SO42− ratio on vinasse treatment performance by two-stage anaerobic membrane bioreactor

Journal of Environmental Management 259 (2020) 110034

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Research article

Influence of COD/SO24 ratio on vinasse treatment performance by two-stage anaerobic membrane bioreactor ~es a, Paulo Vitor Martinelli Cunha a, Ana Fl�avia Rezende Silva a, *, Natalie Cristine Magalha a b Miriam Cristina Santos Amaral , Konrad Koch a b

Department of Sanitary and Environmental Engineering, Federal University of Minas Gerais, Belo Horizonte, Brazil Chair of Urban Water Systems Engineering, Department of Civil, Geo and Environmental Engineering, Technical University of Munich, Munich, Germany

A R T I C L E I N F O

A B S T R A C T

Keywords: Anaerobic digestion Pollutant removal Sugarcane vinasse Sulfidogenesis Membrane fouling

Vinasse is sulfate-rich wastewater due to sulfuric acid dosage in some ethanol production steps. The vinasse sulfate concentration is subject to seasonal variations. A two-stage anaerobic membrane bioreactor (2S-AnMBR) was operated to evaluate the influence of COD/SO24 ratio on vinasse treatment performance by using a real vinasse sample under natural seasonal COD/SO24 variation. This ratio directly affects the sulfidogenesis effi­ ciency, which is responsible for different forms of inhibition in the anaerobic treatment of sulfate-rich waste­ water. The bioreactor presented a stable performance at the highest COD/SO24 ratios (50–94), with high removal of chemical oxygen demand (COD) (97.5 � 0.4%) and volatile fatty acids (VFA) (98.0 � 0.6%), but low removal of sulfate (69.9 � 9.5%), indicating lower sulfate reducing bacteria (SRB) activity. In the lowest COD/SO24 ratios (9–20), a deterioration in the removal of organic matter (87.0 � 1.3%) and VFA (69.8 � 15.5%) was observed, accompanied by sulfate removal increase (92.9 � 2.6%). A significant correlation between COD fractions removed via methanogenesis and sulfidogenesis and the COD/SO24 ratio was found, indicating that the increase of this ratio is beneficial to the methanogenic archaea activity. The occurrence of sulfidogenesis, favored by the lower COD/SO24 ratios, induced the microbial soluble products (SMP) and extracellular polymeric substances (EPS) release and protein/carbohydrate ratio increase in the mixed liquor, contributing to the filtration resis­ tance increase.

1. Introduction The increase in ethanol production in recent decades is being moti­ vated by price instability of oil, associated with the need to preserve the environment. It motivates the search for fuel produced from clean and renewable sources and, thus, reducing greenhouse gas emissions. The bioethanol industry generates a large amount of liquid effluents, among them vinasse (Onodera et al., 2017). 10–15 L of vinasse are produced for each 1 L of ethanol processed (Fuess and Garcia, 2014). Vinasse comes from the distillation stage, leaving the columns at a temperature in the range of 85–90 � C (Moraes et al., 2015), presenting high polluting po­ tential due to the high organic matter concentrations in terms of COD (chemical oxygen demand) and BOD (biochemical oxygen demand), as well as nutrients, mainly potassium (K), nitrogen (N) and phosphorus (P) (Hoarau et al., 2018). Among the most common alternatives adopted for final vinasse

disposal is fertigation, that consists in wastewater application in the sugarcane crops, replacing the chemical fertilizers and water demand (Fuess et al., 2017). The direct application of vinasse in the soil, in large amounts and for long periods, can bring negative impacts such as sali­ nization and elevation of organic matter and nitrogen content in the soil, greenhouse gas emissions, water bodies acidification, unpleasant odor release (Fuess and Garcia, 2014). To optimize the energy potential and sustainability of ethanol pro­ duction, it is important to consider the vinasse as a byproduct and not as a residue. Vinasse contains large amounts of biodegradable organics, then, biological processes can be successfully used (Fuess and Garcia, 2017). Anaerobic digestion allows energy recovery through biogas which is generated as the by-product of the degradation process of organic matter (Albuquerque et al., 2019). Anaerobic membrane bio­ reactors (AnMBR) have been used as an alternative to the conventional anaerobic digestion process. The integration of membrane process and

* Corresponding author. Department of Sanitary and Environmental Engineering, Federal University of Minas Gerais, Av. Ant^ onio Carlos 6627, Bl. 01, 31270-901, Belo Horizonte, Brazil. E-mail address: [email protected] (A.F.R. Silva). https://doi.org/10.1016/j.jenvman.2019.110034 Received 27 August 2019; Received in revised form 21 December 2019; Accepted 22 December 2019 Available online 13 January 2020 0301-4797/© 2020 Elsevier Ltd. All rights reserved.

A.F.R. Silva et al.

Journal of Environmental Management 259 (2020) 110034

anaerobic digestion offer the advantage of reducing the volume of sludge and of the reactor and increasing biogas production (Musa et al., 2018). Christian et al. (2011) evaluated the treatment of raw wastewater from the production of salad dressings and barbeque sauces using full-scale AnMBR, with an influent flow of 300 � 70 m3/d. The sub­ merged membrane units provided a near-absolute barrier to block all suspended solids, resulting in a very high quality effluent compared to conventional technologies. Also, the system operation resulted in reduction in operating process that was directly attributable to the increased system capacity, ability to treat wastewater with higher biomass, and elimination of the need to dewater and disposed dewatered solids. Previous studies have reported the great potential of two-stage anaerobic membrane bioreactor (2S-AnMBR) to treatment vinasse, showing high capacity of removing organic matter (>97%) while pro­ ducing biogas (>6.3 Nm3 of CH4 per m3 of treated vinasse) (Mota et al., 2013; Santos et al., 2017). The two-stage digestion, based on the microorganism separation in two interconnected reactors, provides the acidogenic bacteria growth in the first reactor, maintaining low hy­ draulic retention time (HRT) and pH (between 5 and 6), while the acetogenic/methanogenic microorganism are established in the second reactor maintaining higher HRT and pH (between 7 and 8) (Algapani et al., 2019). The two-stage anaerobic reactor is an attractive alternative for the treatment of wastewater with high organic loads and low pH, such as vinasse, compared to the single-stage anaerobic process (Meng et al., 2017). Also, conventional anaerobic bioreactors are not able to ensure compliance with discharge standards and usually required an additional effluent treatment step (Petta et al., 2017). The integration of two-stage anaerobic digestion with membrane separation process coupled to the methanogenic reactor provides enhanced treatment process, producing high quality effluents free of suspended solids, able to withstand fluctuations in feed quality, complete microorganism retention, increasing its concentration inside the bioreactor (Lin et al., 2013). The AnMBR allows uncoupling the HRT and the sludge retention time (SRT), compensating the slow anaerobic species growth with a SRT increase (Robles et al., 2018). A major drawback that hinders AnMBR performance is membrane fouling, which causes decreased system productivity, increased trans­ membrane pressure (TMP) and frequent cleaning, reducing the mem­ brane lifespan, resulting in higher replacement costs (Robles et al., 2018). The substances that cause membrane fouling may be extracel­ lular polymeric substances (EPS), soluble microbial products (SMP), dissolved organic matter, biopolymers, colloids, sludge flocs, and other organic and inorganic compounds (Lin et al., 2014). Among these, SMP and EPS are often mentioned as important factors in relation to mem­ brane fouling in AnMBR (Charfi et al., 2012), which production is associated with substrate degradation and biomass decay (Wang et al., 2018). Under unfavorable conditions, such as toxic or inhibitory envi­ ronments, as high sulfate concentration, high lysis levels, and cell death are found, causing SMP and EPS release, contributing to intensified fouling process (Lin et al., 2014). Due to the presence of sulfate, sulfite, or thiosulfate, sulfidogenesis also occurs through sulfate-reducing bacteria (SRB) activity, reducing sulfate to sulfide, that can dissolve in the effluent (HS , S2 , H2S) or constitute biogas (H2S) (Jim� enez et al., 2017). SRB compete with other microorganisms for carbon sources and hydrogen, causing a reduction in biogas production, besides the toxicity caused by H2S produced (Hu et al., 2015). The sulfidogenesis efficiency is affected by the electron donor number, is expressed mainly by the COD/SO24 ratio (Sarti and Zaiat, 2011), and at low ratios sulfidogenesis predominates, while methanogenesis prevails in higher values (Madden et al., 2014). Vinasse is a sulfate-rich wastewater (sulfate concentration and COD/ SO24 ratio range from 0.76 to 9 g L 1 and 6.6 to 43.7, respectively), due to the use of sulfuric acid in some ethanol production steps, in order to avoid microbial contamination and yeast flocculation in fermentation vessels (Kiyuna et al., 2017). Contaminant bacteria compete with the

yeasts for available sugars and nutrients, induce yeast flocculation and also produce by-products, such as lactic and acetic acids that inhibit yeast growth and viability, reducing ethanol yields (Dellias et al., 2018). Fuess et al. (2018) presented detailed characterizations of the organic and inorganic vinasse fractions generated in a Brazilian biorefinery over 7 months (May to December). The results indicated a reduction in the sulfate concentration in July and August, when increasing ethanol production levels were reported. The SO24 concentration showed an increasing trend by the end of the harvest, even with an enhancement in ethanol production, suggesting the sulfate accumulation due to the increasing doses of H2SO4 in the fermentation vessels. Consequently, the vinasse sulfate concentration is subject to seasonal variations. In this context, the present work aims to evaluate the influence of COD/SO24 ratio on vinasse treatment performance using a two-stage anaerobic membrane bioreactor (2S-AnMBR) by using real vinasse under natural seasonal COD/SO24 ratio variation. The pollutant removal (organic matter and nutrients), microorganism activity (acidogenic bacteria, methanogenic archaea (MA) and SRB) and mem­ brane fouling were analyzed in two different ranges of COD/SO24 ratio (9–20 and 50–94). COD/SO24 ratio directly affects the sulfidogenesis efficiency, which is responsible for different forms of inhibition in anaerobic wastewater treatment. Although several studies on the influence of the sulfate-reducing process on anaerobic reactors have been reported (Table 1), in­ vestigations have generally been conducted using synthetic wastewaters or real wastewater with additional dosing of sulfate. It can be observed that only Song et al. (2018) evaluated the COD/SO24 ratio in an AnMBR, but using synthetic solution simulating high strength domestic waste­ water. According to the authors’ knowledge, this is the first evaluation of the influence of COD/SO24 ratio on a 2S-AnMBR treating real wastewater rich in sulfate and organic matter. 2. Materials and methods 2.1. Sugarcane vinasse Vinasse was obtained from a distillery located in the state of Minas Gerais (Brazil) producing ethanol from sugarcane juice with a produc­ tion capacity of 1100 m3 day 1 of ethanol, milling up to 2.5 million tons of sugarcane per harvest. All samples were kept refrigerated at a tem­ perature of 4 � C (277.15 K) and protected from light before use to avoid decomposition. The physicochemical characterization of vinasse is presented in Table 2. 2.2. Experimental setup The 2S-AnMBR operated in this study is detailed in Santos et al. (2017), and the schematic diagram is shown in Fig. 1. The system con­ sisted of two reactors placed in series: acidogenic reactor (AR) and methanogenic reactor (MR). The AR was operated in upflow mode, and its volume was maintained at 3.8 L (level 2), with an HRT of 0.48 days. The MR received the AR effluent by gravity, and its volume maintained at 20.4 L (level 3), with an HRT of 2.58 days. MR was equipped with a submerged UF module (PVDF hollow-fiber membrane, average pore size of 0.04 μm, total surface area of 0.065 m2, ZeeWeed 500D – GE) and with a mechanical stirrer (IKA, RW 16 Basic stirrer, 280–300 rpm) to allow complete mixing conditions in this tank. The membrane was connected to a vacuum tank, with the permeate flow maintained con­ stant at 5.1 L h 1 m 2, controlled by a valve to regulate the air inlet. When this tank was filled, it was then discharged into the permeate tank, also used for backwash. The system was operated for 15 min in the filtration mode and for 15 s in the backwash mode, maintained at room temperature (average of 22 � C).

2

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Journal of Environmental Management 259 (2020) 110034

Table 1 Summary of studies that evaluated COD/SO24 on wastewater treatment. Wastewater

Reactor type (monitoring time)

COD/ SO24 ratio

Results

Reference

Synthetic (high strength domestic)

a

60 to 10

COD removal was not affected when the influent COD/ SO24 ratio was maintained higher than 10, and when higher than 30, no notable membrane fouling was observed. Higher COD removal efficiencies at the highest COD/ SO24 ratio. The establishment of a sulfidogenic environment under acidogenic conditions has the potential to enhance sulfate removal prior to methanogenesis. At a COD/SO24 ratio of 7.5, CH4 production was 35% lower compared with a ratio of 12.0. At very high lactate/ sulfate ratio, acetogens and methanogenic were the dominant microbial communities. Methanogenesis appeared unaffected at a COD/SO24 ratio of 8 and was impacted marginally when the ratio was 0.5. COD removal efficiency decreased to 60% after sulfate addition.

Song et al. (2018)

AnMBR

Sulfonation of vegetable oils Synthetic (ethanol and sulfate)

b

AnSBR (130 days)

3.67 to 2.60

c

ASTBR (under thermophilic acidogenic conditions)

2.50, 1.70, 1.25

Vinasse

Batch assays in anaerobic conditions

12.0, 10.0 and 7.5

Synthetic

d

CSTR

20.90 to 0.34

Synthetic

e

EGSB-AF

8 to 0.5

Synthetic (VFA, alcohol and sulfate) Vinasse

Quarterpacked upflow hybrid reactor (500 days)

3.0

f

UASB (set of experiments)

20 to 5

Synthetic (acetate, ethanol and sulfate) Synthetic (sucrose)

UASB (182 days)

1.0

UASB (220 days)

4 and 1

Synthetic starch

UASB (250 days)

10 to 0.5

Synthetic

UASB (330 days)

1.0

Synthetic (acetate,

UASB (375 days)

20 to 0.5

Table 1 (continued ) Wastewater

ethanol and sulfate)

a b c d e

Sarti and Zaiat (2011)

f

Dar et al. (2008)

Madden et al. (2014)

O’Flaherty and Colleran (1999)

Acidification was always complete in the effluent at a COD/SO24 ratio of 1. In COD/SO24 > 2: stable biogas production and COD removal; and <2: deterioration in methane conversion. COD removal > 90%; reduction in methane production The conversion of influent COD to

Lopes et al. (2010)

Results

Reference

methane dropped from 80.5% to 54.4% as the COD/ SO4 2 ratio decreased from 20 to 0.5.

AnMBR: anaerobic membrane bioreactor. AnSBR: anaerobic batch reactor. ASTBR: anaerobic structured-bed reactor. CSTR: continuous stirred tank reactor. EGSB-AF: expanded sludge bed-anaerobic filter. UASB: upflow anaerobic sludge blanket reactor.

The acidogenic and methanogenic reactors were inoculated with granular sludge from a single-stage UASB reactor treating domestic sewage with an initial concentration of 20 gVSS L 1. The biological sludge was initially acclimatized to vinasse treatment in order to make a previous selection of microorganisms present in each of the reactors (acidogenic and methanogenic). The AR and MR were fed with vinasse, and membrane module was fitted to the MR since the beginning of process startup. During the whole period of operation, the pH in AR and MR was monitored, with the pH in the MR maintained above 7.0 by addition of sodium bicarbonate. Membrane cleaning was considered when the TMP reached 50 kPa. Then, the UF module was removed from the reactor and submitted to a cleaning process consisting of three steps, in order to recover membrane permeability: (1) physical cleaning by flushing the membrane surface with tap water for cake layer removal, (2) oxidative chemical cleaning with 1000 ppm NaOCl and (3) chemical cleaning with a citric acid solution (pH 2.5), both for 20 min in the ul­ trasound bath (Unique USC 2800–40 kHz). The system was operated with COD/SO24 ratio range from 9.04 to 93.85 influenced by seasonal variation of the sulfate and COD concen­ tration throughout two harvests. In order to evaluate the 2S-AnMBR system performance in the organic matter and nutrients removal, feed and permeate were analyzed regarding the following physicochemical parameters: COD, SO24 , volatile fatty acids (VFA), solids (total, fixed and volatile), nitrogen (free ammonia and ammonium), pH, conduc­ tivity, cations. The biological activity in the AR was evaluated according to VFA production. Methanogenic reactor mixed liquor was character­ ized in terms of suspended solids (total and volatile) for analysis of biomass activity and microorganism concentration, and in terms of SMP and EPS production by the proteins and carbohydrates concentrations for the membrane fouling monitoring. The microbiological character­ ization of the microorganisms involved in the two reactors was not performed. The use of physicochemical parameters to monitor the bio­ logical process was based on studies in the literature (Musa et al., 2018; Song et al., 2018), as these parameters are important indicator of the biodegradation and microorganism activity in the anaerobic digestion. Samples of feed, AR effluent, methanogenic reactor mixed liquor and permeate were collected periodically with a frequency of twice a week during the operational monitoring.

Kiyuna et al. (2017)

Barrera et al. (2014)

COD/ SO24 ratio

2.3. Operational conditions

Gil-Garcia et al. (2018)

Severe inhibition for MA and SRB at COD/SO24 of 5. COD and sulfate removal above 80% and 30%, respectively.

Reactor type (monitoring time)

Jing et al. (2013)

Lu et al. (2016)

2.4. Analytical methods The procedures described in the Standard Methods for the Exami­ nation of Water and Wastewater (Apha, 2012) were utilized to obtain the following parameters values: COD (5220-D); total solids (2540-B, 2540-E); total suspended solids (2540-D, 2540-E); free ammonia (4500-NH3-B, 4500-NH3-C); sulfate (4500-SO24 -D); total phosphorus (4500-P-B, 4500-P-C); pH (4500-H þ -B - pHmeter Qualxtron QX 1500);

Wu et al. (2018) Hu et al. (2015)

3

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Journal of Environmental Management 259 (2020) 110034

Table 2 Physicochemical characterization of vinasse. Parameter

Unit

pH Conductivity COD a VFA b TS c VTS d FTS e TAN Phosphorus Calcium Magnesium Potassium Sodium Sulfate COD/SO24

– mS cm mg L 1 mg L 1 gL 1 gL 1 gL 1 mg L 1 mg L 1 mg L 1 mg L 1 gL 1 mg L 1 mg L 1 –

a b c d e

1

Minimum

25% quartile

Average

Median

75% quartile

Maximum

3.90 3.09 11,925 1470 5.76 3.57 2.02 0.55 33.33 243 141 2.08 107 232 9.0

4.72 6.64 12,967 2010 10.50 4.79 2.39 0.65 38.35 344 165 2.12 111 243 11.1

4.97 8.05 15,043 2865 13.35 9.79 3.57 114.69 70.52 687 323 2.532 181 682 42.6

5.02 9.28 14,347 2100 11.43 9.04 2.48 170.80 55.89 699 196 2.41 127 256 51.7

5.31 9.65 16,386 4020 18.45 16.06 5.69 191.33 102.67 887 679 2.89 300 1203 69.4

5.93 10.02 22,788 5190 22.74 20.20 6.18 223.07 111.52 1264 740 3.55 314 1743 93.9

VFA: volatile fatty acids. TS: total solids. VTS: volatile total solids. FTS: fixed total solids. TAN: total ammonia nitrogen.

Fig. 1. 2S-AnMBR schematic diagram.

electrical conductivity (2510-B – Conductivity meter Hanna HI 9835). Cations (ammonium, calcium, magnesium, potassium, sodium) were analyzed by ion chromatography (4110 – Dionex ICS-1000 ion chro­ matography, equipped with column type IonPac AS22 and IonPac CS12A). VFA analysis for vinasse and AR effluent were performed ac­ cording to Dilallo method (Dilallo and Albertson, 1961), and for permeate (after UF membrane) by the Kapp method (Buchauer, 1998). For the analysis of SMP and EPS fractions, samples of 50 mL from the methanogenic reactor mixed liquor was collected and centrifuged for 10 min at 5000 rpm, and the supernatant containing SMP was collected. EPS contained in the residual pellet from centrifugation was extracted by resuspension using 50 mL 0.05% NaCl solution. Subsequently, the mixture was heated at 80 � C for 10 min and centrifuged for 10 min at 5000 rpm, and the supernatant containing EPS was collected. Both su­ pernatant samples were passed through 0.45 μm membrane filters (Millipore) and characterized for carbohydrates (Dubois et al., 1956) and proteins (Lowry et al., 1951) concentration.

CODtotal removalð%Þ ¼

� � CODf CODp ⋅100 CODf

CODsulfidogenesis removalð%Þ ¼

� 0:67⋅ SO4f CODf

SO4p

�! ⋅100

(2)

CODsulfidogenesis ð%Þ

(3)

CODp

CODmethanogenesis removalð%Þ ¼ CODtotal ð%Þ

(1)

In Equations (1) to (3), CODf and CODp are the COD concentrations in the feed (vinasse) and 2S-AnMBR permeate, respectively, in mg L 1; SO4f and SO4p are the sulfate concentrations in the feed and permeate, respectively, in mg L 1. According to Lens et al. (1998), the COD removed fraction by sulfidogenesis was calculated considering that to reduce each gram of sulfate, the SRB oxidizes 0.67 g of organic matter. Membrane fouling was investigated by daily monitoring of TMP and permeate flow rate. The resistance-in-series model was used to analyze filtration resistance as follows (Equation (4)), according to Lee et al. (2003).

2.5. Calculations

Rt ¼

The fractions of organic matter removed by methanogenesis and sulfidogenesis were calculated according to Vilela et al. (2014). Equa­ tion (1) shows the total COD removed (%), whereas Equations (2) and (3) determine COD removed by sulfidogenesis (%) and methanogenesis (%), respectively. The calculations assumed that the total amount of organic matter was removed via methanogenesis and sulfidogenesis.

ΔP

μ⋅Jp

Rm þ Rf

(4)

where ΔP is the TMP (Pa); μ the dynamic viscosity of water (Pa.s); Jp the permeate flux (m3 s 1 m 2), Rt the total resistance (m 1), Rm the intrinsic membrane resistance (m 1) and Rf the fouling resistance (m 1), due to cake layer formation, pores blockage and adsorption. Rt was 4

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Journal of Environmental Management 259 (2020) 110034

Polizzi et al. (2018), free ammonia concentrations below 200 mg L 1 are suggested to be beneficial to the anaerobic process, as it serves as an adequate nitrogen supply as a nutrient for anaerobic microorganisms. The inhibitory effects of salts contained in the wastewater on microor­ ganisms are mainly related to cations, as they cause bacterial cell dehydration as well as to have deleterious effects on the enzymatic catalytic rate, leading to a decreased microbial activity (Onodera et al., 2017). Table 3 presents the concentration of main inhibitory com­ pounds, emphasizing that the feed used in this study had concentrations below the typical toxic thresholds, not causing inhibition of the microorganisms. The low vinasse NH3 concentration in the first 150 days of moni­ toring (range I) did not contribute to reduce the organic matter degra­ dation, as will be discussed later, due the high levels of biodegradable organic matter in the samples (97%; Mota et al., 2015) and ammonia release during the degradation of the organic nitrogen present in the vinasse. The average total nitrogen concentration in the vinasse used to feed the 2S-AnMBR during this period was 98 � 8 mg L 1. In range II, the total nitrogen concentration in the vinasse was 210 mg L 1, where 153 mg L 1 were related to the ammonia nitrogen concentration. Therefore, 57 mg L 1 are related to the organic nitrogen concentration, which can be released during anaerobic digestion. Considering that all organic nitrogen is biodegraded, the total nitrogen concentration in the media does not present inhibition potential, since a quantity of this nutrient will still be used for the metabolism of microorganisms. As can be seen from Table 3, the anaerobic digestion process was likely not severely inhibited by any of the listed substance.

obtained by filtration of the methanogenic reactor mixed liquor, and Rm was calculated by filtration of pure water with the virgin membrane. 2.6. Statistical analysis The existence of correlations among COD/SO24 ratio and the others parameters obtained during the operational monitoring of 2S-AnMBR was evaluated. The Spearman correlation coefficient (R) was used with further correlation significance analysis by means of the hypothesis test. Positive R values indicate direct relation (one variable increase leads to the other increase), while negative R values indicate inverse relation (one variable increase leads to the other decrease). The existence of differences between the monitored parameters under the two COD/SO24 ratios ranges was investigated. The MannWhitney U test was used for two independent samples, checking the significance difference by means of the hypothesis test. Besides that, significant differences between multiple independent samples were assessed using the Kruskal-Wallis test, followed by the non-parametric test for multiple comparisons among groups. All statistical analyses were performed using STATISTICA 10.0 software, at a significance level (α) of 5%. 3. Results and discussions 3.1. Variations in the vinasse composition Seasonal variations in the COD and sulfate concentrations can cause dynamic responses in the sulfate reduction process during anaerobic treatment, directly influencing the system performance (Jim�enez et al., 2017). Fig. 2 shows the COD and sulfate concentrations, and the COD/SO24 ratio over the monitoring time, highlighting the two ranges of COD/SO24 ratio (range I and range II). According to the Mann-Whitney U test, significant differences were found between the two ranges for the three variables. The anaerobic digestion process presents a vulnerability to inhibition by the presence of certain substances, being nitrogen and some ions the main inhibitors. The inhibitory concentrations range presented in the literature vary mainly due to the differences in substrates and inoculum, environmental conditions and biomass acclimatization (Shi et al., 2017). Ammonium (NHþ 4 ) and free ammonia (NH3) are considered the two principal forms of total nitrogen, with NH3 being indicated as the main agent causing the inhibition process (Dai et al., 2017). According to

Table 3 Substances concentration and inhibition threshold (mean � std. dev.). Parameter

Unit

TAN

mg L

Calcium

mmol L 1 mmol L 1 mmol L 1 mmol L 1

Magnesium Potassium Sodium

1

Range I

Range II

Limits

References

0.6 � 0.0

153.5 � 70.0 16.2 � 0.5

200

6.4 � 0.0

80

Polizzi et al. (2018) Onodera et al. (2017)

69.3 � 3.3

280–600

0.7 � 0.2

320

6.3 � 0.2 29.2 � 1.2 81.0 � 6.8 5.2 � 0.5

Fig. 2. COD and sulfate concentrations, and COD/SO24 ratio in vinasse over the monitoring time. 5

120

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Journal of Environmental Management 259 (2020) 110034

3.2. Effect of COD/SO24 ratio on biological activity of acidogenesis

Table 4 COD and sulfate concentrations in feed and acidogenic reactor and its removal during range II (mean � std. deviation).

In this microbial consortium, the SRB depend on the hydrolytic and acidogenic bacteria metabolism to hydrolyze the particulate COD. Since they do not degrade natural biopolymers (starch, glycogen, proteins, lipids), they do not presenting competition with these microorganism groups (Sarti and Zaiat, 2011). Fig. 3 shows the VFA concentration in the feed and AR effluent. It is observed that the VFA concentration in the vinasse presents high values, however, these values were even higher in range II. The VFA concentration in the AR effluent presented higher values in relation to the feed, due to the VFA production by acidogenic bacteria. The biological activity of the microorganisms in the acidogenic phase was analyzed for VFA production per unit VSS per day. The values ob­ tained in the ranges I and II were 175.6 � 137.8 and 197.5 � 123.5 1 mgVFA gVSS d 1, respectively, with no significant differences in the VFA production among ranges. These results indicate that there was no in­ hibition of acidogenesis by SRB activity in the two ranges of COD/SO24 ratios evaluated. The COD and sulfate concentrations in the acidogenic reactor were evaluated during the monitoring in the lowest COD/SO24 ratios (range II), and the results are presented in Table 4. It was observed low COD removal, since the acidogenic phase consists of organic matter conver­ sion into VFA. The COD and sulfate removal percentages were consis­ tent, since to reduce 1 g of sulfate, SRB oxidized 0.67 g of organic matter (Lens et al., 1998).

Parameter

Unit

Feed Acidogenic reactor Removal

mg L mg L %

1 1

COD

Sulfate

13,147.2 � 952.2 12,852.3 � 972.8 3.8 � 2.0

1167.9 � 232.5 702.3 � 325.7 44.5 � 16.7

between COD/SO24 ratio and VFA concentration in the permeate with correlation coefficient R of 0.63. This means that a decreasing COD/SO24 ratio leads to an increasing VFA concentration, as observed. The VFA removal percentage in the range I presented stable values, around 98%. Range II showed a variation in the removal efficiencies, with an average removal of 70%, indicating an inhibition caused by the sulfide on the MA and SRB. According to Wang et al. (2009), the methanogenic activity inhibi­ tion by VFA presence varies according to the compound being 2,400, 1800 and 900 mg L 1 the inhibitory limits of acetic, butyric and pro­ pionic acid, respectively. Annachhatre and Suktrakoolvait (2001) observed a COD removal reduction in COD/SO24 ratios lower than 15, being accompanied by an increase in sulfide and VFA concentrations in the treated effluent, indicating a drop in methanogenesis activity. Similarly, Kumar et al. (2007) evaluated a lab-scale anaerobic hybrid reactor with COD/SO24 ratio around 18 and found VFA concentration increase inside the reactor with increased HRT, indicating an incomplete conversion of VFA in methane due to sulfide toxicity over MA. The H2S concentrations in the 2S-AnMBR permeate in the range II was 166 � 15 mg L 1. Since the UF membrane coupled to the MR is not able to prevent the passage of H2S through its pores, the H2S concentration in the mixed liquor methanogenic reactor is the same as that found in the permeate. Yamaguchi et al. (1999) found a significant reduction in COD removal due to an accumulation of acetate in the presence of 107 � 69 mgH2S L 1 of undissociated sulfide in a UASB reactor fed with sulfate-rich waste­ water. As the sulfide concentration reduced to 90 � 24 mg L 1, the COD removal increased from 33% to 77%. The TSS concentrations in the MR in the ranges I and II were 12.4 � 4.9 and 12.5 � 2.3 gTSS L 1, respectively (Fig. 5). Another factor analyzed was the VSS/TSS ratio, a biomass activity indicator in the degradation process, with values of 0.69 � 0.04 and 0.66 � 0.03 in ranges I and II, respectively. These results were similar to those found by Song et al. (2018), who evaluated the COD/SO24 ratio reduction from 60 to 10 by increasing sulfate concentration. The results indicated that the biomass concentration was not affected, and the VSS/TSS ratio remained stable at around 0.6. Fig. 6 (a) and (b) show COD and sulfate concentrations in the 2SAnMBR permeate and its removal percentage in the two ranges. Higher removal efficiencies of COD were observed in the range I, with an average of 97.5 � 0.4%. According to Fuess and Garcia (2015), when optimal conditions are established within anaerobic reactors, COD removal efficiency can be as high as 90–95%. In range II, a reduction in removal efficiency was observed as the COD/SO24 ratio approached 10, showing instability in the values, with an average of 87.0 � 1.3%. The Spearman test indicated a significant positive correlation between COD/SO24 ratio and COD removal efficiency, with the R coefficient of 0.8726. The removal efficiency decrease is associated with inhibition arising from the competition between the MA and SRB, either from substrate competition or by sulfide toxicity, since the other methano­ genesis inhibitory compounds, such as NH3 and cations, were below the inhibition limits, according to Table 3. The biological activity of the methanogenic microorganisms was also analyzed according to COD removal per unit VSS per day. The values obtained in ranges I and II were 654.2 � 243.9 and 549.5 � 84.6 mgCOD 1 gVSS d 1, respectively, and significant differences in COD removed in both ranges were found. The results indicated a possible methanogenesis inhibition by SRB activity in range II. For a more specific analysis within

3.3. Effect of COD/SO24 ratio on biological activity of methanogenesis The methanogenesis step involves organic substrate conversion (acetate, methanol, H2, CO2, and others) into methane through the ac­ tivity of different MA species (Wu et al., 2018). The sulfate-rich waste­ water treatment stimulates SRB populations, leading to reduction of the methanogenic activity either by competition for substrates or by increased inhibition by sulfide formed as a sulfate reduction product (Fuess and Garcia, 2015). Among the problems caused by the competition between the MA and SRB is a buildup of metabolic intermediates such as VFA, leading to MR acidification, reducing COD removal efficiency, and eventually a collapse in the process (Wu et al., 2018). Fig. 4 presents the VFA con­ centration in permeate and its removal. The VFA removal concerns the consumption of these acids by acetogenic bacteria and methanogenic archaea in the last two stages of the anaerobic digestion process. The values of VFA concentration in the 2S-AnMBR permeate were clearly higher in range II (1265 � 862 mg L 1) in relation to the range I (55 � 13 mg L 1). Since the UF membrane is not able to retain VFA, the VFA concentration in the permeate is expected to be equivalent to the VFA concentration in the mixed liquor of the methanogenic reactor. Corroborating these results, a significant negative correlation was found

Fig. 3. VFA concentration in feed and acidogenic reactor effluent and VFA production per unit VSS per day over two ranges of COD/SO24 ratio. 6

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Journal of Environmental Management 259 (2020) 110034

Fig. 4. VFA concentration in 2S-AnMBR permeate and its removal over two ranges of COD/SO24 ratio.

Fig. 5. MLVSS and VSS/TSS relation as a function of COD/SO24 ratio.

significant negative correlation between COD/SO24 ratio and sulfate removal efficiency, with the R coefficient of 0.6379. These results indicate the higher SRB activity in the lowest COD/SO24 ratio, which is in agreement with what was observed in relation to COD. The fractions of COD removed by methanogenesis and sulfidogenesis were calculated, and the results are presented in Fig. 8. According to Spearman correlation, the COD/SO24 ratio showed a significant corre­ lation with the two removal routes, with coefficients R of 0.97 and 0.99 for the fraction degraded by MA and SRB. This is confirmed by Lu et al. (2016), whose results indicated that organic matter degradation pathways are closely related to the COD/SO24 ratio. In range I, a stable COD removal was observed by both routes, being 96.8 � 0.4% via methanogenesis and 0.7 � 0.2% via sulfidogenesis. In range II, a gradual increase in sulfidogenesis removal was observed as the COD/SO24 ratio approached 10 to the detriment of the methanogenic pathway, evidencing a competition between the MA and SRB, being 80.6 � 2.6% via methanogenesis and 6.4 � 1.5% via sulfidogenesis. These values were similar to those found by Hu et al. (2015) and Jim�enez et al. (2017) in COD/SO24 ratios ranging from 10 to 20.

the two ranges (ranges I and II), these were divided into sub-regions of COD/SO24 (9.0–10.0, 10.0–14.0, 16.0–20.0, 51.0–73.0 and > 93.0) as depicted in Fig. 7. Using the Kruskal-Wallis statistical test, following the multiple comparison analysis, the existence of significant differences between these five intervals was evaluated. The first two intervals did not present significant differences between them; however, they were statistically different from the other intervals. This confirms the increased inhibition of methanogenic archaea activity in COD/SO24 ratios close to 10, and this effect is reduced as the values are higher than 20. The feed sulfate concentrations presented much higher values in range II (Fig. 2), showing that the COD/SO24 ratio reduction was mainly due to the increase of the vinasse sulfate concentration. The removal efficiency presented higher values in range II, with an average of 92.9 � 2.6%, while in the range I the values presented an average of 69.9 � 9.5%. Erdirencelebi et al. (2007), operating an UASB reactor fed with a synthetic glucose solution, found COD and sulfate removals of 95% and 80%, respectively, in COD/SO24 ratios ranging from 10 to 20. These results were close to those found in the range I, both in terms of COD and sulfate, even in lower COD/SO24 ratios, indicating better process sta­ bility in relation to sulfidogenesis. The Spearman test indicated a 7

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Journal of Environmental Management 259 (2020) 110034

(a)

(b) Fig. 6. (a) COD and (b) sulfate concentrations in 2S-AnMBR permeate and its removal.

3.4. Effect of COD/SO24 on membrane fouling

and adsorption of EPS and SMP on the membrane surface and pores, whereas organic and inorganic fouling refer to the biopolymers and scalants presence, and the inorganic precipitation depends on the cat­ ions existence in the effluent and sludge suspension (Lin et al., 2013).

Membrane fouling can be classified into biological (biofouling), organic and inorganic fouling. Biofouling is related to the accumulation 8

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Journal of Environmental Management 259 (2020) 110034

microbial products in the cake layer, with higher ratios resulting in higher SMP and EPS amounts, contributing to more severe fouling. In 1 d 1 in the range I this study, the F/M ratio was 204.7 � 71.5 mgCOD gVSS 1 1 and 631.4 � 93.2 mgCOD gVSS d in range II, with significant differences between the ranges. The influence of ionic strength due to the industrial effluent salinity can cause diverse effects in membrane fouling, being able to affect the cake layer formed (Wang et al., 2014). According to Lin et al. (2014), EPS containing ionizable groups can react with divalent cations (Ca2þ, Mg2þ) to form complex, strengthening the cake layer structure, facili­ tating the foulants adhesion. As can be seen from Table 7, a considerable reduction in calcium and potassium concentrations in the 2S-AnMBR permeate was observed in range II. This can be associated with these ions retention in the cake layer formed on the membrane surface, contributing to the fouling resistance increase, according to the values presented in Table 6. Lin et al. (2011) evaluated a lab-scale submerged anaerobic membrane bioreactor treating thermomechanical pulping whitewater. The composition analyzes performed on the cake layer formed on the membrane surface showed a composition containing,

Fig. 7. COD removal rate in sub-regions of COD/SO24 ratio.

Table 5 presents protein and carbohydrate concentrations as well as the protein to carbohydrate ratio (PT/CB ratio) in the SMP and EPS fractions, showing that all parameters evaluated had higher values in range II. According to Mann-Whitney U test, significant differences were found in PT/CB ratio between the two ranges, both to SMP and EPS. All these factors contribute to the filtration resistance increase in range II, as presented in Table 5. Due to their hydrophobicity, proteins generally can cause greater adhesion in the hydrophobic membranes than the carbo­ hydrates with their hydrophilic characteristics (Chen et al., 2017). Ac­ cording to Lin et al. (2011), sludge with higher PT/CB ratio is generally considered to have a higher viscosity, favors the cake layer formation. According to Table 6, significant correlations between COD/SO24 ratio and protein and carbohydrate production in both SMP and EPS was found. These correlations presented negative values, indicating that reducing COD/SO24 ratio promotes an increase in these components production and, consequently, in the SMP and EPS concentration. In Table 5, range II, represented by lower COD/SO24 ratios, presents higher values of PT and CB, corroborating the results found by the Spearman correlation. The reduction of COD/SO24 ratio induces toxicity to the media, contributing to the microbial cell lysis, with the conse­ quent SMP and EPS release. Song et al. (2018) observed an increase in membrane fouling with the sulfate addition (favoring sulfidogenesis), possibly due to the higher concentrations of SMP and EPS in the mixed liquor. Kobayashi et al. (2015) found that the sulfide concentration in­ crease in the feed causes higher carbohydrates and proteins release, which are important SMP and EPS constituents. Another factor pre­ sented by Liu et al. (2012) was the food to microorganism ratio (F/M) in the reactor. It has an important effect on the content and proportion of

Table 5 Characterization of SMP and EPS fractions and membrane fouling resistance (Rf) in ranges I and II (mean � std. deviation). Parameter

Unit

Proteins Carbohydrates PT/CB ratio Rf

mg L 1 mg L 1 – m 1

Range I

Range II

SMP

EPS

SMP

282.1 � 90.8 29.4 � 6.0

17.7 � 14.0 5.3 � 2.4

2878 � 518.6 � 569 86.7 137.4 � 32.4 � 9.4 17.0 21.2 � 4.8 17.7 � 7.1 2.3⸳1013 � 7.6⸳1012

9.9 � 2.1 4.4 � 2.0 3.6⸳1012 � 8.1⸳1011

EPS

Table 6 Spearman correlation coefficient (R) and correlation significance (p-value). Variable pair COD/SO24 COD/SO24 COD/SO24 COD/SO24

and SMPproteins and SMPcarbohydrates and EPSproteins and EPScarbohydrates

Fig. 8. COD removal by methanogenesis and sulfidogenesis. 9

Spearman (R) 0.4875 0.6434 0.5744 0.7222

p-value <0.0005 <0.0005 <0.0005 <0.0005

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Journal of Environmental Management 259 (2020) 110034

5. Author contributions section

Table 7 Cations concentration in feed and 2S-AnMBR permeate (mean � std. deviation). Parameter

Unit

Calcium Magnesium Potassium Sodium

mg L mg L mg L mg L

Range I 1 1 1 1

� via Rezende Silva, Natalie Cristine Magalha ~es, Paulo Ana Fla Vitor Martinelli Cunha, Miriam Cristina Santos Amaral, Konrad Koch: Conceptualization; Methodology; Formal analysis; Investigation; Resources; Writing - Original Draft; Writing - Review & Editing; Visualization.

Range II

Feed

Permeate

Feed

Permeate

254 � 7 711 � 29 3399 � 172 118 � 11

220 � 30 622 � 49 3166 � 267 362 � 25

831 � 214 182 � 27 2320 � 232 305 � 14

242 � 70 164 � 92 2257 � 158 363 � 3

Acknowledgements

among other elements, 4.45% Ca and 0.30% K. These analyses indicated certain amounts of inorganic elements were accumulated in the cake layer, which may exert significant effects on the formation of the cake layer. Through charge neutralization and bridging effect, clusters and metal ions can be caught by the sludge flocs or biopolymers, increasing the filtration resistance. Meng et al. (2007), operating an aerobic membrane bioreactor for synthetic wastewater treatment, found an inorganic contribution of 23% in the cake layer formed on the mem­ brane surface, with Ca2þ being one of the major constituents. In addi­ tion, CO2 produced by microorganisms can affect the supersaturation of carbonates in the media, increasing the potential of membrane scaling due to the carbonate precipitation of metals such as Ca2þ.

This research was supported by the National Council of Technolog­ ical and Scientific Development (CNPq), the Research Support Foun­ dation of the State of Minas Gerais (FAPEMIG) and the Coordination of Improvement of Higher Level Personnel (CAPES). References Albuquerque, J.N., Ratusznei, S.M., Rodrigues, J.A.D., 2019. Biomethane production by thermophilic co-digestion of sugarcane vinasse and whey in an AnSBBR: effects of composition, organic load, feed strategy and temperature. J. Environ. Manag. 251. Algapani, D.E., Qiao, W., Ricci, M., Bianchi, D., Wandera, S.M., Adani, F., Dong, R., 2019. Bio-hydrogen and bio-methane production from food waste in a twostage anaerobic digestion process with digestate recirculation. Renew. Energy 130, 1108–1115. https://doi.org/10.1016/j.renene.2018.08.079. Annachhatre, A.P., Suktrakoolvait, S., 2001. Biological sulfate reduction using molasses as a carbon source. Water Environ. Res. 73 (1), 118–126. https://doi.org/10.2175/ 106143001X138778. Apha, 2012. Standard Methods for the Examination of Water and Wastewater, 22a ed. American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USA. Barrera, E.L., Spanjers, H., Romero, O., Rosa, E., Dewulf, J., 2014. Characterization of the sulfate reduction process in the anaerobic digestion of a very high strength and sulfate rich vinasse. Chem. Eng. J. 248, 383–393. https://doi.org/10.1016/j. cej.2014.03.057. Buchauer, K., 1998. A comparison of two simple titration procedures to determine volatile fatty acids in influents to waste-water and sludge treatment processes. Water S.A. 24, 49–56. Ceccato-Antonini, S.R., 2018. Conventional and nonconventional strategies for controlling bacterial contamination in fuel ethanol fermentations. World J. Microbiol. Biotechnol. 34 (6), 1–11. https://doi.org/10.1007/s11274-018-2463-2. Charfi, A., Amar, N.B., Harmand, J., 2012. Analysis of fouling mechanisms in anaerobic membrane bioreactors. Water Res. 46, 2637–2650. https://doi.org/10.1016/j. watres.2012.02.021. Chen, R., Nie, Y., Hu, Y., Miao, R., Utashiro, T., Li, Q., Xu, M., Li, Y., 2017. Fouling behaviour of soluble microbial products and extracellular polymeric substances in a submerged anaerobic membrane bioreactor treating low-strength wastewater at room temperature. J. Membr. Sci. 531, 1–9. https://doi.org/10.1016/j. memsci.2017.02.046. Christian, S., Grant, S., McCarthy, P., Wilson, D., Mills, D., 2011. The first two years of full-scale Anaerobic membrane bioreactor (AnMBR) operation treating high-strength industrial wastewater. Water Pract. Technol. 6 https://doi.org/10.2166/ wpt.2011.032. Costa, M.A.S., Cerri, B.C., Ceccato-Antonini, S.R., 2017. Ethanol addition enhances acid treatment to eliminate Lactobacillus fermentum from the fermentation process for fuel ethanol production. Lett. Appl. Microbiol. 66 (1), 77–85. https://doi.org/10.1111/ lam.12819. Dai, X., Hu, C., Zhang, D., Dai, L., Duan, N., 2017. Impact of a high ammoniaammonium-pH system on methane-producing archaea and sulfate-reducing bacteria in mesophilic anaerobic digestion. Bioresour. Technol. 245, 598–605. https://doi. org/10.1016/j.biortech.2017.08.208. Dar, S.A., Kleerebezem, R., Stams, A.J.M., Kuenen, J.G., Muyzer, G., 2008. Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a labscale anaerobic bioreactor as affected by changing substrate to sulfate ratio. Appl. Microbiol. Biotechnol. 78, 1045–1055. https://doi.org/10.1007/s00253-008-13918. Dellias, M. de T.F., Borges, C.D., Lopes, M.L., Cruz, S.H., de Amorim, H.V., Tsai, S.M., 2018. Biofilm formation and antimicrobial sensitivity of lactobacilli contaminants from sugarcane-based fuel ethanol fermentation. Antonie Leeuwenhoek 111 (9), 1631–1644. https://doi.org/10.1007/s10482-018-1050-8. Dilallo, R., Albertson, O.E., 1961. Volatile acids by direct titration. J. Water Pollut. Control Fed. 33 (4), 356–365. https://doi.org/10.1016/j.desal.2008.10.010. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28 (3), 350–356. https://doi.org/10.1021/ac60111a017. Erdirencelebi, D., Ozturk, I., Cokgor, E.U., 2007. System performance in UASB reactors receiving increasing levels of sulfate. Clean 35 (3), 275–281. https://doi.org/ 10.1002/clen.200700043. Fuess, L.T., Garcia, M.L., 2014. Implications of stillage land disposal: a critical review on the impacts of fertigation. J. Environ. Manag. 145, 210–229. https://doi.org/ 10.1016/j.jenvman.2014.07.003.

4. Conclusions 2S-AnMBR performance at the highest COD/SO24 ratios showed stable COD removal efficiencies above 97%. A sulfate concentration increase, leading to a lower COD/SO24 ratio, negatively impacted the methanogenic archaea activity by fostering sulfidogenesis. This led to VFA accumulation in the methanogenic reactor, with a consequent reduction of organic matter removal. The increased concentration of SMP and EPS, caused by the sulfide toxicity on the microorganisms, caused an increase in membrane filtration resistance. In relation to the 2S-AnMBR permeate, it is composed by high amounts of organic matter and salts, hindering the reuse of this effluent in boilers or cooling sys­ tems. An alternative is its use in cane washing, as the volume needed for this step in the industrial process is very high and does not require water with high quality standards. The H2S removal in permeate is important as it is a compound which presence causes equipment and pipelines corrosion, damage to the environment, health and safety problems. Detrimental effects caused by the increase of sulfate concentration in the vinasse when undergoing the anaerobic treatment were observed. According to the results, the reduction of COD/SO24 ratio values close to 10 favored the sulfidogenesis occurrence. Therefore, it is important to reduce the sulfate concentration in the wastewater, which can be ach­ ieved through improvements in the ethanol production process, since the sulfate comes from the sulfuric acid addition in the fermentation stage, in order to avoid microbial contamination of the medium. Replacing sulfuric acid with other products, such as antibiotics, reduces the concentration of sulfate present in vinasse. Thus, the sulfate present in vinasse will come only from feedstock, which corresponds to low concentrations, leading to increased COD/SO24 ratio, benefiting the anaerobic process, increasing methane production, enriching biogas (there will be no H2S production), and reducing the fouling of the membrane coupled to the bioreactor. However, this treatment may not be effective due to the contami­ nation level and type of bacteria in addition to the damages caused to the yeast cells and the raise of the production costs relative to the in­ crease of sulfuric acid consumption (Costa et al., 2017). In order to avoid problems concerning antibiotic resistance and the risks of the sulfuric acid use, natural products with antimicrobial properties should be tested. Numerous antimicrobial compounds are found in herbs, spices, fruits, vegetables, seeds, and leaves, exerting direct or indirect effects, offering a safer option against bacterial contamination in the ethanol production (Ceccato-Antonini, 2018).

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