrate
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18 utilization rates (robs), with a slight decrease at higher LAS values, as observed in assay
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II. Macedo et al. [17], using ethanol to replace sucrose as the co-substrate, observed an
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23 improvement in the efficiency of LAS removal in a FBR. Those authors reported the
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25 importance of ethanol to reactivate and favor the diversity of bacteria capable of

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degrading LAS. Under inhibitory conditions, when the substrate requires several
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30 transformation steps, the complexity of the biotransformation reactions increases
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32 significantly. Therefore, simple organic compounds such as ethanol could be useful for
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34 reactivating bacteria capable of degrading LAS [36,17]. Thus, in this sense, ethanol
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37 probably favored R4’ and R5’ in assay II, once the inhibitory parameters of degradation
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39 were less than those in assay I (Table 5).

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41 The minimum residual LAS was measured in the reference assay (0.3 mg L-1),
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and the maximum value was observed in assay I of R5 (27.9 mg L-1) (Table 6). The
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46 final LAS removal efficiency was significantly lower in assay I of R4 and R5 (without
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48 ethanol) (77.5% and 56.1%, respectively). In both assays, lower robs values were
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50
obtained for higher LAS concentrations.
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53 Andrade et al. [19] investigated LAS degradation under anoxic conditions using
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55 central composite design. Although they verified that ethanol (co-substrate) and nitrate
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57 (electron acceptor) were statistically significant factors (p < 0.05) in LAS removal, they
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did not observe an improvement in LAS removal with the addition of ethanol and
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7 nitrate at the highest LAS concentration (kLAS decreased from 0.054 h−1 to 0.011 h−1 for
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9 LAS concentration of 27 mg LAS L−1 and 60 mg LAS L−1, respectively). These authors
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11 attributed this decrease to a supposed inhibition in the presence of excess substrate.
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14 The volume of the laundry wastewater increased with increasing LAS
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16 concentration (R1/R1’ to R5/R5’). In the experiments with different LAS
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18 concentrations, there is a combined inhibitory effect: the toxic effect generated by the
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presence of LAS and the inhibitory effect of other inhibitory compounds present in the
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23 wastewater composition, mainly the laundry wastewater. Braga and Varesche [2]
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25 detected thirty-three different xenobiotic organic compounds in commercial laundry

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wastewater through qualitative screening. The major groups of these compounds were
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30 fragrances, preservatives, solvents and surfactants. The majority were classified as
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32 inhibitory to various microorganisms. These compounds present in laundry wastewater

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34 are also diluted in raw sewage, which includes other inhibitory compounds such as
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37 pesticides, fertilizers, antibiotics and pharmaceutical products that could be toxic to
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39 microorganisms. In the present study, the dilution of laundry wastewater with domestic
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41 sewage likely results in a large amount of these inhibitory compounds.
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Motteran et al. [15] evaluated LAS degradation and the kinetics of COD
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46 degradation and methane production for different inoculum and LAS sources
47
48 (commercial standard LAS and laundry wastewater). The authors concluded that other
49
50
compounds in laundry wastewater directly influence the LAS degradation kinetics and
51
52
53 methane production.
54
55 3.2. Organic matter kinetic degradation
56
57
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14
1
2
3
4
For organic matter degradation in terms of filtered COD, the robs values were
5
6
7 higher in assay II (with ethanol) than in assay I (without ethanol) for all reactors (Table
8
9 7).
10
11 As an increased LAS concentration was associated with an increase in the
12
13
14 volume of laundry wastewater, which presents a higher COD than domestic sewage, the
15
16 initial COD in the reactors increases with LAS concentration. In general, ethanol
17
18 addition (assay II) also contributes to a higher COD.
19
20
21
Comparing the reactors of assay II (with ethanol) and assay I (without ethanol),
Fo

22
23 even with the higher initial COD, higher values of robs were observed in assay II (with
24
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25 ethanol). The initial LAS concentration likely affected the COD degradation rate more
26
27
than the initial COD concentration. In this sense, in an unconventional method for unit-
28
ee

29
30 of-measurement, the change in the specific substrate utilization rate was a function of
31
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32 the LAS concentration in terms of COD. The behavior of the initial observed specific
33
34 substrate utilization rate (robs) as a function of LAS concentration in terms of COD
35
ev

36
37 (COD-LAS) for each reactor was investigated to verify whether excess LAS provided
38
iew

39 an inhibitory effect (Figure 2). The values of robs shown in Tables 4 and 5 were
40
41 calculated for a biomass concentration (X) of 24.4 mg VSS L-1. Herein, instead of
42
43
44
employing the initial COD concentration, the initial LAS concentration was applied in
45
46 terms of COD to verify the substrate inhibition.
47
48 Data of S0/robs were plotted as a function of LAS concentration in terms of COD
49
50
(COD-LAS) without (assay I) or with ethanol (assay II), and the rearranged inhibition
51
52
53 expression [27] was fitted to the experimental data (Figure 1(a)), thus permitting the
54
55 estimation of the apparent kinetic parameters (Table 5).
56
57
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Page 15 of 40 Bioprocess and Biosystems Engineering

15
1
2
3
4
Ethanol addition favored higher values of kapp and r* in assay II (Table 8), likely
5
6
7 due to the higher initial COD. However, Sbm was slightly lower in the presence of
8
9 ethanol (Table 5). The higher the LAS concentration, the more the presence of ethanol
10
11 favored higher values of robs for COD degradation (reaching values 46.2% higher for
12
13
14 24.8 mg LAS L-1) (Figure 2 (b)).
15
16 The inhibition resulting from increased LAS concentration affects the COD
17
18 removal efficiency. Lower COD removal was observed for higher LAS concentrations.
19
20
21
Oviedo et al. [7] reported that the presence of LAS affects the degradation capacity of
Fo

22
23 organic matter and LAS in activated sewage systems. In the present study, COD
24
rP

25 removal efficiencies of approximately 80% or higher were observed. In the absence of

26
27
ethanol, the COD removal efficiency decreased to approximately 70% in R4 and R5
28
ee

29
30 (40.4 mg LAS L-1 and 63.5 mg LAS L-1 of the LAS concentration in R4 and R5,
31
rR

32 respectively). In the presence of ethanol, the removal efficiency in R5’ was less than
33
34 80% (73% removal efficiency for 66.9 mg LAS L-1; Table 9).
35
ev

36
37 Decreased organic matter degradation in the presence of LAS is commonly
38
iew

39 reported in assay experiments. Duarte et al. [37] observed that 22 mg LAS L-1 resulted
40
41 in a decrease in the COD removal efficiency of 74±19% to 44±14% employing ASBR.
42
43
44
Souza et al. [38] observed that the conversion of organic matter was strongly inhibited
45
46 by increasing the initial LAS concentration (up to 100 mg L-1) when anaerobic sludge
47
48 was used as the inoculum in assay flasks containing acetic, propionic and butyric acids,
49
50
corresponding to a total COD of 3 g L-1 and COD removal efficiencies of 95.5%, 95%,
51
52
53 79%, 59%, 25%, and 7% for 0, 10, 30, 50, 75, and 100 mg LAS L-1, respectively.
54
55 Oviedo et al. [7] and Karahan [5] also reported that LAS influenced the
56
57 degradation capacity of both organic matter and itself in aerobic processes. According
58
59
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 Bioprocess and Biosystems Engineering Page 16 of 40

16
1
2
3
4
to Karahan [5], LAS exhibits non-competitive inhibition on the hydrolysis mechanism
5
6
7 of synthetic sewage and imposes competitive inhibition on the growth of biomass [5]. In
8
9 the present study, COD degradation inhibition was observed as the LAS concentration
10
11 increased. However, ethanol addition weakened such inhibition.
12
13
14 3.3. pH and VFAs
15
16 Initial pH values between 7.4 and 7.8 were observed in all flasks (Table 10),
17
18 which underwent a modest decrease, reaching final values between 6.4 and 6.8.
19
20
21
Microorganisms can be inhibited by the product or substrate, the physical characteristics
Fo

22
23 of the medium (pH or temperature), or inhibitory substances, including organic or
24
rP

25 inorganic substances [16]. In the present study, the pH of the medium is not low
26
27
enough to be inhibitory, and thus, the inhibition in this assay must be due to the
28
ee

29
30 surfactant.
31
rR

32 There was no significant accumulation of VFA in the system. The final VFA
33
34 concentrations were lower than the initial concentrations. The highest final values were
35
ev

36
37 observed in R4 and R5 for assay I and in R4’ for assay II (60 mg HAc L-1, 91 mg HAc
38
iew

39 L-1 and 94 mg HAc L-1, respectively). The production of VFA is a key factor in the
40
41 degradation of LAS. Lobner et al. [39] operating an upflow anaerobic sludge blanket
42
43
44
(UASB) reactor with an HRT of 48 h with 10 mg LAS L-1 influent observed greater
45
46 LAS removal for concentrations of volatile organic acids below 50 mg HAc L-1. Okada
47
48 et al. [40] reported that the presence of VFAs affected the LAS removal stability when
49
50
UASB and EGSB reactors were used. These authors observed that in the UASB reactor,
51
52
53 the LAS removal decreased (20–25%) in the presence of 2–45 mg L−1 acetic acid,
54
55 whereas in the EGBS reactor, the LAS removal increased (40–80%) at low
56
57 concentrations of acetic acid (1–22 mg L−1), probably because of the EGSB
58
59
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Page 17 of 40 Bioprocess and Biosystems Engineering

17
1
2
3
4
recirculation rate. In the present study, despite that the VFA values in terms of acetic
5
6
7 acid were higher than the concentrations reported as inhibitory in R1, R4 and R5 in
8
9 assay I and in R3 and R4 in assay II, the VFA value was not correlated with reduced
10
11 LAS removal.
12
13
14 3.4. LAS adsorption
15
16 Figure 3 illustrates the LAS mass balance at the end of the experiments. In assay
17
18 I, LAS adsorption reached the highest value in R4 (8%), and the biodegradation
19
20
21
percentage was only 72% in that reactor (Figure 3(c)). In assay II, the lowest
Fo

22
23 biodegradation percentage was also observed in R4’ (88%) with a high percentage of
24
rP

25 LAS adsorption (3.2%). A greater volume of laundry wastewater was added in R4 and
26
27
R4’ than in the other reactors in both assays. According to Westall et al. [41], ions such
28
ee

29
30 as Ca2+ and Mg2+ influence LAS adsorption. The presence of such ions in the laundry
31
rR

32 wastewater composition may have promoted LAS adsorption due to the reduced
33
34 electrostatic repulsion between ionic parts of the surfactant and sludge [32]. In R2’, a
35
ev

36
37 high LAS adsorption (5.5%) was also observed, and LAS biodegradation reached
38
iew

39 92.3%. In the other reactors, the LAS adsorption was below 0.74% of the initial LAS
40
41 concentration. Oliveira et al. [8] studied the adsorption capacities of different support
42
43
44
materials (activated carbon, expanded clay, glass beads and sand) and reported lower
45
46 surfactant adsorption in sand (0.2%), suggesting that use of sand is beneficial because it
47
48 favors the development of biofilms capable of supporting LAS degradation.
49
50
3.5. Effect of LAS concentration and ethanol addition on the microbial
51
52
53 community structure
54
55 According to the Jaccard similarity coefficient of the PCR–DGGE banding
56
57 patterns, a higher initial LAS concentration resulted in a higher microbial population
58
59
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 Bioprocess and Biosystems Engineering Page 18 of 40

18
1
2
3
4
differentiation (Figure 4). For the Bacteria domain, in relation to the initial facultative
5
6
7 biomass, which was previously adapted in a FBR, a higher differentiation was observed
8
9 in R4 and R5 in both assays (68% and 56% similarity, respectively). These reactors
10
11 were subjected to the most extreme LAS and COD concentrations and presented a
12
13
14 different behavior (lesser robs) than the reactors under other conditions.
15
16 Delforno et al. [32] reported that the granular biomass of an EGSB reactor
17
18 treating laundry wastewater with low LAS concentration (~10 mg LAS L-1), previously
19
20
21
adapted to standard LAS (Sigma-Aldrich), showed higher coefficient similarity to
Fo

22
23 inoculum and to biomass not adapted (80%). The observed LAS removal efficiency was
24
rP

25 similar to the adapted and not adapted biomass. In the present study, despite biomass
26
27
adaptation, a high LAS concentration favored biomass differentiation and resulted in
28
ee

29
30 different LAS removals and kinetic parameters. Andrade et al. [18] evaluated LAS from
31
rR

32 laundry wastewater degradation in optimized conditions by anoxic batch reactors.

33
34 Under these conditions, the LAS concentration affected the selection of the microbial
35
ev

36
37 population, and a similarity coefficient of 54% was found between the biomass of the
38
iew

39 reactors with 27 mg LAS L-1 and 60 mg LAS L-1.

40
41 The biomass samples taken from R5 and R5’ also presented the lowest similarity
42
43
44
coefficient in relation to the biomass samples taken from the other reactional conditions,
45
46 with the differentiation being higher in the presence of ethanol: 66% between the
47
48 biomass of R1 and R5, R3 and R5, and R4 and R5; 68% between the biomass of R2 and
49
50
R5 in assay I; and 57% between R1’ and R5’, R2’ and R5’, R3’ and R5’, and R4’ and
51
52
53 R5’ in assay II. For the other reactors, the similarity coefficients between biomass
54
55 samples were greater than or equal to 70%.
56
57
58
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Page 19 of 40 Bioprocess and Biosystems Engineering

19
1
2
3
4
Motteran et al. [42] evaluated the degradation of high concentrations of nonionic
5
6
7 surfactant (linear alcohol ethoxylate) in an anaerobic FBR. The authors observed that
8
9 the absence of sucrose altered the microbial community. The similarity coefficient
10
11 between the phases with and without sucrose was 59% for the Bacteria domain. Unlike
12
13
14 in the present work, the authors obtained higher LAE removal (98%) in the absence of
15
16 co-substrate. Macedo et al. [17] reported that the microbial community that developed
17
18 in response to an ethanol-only co-substrate improved LAS degradation more so than the
19
20
21
community that developed in response to a mixture of sucrose and ethanol, suggesting
Fo

22
23 that ethanol is a better option for enriching a LAS-degrading microbial community.
24
rP

25 The addition of ethanol resulted in a higher biomass differentiation between

26
27
assays to higher LAS concentrations in R5. The similarity coefficients were 88%, 83%,
28
ee

29
30 90%, 82%, and 57% between R1 and R1’, R2 and R2’, R3 and R3’, R4 and R4’, and R5
31
rR

32 and R5’, respectively. In fact, the kinetic constant of LAS degradation was two times
33
34 higher in R5’ than in R5, and the LAS removal efficiency was much higher in R5’ with
35
ev

36
37 ethanol (56.1% and 98.8% LAS removal efficiency in assays I and II, respectively).
38
iew

39 Although a higher degradation rate was observed in R4’ in the presence of ethanol,
40
41 there was no such significant differentiation among populations, and the similarity
42
43
44
coefficient was higher than that in R5’. The LAS removal efficiencies between assays
45
46 were closer in R4’ than in R5’ (77.5% and 95.6% LAS removal efficiency in assays I
47
48 and II, respectively).
49
50
Figure 5 illustrates the effects of increased LAS concentration and ethanol
51
52
53 addition on the biomass dominance (D) and diversity (Shannon_H).
54
55 In the presence of ethanol, changes in the microbial α-diversity at approximately
56
57 24 mg LAS L-1 were observed. The dominance index (0.09541) was high in R3’ (24.2
58
59
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 Bioprocess and Biosystems Engineering Page 20 of 40

20
1
2
3
4
mg LAS L-1). In that condition, a low diversity value was also observed (Shannon
5
6
7 diversity index, 2.369). In R3, for both assays, the highest robs value for LAS
8
9 degradation was observed, followed by a decrease in the robs value (graph inflection
10
11 point; Figure 1(b)). After that, at higher initial LAS concentrations (R4 and R5), the α-
12
13
14 diversity indexes did not change significantly. In fact, the substrate concentration that
15
16 provides the maximum substrate utilization rate by the biomass (Sbm) was 18.98 mg
17
18 LAS L-1 in the presence of ethanol (assay II), and the resulting adjusted kinetic model
19
20
21
shows a slight decrease in that rate for a large range of substrate concentrations.
Fo

22
23 Therefore, a microbial community that was adapted to the surfactant and capable of
24
rP

25 LAS degradation was possibly selected when the diversity was lower and the
26
27
dominance was higher.
28
ee

29
30 Andrade et al. [18] evaluated LAS removal in anoxic assay reactors and
31
rR

32 observed that the dominance rate increased and the diversity rate decreased with
33
34 increasing LAS. According to these authors, the LAS concentration in the reactors may
35
ev

36
37 have selected microbial populations that were better adapted to the surfactant. Indeed,
38
iew

39 some of these populations may have been related to the microbiota involved in the
40
41 degradation of this compound. In the absence of ethanol, the dominance index
42
43
44
decreased until the LAS concentration was approximately 24 mg L-1, while the diversity
45
46 value increased. After that point, regular behavior was not observed; that is, the
47
48 diversity changed with increased LAS concentration (>40 mg L-1). In fact, Sbm was
49
50
much lower without ethanol (2.39 mg LAS L-1). Furthermore, the LAS removal
51
52
53 efficiency at higher LAS concentrations was lower in assay I (77.5% and 95.6% for R4
54
55 in assay I and assay II, respectively, and 56.1% and 98.8% in assay I and assay II,
56
57 respectively).
58
59
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Page 21 of 40 Bioprocess and Biosystems Engineering

21
1
2
3
4
4. Conclusion
5
6
7
8 A substrate inhibition model was used to fit the data. Ethanol had a positive effect on
9
10 the LAS degradation kinetics at concentrations higher than 14.4 mg LAS L-1. The
11
12 substrate concentration that provided the maximum LAS utilization rate by the biomass
13
14
(Sbm) was 2.4 mg LAS L-1 without ethanol and 18.9 mg LAS L-1 with ethanol. For COD
15
16
17 degradation, robs was higher in the presence of ethanol. Microbial selection occurred
18
19 with increased LAS concentration and ethanol addition. With ethanol, the dominance
20
21 index increased at a LAS concentration near Sbm and was almost constant at higher
Fo

22
23
24 concentrations, indicating the predominance of populations able to tolerate higher
rP

25
26 surfactant concentrations. Without ethanol, the bacterial population showed irregular
27
28 behavior.
ee

29
30
31
Acknowledgments
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32
33
34
35
The authors gratefully acknowledge the support provided for this study by the São
ev

36
37 Paulo Research Foundation (FAPESP; grants 2015/02640-2 and 2015/06246-7).
38
iew

39
40 References
41
42
43 [1]Mungray AK, Kumar P (2009) Fate of linear alkylbenzene sulfonates in the environment: A
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46 [2]Braga JK, Varesche MBA (2014) Commercial Laundry Water Characterisation. Am J of
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Anal Chem. 5:8-16
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[3]Bergé A, Wiest L, Baudot R et al (2017) Occurrence of multi-class surfactants in urban
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54 [4]Yuan CL, Xu ZZ, Fan MX et al (2014)Study on characteristics and harm of surfactants. J
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13 [8]Oliveira LL, Costa RB, Duarte ICS et al (2010) Anaerobic degradation of linear
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alkylbenzene sulfonate in fluidized bed reactor. Braz J Chem Eng 27(04):539-543
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20 [10]Macedo TZ, Delforno TP, Braga JK et al (2017) Robustness and Microbial Diversity of a
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24 [11]Centurion VB, Moura AGL, Delforno TP, et al (2018) Anaerobic co-digestion of
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39 [14]Delforno TP, Moura AGL, Okada DY et al (2015) Microbial diversity and the implications
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43 [15]Motteran F, Braga JK, Silva EL et al (2016) Kinetics of methane production and
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45 biodegradation of linear alkylbenzene sulfonate from laundry wastewater. J Environ Sci Heal -
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48 [16]Garcia-Morales JL, Nebot E, Romero LI et al (2001) Comparison between acidogenic and
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52 [17]Macedo TZ, Okada DY, Delforno TP et al (2015) The comparative advantages of ethanol
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Fo

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effluent by online column-switching liquid chromatography/tandem mass spectrometry. Sci
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39 anaerobic fixed-bed reactors for wastewater treatment. ICFAI J Environ Sci 2007(2):58-67
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41 [28]Andrews JF (1968) A mathematical model for the continuous culture of microorganisms
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44 [29]Griffiths RI, Whiteley AS, Donnell AG, Bailey MJ (2000) Rapid method for coextraction of
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DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based
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49 [30]Nübel U, Engelen B, Felsre A, et al (1996) Sequence heterogeneities of genes encoding 16S
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53 [31]Delforno TP, Moura a GL, Okada DY, Varesche MBA (2014)Effect of biomass adaptation
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to the degradation of anionic surfactants in laundry wastewater using EGSB reactors. Bioresour
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4 [32]Okada DY, Delforno TP, Etchebehere C et al (2014)Evaluation of the microbial community
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6 of up fl ow anaerobic sludge blanket reactors used for the removal and degradation of linear
7 alkylbenzene sulfonate by pyrosequencing Int Biodeter Biodegr 96:63-70
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9 [33]Mösche M, Meyer U (2002) Toxicity of linear alkylbenzene sulfonate in anaerobic
10 digestion: Influence of exposure time Water Res 36(13):3253-3260
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12 [34]Garcia MT, Campos E, Sánchez-Leal J et al (2006) Effect of linear alkylbenzene
13 sulphonates (LAS) on the anaerobic digestion of sewage sludge Water Res 40(15):2958-2964
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15 [35]Schörberl, Marl P (1989) Basic PrincipIes of LAS. Tenside Surfactants Deterg 26(2):86-94
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17 [36]Duarte ICS, Oliveira LL, Saavedra NK, et al (2010) Treatment of linear alkylbenzene
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19 sulfonate in a horizontal anaerobic immobilized biomass reactor Bioresour Technol 101(2):606-
20 612
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Fo

22 [37]Souza LFC, Florencio L, Gavazza S, Kato MT (2016)Methanogenic activity inhibition by

23 increasing the linear alkylbenzene sulfonate (LAS) concentration. J Environ Sci Heal - Part A
24 Toxic/Hazardous Subst Environ Eng 51(8):656-660
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26 [38~]Lobner T, Torang L, Batstone DJ et al (2005) Effects of process stability on anaerobic
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28
biodegradation of LAS in UASB reactors. Biotechnol Bioeng 89(7):759-765
ee

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30 [39]Okada DY, Delforno TP, Esteves AS, et al (2013) Influence of volatile fatty acid
31 concentration stability on anaerobic degradation of linear alkylbenzene sulfonate J Environ
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32 Manage 128:169-172
33
34 [40]Westall JC, Chen H, Zhang W et al (1999) Adsorption of linear alkylbenzenesulfonates on
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sediment materials. Environ Sci Technol 33(18):3110-3118

36
37 [41]Motteran F, Braga JK, Sakamoto IK et al (2014) Degradation of high concentrations of
38
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39
nonionic surfactant (linear alcohol ethoxylate) in an anaerobic fluidized bed reactor Sci Total
40 Environ 481C:121-128
41
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1
2
3
4
5
6
7
8
9
10
11
12
13
Tables
14
15
16
17 Table 1 Stages of FBR operation to support material colonization and biomass
18
19 adaptation
20
21 Time of
Fo

22
23
24 Operational stage operation Circuit Feeding composition
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25
26 (days)
27
28 Activated sludge (30% of reactor volume),
ee

29
30
31
yeast extract (200 mg L-1), ethanol (50 mg
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32
33 I – Inoculation 12 Closed L-1), salt solution (Torres, 1992), and
34
35
ev

sodium bicarbonate (Macedo et al., 2015b)

36
37
38
iew

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40 Domestic sewage (191±6 mg COD L-1),
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42 laundry wastewater (658±8 mg COD L-1)
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44 II –Biomass adaptation 26 Closed (~20 mg LAS L-1) and 200 mg L-1 sodium
45
46
47 bicarbonate
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49
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51 Domestic sewage (191±6 mg COD L-1),
52
53
54
III – Reactor feeding 7 Open laundry wastewater (658±8 mg COD L-1)
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56 (~20 mg LAS L-1) and 200 mg L-1 sodium
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 Bioprocess and Biosystems Engineering Page 26 of 40

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18 Table 2 Commercial laundry wastewater and domestic sewage characterization
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20 Commercial
21 Variable Domestic sewage
Fo

22
23
laundry wastewater
24
COD (mg L-1) 658±8 191±6
rP

25
26
27 LAS (mg L-1) 60 2.5
28
ee

29
30
pH 7.0 7.3
31
rR

32 Partial alkalinity (mg L-1) 130 120

33
34 Total alkalinity (mg L-1) 184 174
35
ev

36
37
Solids (mg SSV L-1) 0.1135 0.0825
38
iew

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Page 27 of 40 Bioprocess and Biosystems Engineering

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17 Table 3 Feed composition of the assays
18
19
20 Volume proportion LAS
21
Fo

22 (mL) (mg L-1)

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24
Laundry Domestic Assay I Assay II
rP

25
26
27 wastewater sewage without ethanol with ethanol
28
ee

29 85 915 8.7 (R1) 8.3 (R1’)

30
31 250 750 13.1 (R2) 13.2 (R2’)
rR

32
33
34 500 500 24.0 (R3) 24.2 (R3’)
35
ev

36 835 165 40.4 (R4) 34.8 (R4’)

37
38 947a 53 63.5 (R5) 66.9 (R5’)
iew

39
40 aCommercial
41 laundry wastewater with standard LAS addition (Sigma-Aldrich).
42
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 Bioprocess and Biosystems Engineering Page 28 of 40

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17 Table 4 Values of specific substrate utilization rates (robs) for each profile
18
19
20 corresponding to different initial LAS concentrations (S0) (assay I without ethanol) and
21
Fo

22 in the presence of 50 mg L-1 ethanol (assay II).

23
24
rP

25
(mg LAS L-1)
26
27 (mg LAS mg-1 SSV h-1)
28
ee

29 R1 8.7 0.0270
30
31
R2 13.1 0.0323
rR

32
33
34 Assay I R3 24.0 0.0406
35
ev

36 R4 40.4 0.0156
37
38
iew

39
R5 63.5 0.0137
40
41 R1’ 8.3 0.0301
42
43 R2’ 13.2 0.0326
44
45
46 Assay II R3’ 24.2 0.0381
47
48 R4’ 34.8 0.0297
49
50 R5’ 66.9 0.0259
51
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Page 29 of 40 Bioprocess and Biosystems Engineering

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17 Table 5 Kinetic parameters of the excess-substrate inhibition model (Andrews, 1968).
18
19
20 Assay I Assay II
21
Fo

22 Parameter (without (with

23
24
ethanol) ethanol)
rP

25
26
27 kapp (mg LAS mg-1 SSV h-1) 0.069 0.051
28
ee

29 Ksapp (mg LAS L-1) 0.417 4.821

30
31
Kiapp (mg LAS L-1) 13.73 74.76
rR

32
33
34 R² 0.97 0.99
35
ev

36 2.39 18.98
37
38
iew

39 r*(mg LAS mg-1 SSV h-1) 0.051 0.033

40
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 Bioprocess and Biosystems Engineering Page 30 of 40

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15 Table 6 Values of the initial LAS concentration (LAS0), final LAS concentration
16
17
18
(LASf) and removal efficiency of LAS
19
20 Assay I Assay II
21
Fo

22 LAS0 LASf Removal LAS0 LASf Removal

23
24
(mg L-1) (mg L-1) efficiency (mg L-1) (mg L-1) efficiency of
rP

25
26
27 of LAS (%) LAS (%)
28
ee

29 8.7 0.4 95.4 8.3 ≤0.3 a 96.4

30
31
13.1 ≤0.3a 97.7 13.2 ≤0.3 a 97.7
rR

32
33
34 24.0 ≤0.3 a 98.8 24.2 ≤0.3 a 98.8
35
ev

36 40.4 9.1 77.5 34.8 1.5 95.6

37
38
iew

39
63.5 27.9 56.1 66.9 0.8 98.8
40
41 a limit of detection (LOD) of the analytical method
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Page 31 of 40 Bioprocess and Biosystems Engineering

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13 Table 7 Values of specific substrate utilization rates (robs) for each profile correspondent
14
15
16
to different LAS initial concentrations ( ) (Assays I and II)
17
18
19 COD-LAS
20 (mg L-1)
21 (mg L-1) (mg COD mg-1 SSV h-1)
Fo

22
23
24 R1 8.7 19.0 0.6311
rP

25
26 R2 13.1 28.6 0.7910
27
28 Assay I R3 24.0 52.3 0.6762
ee

29
30
31 R4 40.4 88.1 0.7828
rR

32
33 R5 63.5 138.4 0.7172
34
35
ev

R1’ 8.3 18.1 0.9672

36
37
38 R2’ 13.2 28.8 1.0410
iew

39
40 Assay II R3’ 24.2 52.8 1.0000
41
42 R4’ 34.8 75.9 1.0738
43
44
45 R5’ 66.9 145.8 0.8893
46
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 Bioprocess and Biosystems Engineering Page 32 of 40

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14 Table 8 Kinetic parameters of the excess-substrate inhibition model (Andrews, 1968).
15
16 Parameter Assay I Assay II
17
18
19
kapp (mg COD-LAS mg-1 SSV h-1) 0.91 1.51
20
21 Ksapp (mg COD-LAS L-1) 7.33 9.78
Fo

22
23 Kiapp (mg COD-LAS L-1) 635.83 234.41
24
rP

25
26
R² 0.99 0.99
27
28 68.29 47.87
ee

29
30
r* 0.754 1.07
31
rR

32
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ev

36
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iew

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Page 33 of 40 Bioprocess and Biosystems Engineering

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13 Table 9 Values of initial COD concentration (COD0), final COD concentration (CODf)
14
15 and removal efficiency of COD
16
17
18 Assay I Assay II
19
20 COD0 CODf Removal COD0 CODf Removal
21
Fo

22 (mg L-1) (mg L-1) efficiency (mg L-1) (mg L-1) efficiency of
23
24
of COD (%) COD (%)
rP

25
26
27 171 31 82 238 46 80
28
ee

29 259 33 87 297 36 88
30
31
rR

32 289 58 80 382 82 79
33
34 465 152 67 482 87 81
35
ev

36 520 160 69 519 139 73

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iew

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 Bioprocess and Biosystems Engineering Page 34 of 40

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13 Table 10. Initial and final pH values and VFA concentrations for assay I and assay II
14
15
16 Assay I Assay II
17
18 pH VFAs (mg HAc L-1) pH VFAs (mg HAc L-1)
19
20 Initial Final Initial Final Initial Final Initial Final
21
Fo

22
R1 7.6 6.6 85 80 R1’ 7.6 6.5 135 14
23
24
R2 7.8 6.8 104
103,9 ≤5 a R2’ 7.5 6.7 173 15
rP

25
26
27 R3 7.7 6.8 182
181,7 26 R3 7.4 6.7 194 64
28
ee

29 R4 7.6 6.8 169

168,8 60 R4’ 7.4 6.6 1681 94
30
31 R5 7.5 6.7 203 91 R5’ 7.5 6.4 123 ≤5 a
202,7
rR

32
33 a limit of detection (LOD) of the analytical method
34
35
ev

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iew

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Page 35 of 40 Bioprocess and Biosystems Engineering

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14 Figures captions
15
16
17 Fig. 1 (a) Adapted expression of inhibition mode fitted to the experimental data to
18
19 estimate the apparent kinetic parameters. (b) Data generated by the Haldane expression
20
21
with adjusted parameters for a range of substrate concentrations
Fo

22
23
24 Fig. 2 (a) Adapted expression of inhibition mode fitted to the experimental data to
rP

25
26
27 estimate the apparent kinetic parameters. (b) Data generated by the Haldane expression
28
ee

29 with adjusted parameters for a range of substrate concentrations

30
31 Fig. 3 Mass balance at the end of the experiments: (a) adsorbed, removed, and final
rR

32
33
34 LAS concentrations (mg) in assay I (without ethanol); (b) adsorbed, removed, and final
35
ev

36 LAS concentrations (mg) in assay II (with ethanol); and (c) LAS biodegradation
37
38 efficiency (%) with and without ethanol
iew

39
40
Fig. 4 Dendrogram of the similarity coefficient (Pearson correlation) based on the
41
42
43 DGGE profiles for bacterial domains in the initial biofilm attached to sand and samples
44
45 taken from the final reactor for R1 (8.7 mg LAS L-1), R2 (13.1 mg LAS L-1), R3 (24.0
46
47 mg LAS L-1), R4 (40.4 mg LAS L-1) and R5 (63.5 mg LAS L-1) without ethanol
48
49
50 addition (assay I) and R1’ (8.3 mg LAS L-1), R2’ (13.2 mg LAS L-1), R3’ (24.2 mg LAS
51
52 L-1), R4’ (34.8 mg LAS L-1) and R5’ (66.9 mg LAS L-1) with ethanol addition (assay II)
53
54 Fig. 5 Effect of LAS concentration on bacterial dominance and diversity in the presence
55
56
57 (assay I) or absence of ethanol (assay II)
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 Bioprocess and Biosystems Engineering Page 36 of 40

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ee

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rR

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ev

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iew

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45 Fig. 1 (a) Adapted expression of inhibition mode fitted to the experimental data to estimate the apparent
46 kinetic parameters. (b) Data generated by the Haldane expression with adjusted parameters for a range of
47 substrate concentrations
48 99x137mm (300 x 300 DPI)
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Page 37 of 40 Bioprocess and Biosystems Engineering

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ee

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rR

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ev

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iew

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45 Fig. 2 (a) Adapted expression of inhibition mode fitted to the experimental data to estimate the apparent
46 kinetic parameters. (b) Data generated by the Haldane expression with adjusted parameters for a range of
47 substrate concentrations
48 99x152mm (300 x 300 DPI)
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 Bioprocess and Biosystems Engineering Page 38 of 40

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ee

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rR

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ev

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iew

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45 Fig. 3 Mass balance at the end of the experiments: (a) adsorbed, removed, and final LAS concentrations
46 (mg) in assay I (without ethanol); (b) adsorbed, removed, and final LAS concentrations (mg) in assay II
47 (with ethanol); and (c) LAS biodegradation efficiency (%) with and without ethanol
48 90x169mm (300 x 300 DPI)
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Page 39 of 40 Bioprocess and Biosystems Engineering

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Fo
20
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22 Fig. 4 Dendrogram of the similarity coefficient (Pearson correlation) based on the DGGE profiles for bacterial
23 domains in the initial biofilm attached to sand and samples taken from the final reactor for R1 (8.7 mg LAS
rP

24 L-1), R2 (13.1 mg LAS L-1), R3 (24.0 mg LAS L-1), R4 (40.4 mg LAS L-1) and R5 (63.5 mg LAS L-1) without
25 ethanol addition (assay I) and R1’ (8.3 mg LAS L-1), R2’ (13.2 mg LAS L-1), R3’ (24.2 mg LAS L-1), R4’
26 (34.8 mg LAS L-1) and R5’ (66.9 mg LAS L-1) with ethanol addition (assay II)
ee

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rR

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ev

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iew

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 Bioprocess and Biosystems Engineering Page 40 of 40

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Fo
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rP

24
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ee

27
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29 Fig. 5 Effect of LAS concentration on bacterial dominance and diversity in the presence (assay I) or absence
rR

30 of ethanol (assay II)

31
99x68mm (300 x 300 DPI)
32
ev

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iew

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