Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability

Bioresource Technology xxx (2016) xxx–xxx

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Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability Leandro Janke a,b,⇑, Athaydes F. Leite c, Karla Batista c, Witan Silva a, Marcell Nikolausz c, Michael Nelles a,b, Walter Stinner a a

Department of Biochemical Conversion, Deutsches Biomasseforschungszentrum gemeinnützige GmbH, Torgauer Straße 116, 04347 Leipzig, Germany Faculty of Agricultural and Environmental Sciences, Chair of Waste Management, University of Rostock, Justus-von-Liebig-Weg 6, 18059 Rostock, Germany c Department of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany b

h i g h l i g h t s
A new method to stabilize the anaerobic digestion of sugarcane vinasse is proposed.
Urea addition increased alkalinity but not prevented the process failure.
The addition of KH2PO4 induced volatile fatty acids accumulation.
Urea and trace elements addition improved the process stability in UASB reactor.
A stable specific methane production of 239 mL g COD

a r t i c l e

i n f o

Article history: Received 3 December 2015 Received in revised form 26 January 2016 Accepted 28 January 2016 Available online xxxx Keywords: Sugarcane vinasse Anaerobic digestion Process stability Biogas productivity

1

was achieved.

a b s t r a c t In this study, the effects of nitrogen, phosphate and trace elements supplementation were investigated in a semi-continuously operated upflow anaerobic sludge blanket system to enhance process stability and biogas production from sugarcane vinasse. Phosphate in form of KH2PO4 induced volatile fatty acids accumulation possibly due to potassium inhibition of the methanogenesis. Although nitrogen in form of urea increased the reactor’s alkalinity, the process was overloaded with an organic loading rate of 6.1 g COD L1 d1 and a hydraulic retention time of 3.6 days. However, by supplementing urea and trace elements a stable operation even at an organic loading rate of 9.6 g COD L1 d1 and a hydraulic retention time of 2.5 days was possible, resulting in 79% higher methane production rate with a stable specific methane production of 239 mL g COD1. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Sugarcane is one of the most used feedstocks for sugar and bioethanol production in the world (Elbehri et al., 2013). During the ethanol distillation, large amounts of sugarcane vinasse (SCV), also called stillage, are produced. In countries like Brazil SCV is directly used on the fields to partially replace mineral fertilizers during the sugarcane cultivation. This practice has been accused to cause several environmental problems, such as leaching of metals to groundwater, changes in soil quality, increase of phy⇑ Corresponding author at: Department of Biochemical Conversion, Deutsches Biomasseforschungszentrum gemeinnützige GmbH, Torgauer Straße 116, 04347 Leipzig, Germany. Tel.: +49 (0) 341 2434 793; fax: +49 (0) 341 2434 133. E-mail address: [email protected] (L. Janke).

totoxicity, unpleasant odor, as well as leading to considerable methane emissions during temporary storage, transportation and also nitrous oxide emissions after application of SCV to the soil (Christofoletti et al., 2013; De Oliveira et al., 2013). Anaerobic digestion (AD) is a potential solution to reduce such type of environmental problems, since the organic matter content of SCV would be converted to methane and/or platform chemicals for value-added products as a result of the different syntrophic biochemical phases: hydrolysis, acidogenesis, acetogenesis and methanogenesis (Cysneiros et al., 2012; Janke et al., 2015a; A. Leite et al., 2015). The integration of such process into the current sugar and bioethanol production would diversify the product portfolio of the sugarcane plants in a biorefinery concept and improve their robustness against market fluctuations of the main products (Mariano et al., 2013).

http://dx.doi.org/10.1016/j.biortech.2016.01.110 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110

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However, despite of its proven biogas potential (Janke et al., 2014), the use of SCV for biogas production is challenging by different reasons. Due to the sulfating process used in raw sugar production and the addition of sulfuric acid to prevent bacterial contamination during the alcoholic fermentation, usually high amounts of sulfate are found in SCV (Moraes et al., 2015; Wilkie et al., 2000). The AD of sulfur-rich substrates can lead to various undesirable effects: (a) sulfate reducing bacteria (SRB) outcompete methanogens for hydrogen and acetate due to thermodynamic advantages, resulting in sulfides and less methane production; (b) high sulfide concentrations has a direct toxic effect on certain anaerobic microorganisms; and (c) sulfide production and metalsulfide precipitation is known as one of the most important processes limiting the availability of trace metals for microbial uptake, thus negatively affecting the efficiency and stability of the AD process (Chen et al., 2008; Demirel and Scherer, 2011; Shakeri Yekta et al., 2014). Previous studies on main characteristics of SCV in different types of sugarcane biorefineries (Janke et al., 2015b), showed that regardless of the different feedstocks used for bioethanol production (sugarcane juice and/or molasses), some important trace elements (TE) as Ni and Cu were found below the optimum values for AD, or in case of Fe, Co, Mo, W, Mn, Se and Zn concentrations were close to the minimum range recommended by Oechsner et al. (2008) and Kayhanian and Rich (1995). Although the benefits of TE supplementation on biogas production from other types of vinasse, as sugar beet, wheat and dried distillers grains with solubles (DDGS) have already been studied (Gustavsson et al., 2013; Moraes et al., 2015; Schmidt et al., 2014), to our knowledge no recent study was done by using SCV. It is well known that the characteristics of vinasse are highly dependent on the different feedstocks used for bioethanol production (Wilkie et al., 2000), thereby different supplementation requirements can be expected for an optimized AD system using SCV as substrate. Furthermore, the low alkalinity and high organic acids concentration of SCV results in an unfavorable pH (between 3 and 5) for methanogenesis, which in turn requires the addition of alkaline compounds to counteract such acidity by buffering the pH to an optimum value for AD (Mota et al., 2013; Wang et al., 2014). As a result, the costs for alkalinity supplementation can be a major expense for anaerobic treatment of some industrial wastewaters, possibly exceeding the value of the methane produced (Speece, 1996). In such cases, the addition of urea to the AD of SCV followed by fertigation of the digestate on the sugarcane fields is a promising strategy to increase the alkalinity of the AD system (via urea conversion into OH + NH+4), as well as reducing the costs of alkalinity supplementation, since urea is frequently used as a source of nitrogen during the sugarcane cultivation. However, such concept has already been tested under low urea concentrations (0.15– 0.20 g L1) in a mesophilic upflow anaerobic sludge blanket (UASB) system with unsatisfactory results (Boncz et al., 2012). The addition of urea in higher concentrations would enhance its buffering effects, but due to the C:N ratio of SCV (13–24:1) below or close to the lower limit recommended for AD (20–40:1) (FNR, 2010; A. F. Leite et al., 2015), a higher risk of process failure by free ammonia inhibition may appear (Lv et al., 2014). Therefore, the supplementation of urea in higher concentrations (2 g L1), phosphate and TE based on the substrate elemental composition were investigated in a semi-continuously operated UASB system in order to assess whether the increase of alkalinity provided by urea conversion into OH + NH+4 could stabilize the process and enable the application of high organic loading rates (OLR) and low hydraulic retention times (HRT). Such approach could considerably reduce the capital/operational expenditures of

biogas production from SCV and encourage the development of a bio-based economy. 2. Methods 2.1. Substrate and inoculum Two different samples of SCV (sample 1 and sample 2) were obtained from a distillery plant in the State of Goiás (Brazil) during the 2014/2015 season, transported to Germany in sealed plastic gallons and stored at 4 °C until its use. A large-scale expanded granular sludge blanket (EGSB) reactor treating wastewater from a pulp and paper industry provided the seed sludge used as inoculum for the semi-continuous experiment. 2.2. Semi-continuous experiment Two lab-scale UASB reactors (R1 and R2) with 1.5 L total volume each (1.3 L working volume) were used for this experiment. The operation temperature was kept under mesophilic conditions (40 °C) by recirculating hot water through the double-walled reactors. To improve the substrate contact with the granular biomass, digestate was continuously re-circulated (5 mL min1) by a peristaltic pump TU 200 (Medorex, Germany). Details about the schematic design of the UASB system are found in Fig. 1. Prior to the start of the experiment the reactors were operated for 150 days with the same type of substrate (SCV) buffered with NaHCO3 (0.5 g g COD1). The current experiment was carried out over 190 consecutive days with the same feeding regime (20 times per day) by using a peristaltic pump PD 5201 (Heidolph, Germany). For the phase I (days 0–65) the HRT was set to 16.3 days and gradually decreased to 3.6 days and the OLR increased from 1.5 to 6.1 g COD L1 d1 in order to assess the effects of urea (2 g L1) and KH2PO4 (0.34 g L1) supplementation in reactor R1 under higher load conditions. During the phase II (days 66–117) both reactors (R1 and R2) were daily supplemented with a TE solution composed of 5 mg Co, 2 g Fe, 40 mg Cu, 700 mg Mn, 7 mg Mo, 15 mg Ni, 1 mg W and 150 mg Zn per kg of TS according to average values suggested by previous studies (Kayhanian and Rich, 1995; Oechsner et al., 2008). Furthermore, NaHCO3 (1 g g COD1) was also added to recover the pH of both reactors from an inhibited state during the days 76–117. During the phase III (days 118– 190) urea (2 g L1) combined with TE were only added to the reactor R1, while only TE added to the reactor R2 in the same concentrations as used for the phase II. Detailed information about different feeding rates, OLR and HRT are listed in Table 1. 2.3. Analytical methods Total solids (TS) and volatile solids (VS) were determined by drying the samples for 24 h at 105 °C in a drying oven (Binder, Germany) and further reducing the organic content to ashes for 2 h at 550 °C in a high temperature oven (Carbolite, UK). The COD of noncentrifuged substrate samples was analyzed using a LCK 014 COD kit (Hach-Lange, Germany) according to the manufacturer’s protocol. To determine the major elements contained in SCV, dried samples were pre-treated with a mixture of HNO3/H2O2/HF and latter neutralized with H3BO3, and the resulting clear solution was analyzed by inductively coupled plasma atomic spectrometry (ICPOES, ThermoFisher iCAP6200). The daily biogas production in each UASB reactor was measured by a milligascounter type MGC-10 (Ritter, Germany), and corrected to standard temperature and pressure conditions (273.15 K and 101.325 kPa). The composition of the biogas (CH4, CO2, O2 and H2S) collected in a gas bag after being recorded by

Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110

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Fig. 1. Schematic design of the UASB system.

the milligascounter was measured twice a week using a GA2000 Landfill Gas Analyzer (Geotechnical Instruments Ltda., UK). Specific methane production (SMP) was presented in norm milliliters per g of COD added (mL g COD1) and the methane production rate (MPR) calculated in norm liters of methane per liter of reactor per day (L L1 d1). Every day the pH value of the digestate was measured directly in the digestate storage of the UASB reactors with a pH-electrode Sentix 41 (WTW, Germany). Two times a week digestate samples

were centrifuged for 10 min at 10,000 rpm and 10 °C. The supernatant was used after filtration for subsequent analysis, including the measurement of volatile organic acids (VOA) and the ratio of total inorganic carbonate to calcium carbonate (VOA/TIC, gVOA =gCACO3 ) by using a Titration Excellence T90 titrator (MettlerToledo GmbH, Switzerland). Volatile fatty acids (VFA), including acetic, propionic, n-butyric, iso-butyric, n-valeric, iso-valeric and hexanoic acid, were determined using a 5890 series II gas chromatograph (Hewlett Packard, USA) equipped with an HS40

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Table 1 Overview of the reactors setup during the AD experiment with SCV. Phase

Period (day)

SCVa (mL d1)

HRTb (d)

OLRc (g COD L1 d1)

Reactor 1

Reactor 2

I

0–8 9–19 20–28 29–43 44–49 50–65

80 160 240 280 320 360

16.3 8.1 5.4 4.6 4.1 3.6

1.5 2.7 4.1 4.7 5.4 6.1

w/ urea

w/o urea

w/ urea + KH2PO4 (days 35–62)

w/o urea + KH2PO4 (days 35–62)

II

66–70 71–75 76–117

360 360 80

3.6 3.6 16.3

6.1 6.1 1.4

w/ urea w/ urea + TE w/ urea + TE + NaHCO3

w/o urea w/o urea, w/ TE w/o urea, w/ TE + NaHCO3

III

118–138 139–153 154–169 170–180 181–190

160 240 320 380 520

8.1 5.4 4.1 3.4 2.5

2.7 4.5 5.9 7.0 9.6

w/ urea + TE

w/o urea, w/ TE

Note: w/ means with; w/o means without. a Sugarcane vinasse. b Hydraulic retention time. c Organic loading rate.

automatic headspace sampler (Perkin Elmer, USA) and an Agilent HP-FFAP column (30 m  0.32 mm  0.25 lm) according to a previously described method (Sträuber et al., 2015). Total ammonianitrogen concentration (TAN) was determined from 500 lL filtered supernatant diluted with distilled water (1:500) with the Nessler method using a benchtop spectrophotometer DR 3900 (HachLange, Germany). Free ammonia-nitrogen concentration (FAN) was calculated according to the equation previously described by Nie et al. (2015):

FAN ¼ TAN  1 þ

10pH ð0:09018þ2729:92 Þ TðKÞ

10

!1 ð1Þ

where FAN is the concentration of free ammonia-nitrogen in g L1; TAN is the concentration of measured total ammonia-nitrogen in g L1; T(K) is the temperature in kelvin. 2.4. Data analysis To assess the relationship among the adopted measures and their effects on the monitored parameters, a Spearman’s rankorder correlation analysis was performed separately for each reactor (R1 and R2), and independently for each phase of the experiment. The analysis was run with the software Statistica 6.0 (Statsoft, Tulsa, OK, USA). The adjustment of the experimental data to the Spearman’s correlation was determined by the following equation:

P 2 6 d q¼1 2 i nðn  1Þ

ð2Þ

where q is the Spearman rank correlation; di is the difference between the ranks of corresponding values; Xi and Yi and n is the number of value in each data set.

Table 2 Main characteristics of the SCV. Parameters

Sample 1

Sample 2

Units

Total solids (TS) Volatile solids (VS) Carbon (C) Nitrogen (N) Phosphorus (P) Sulfur (S) Iron (Fe) Calcium (Ca) Sodium (Na) Potassium (K) Magnesium (Mg) Nickel (Ni) Cobalt (Co) Molybdenum (Mo) Tungsten (W) Manganese (Mn) Copper (Cu) Selenium (Se) Zinc (Zn) Chemical oxygen demand (COD) Total ammonia-nitrogen (TAN) Acetic acid Ethanol Lactic acid Phenylacetic acid Propionic acid Minor organic acids and estersb Sum of organic acids

1.24 ± 0.01 80.98 ± 0.11 40.0 ± 0.15 2.69 ± 0.02 0.13 ± 0.01 1.37 ± 0.02 283 ± 5.85 4870 ± 49.43 229 ± 1.93 18,834 ± 52.38 2857 ± 39.10 0.47 ± 0.07 0.53 ± 0.01 0.48 ± 0.02 n.d. 55.4 ± 0.69 3.72 ± 0.31 n.d. 29.4 ± 0.47 22.72 ± 0.96

1.94 ± 0.02 84.75 ± 0.44 39.3 ± 1.05 3.53 ± 0.16 0.47 ± 1.00 1.58 ± 0.06 546 ± 3.80 7735 ± 20.30 491 ± 3.87 43,227 ± 872 5851 ± 159 0.79 ± 0.04 0.84 ± 0.03 0.86 ± 0.05 n.d. 93.7 ± 1.45 9.58 ± 0.11 n.a. 81.3 ± 0.49 23.07 ± 1.46

% FMa % TS % TS % TS % TS % TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS mg kg1 TS g L1

17.92 ± 0.29

26.46 ± 0.15

mg L1

942 ± 11.4 1259 ± 20.9 682 ± 45.8 n.d. 39.7 ± 2.0 53.8 ± 0.42

931 ± 21.6 1626 ± 16.1 1304 ± 33.2 188 ± 5.95 120 ± 5.26 74.2 ± 2.07

mg L1 mg L1 mg L1 mg L1 mg L1 mg L1

2978 ± 72.7

4246 ± 34.1

mg L1

n.a.: not analyzed. n.d.: not detected. Note: Sample 1 was used during the period of 0–94 days and sample 2 used during the period of 95–190 days. a Fresh matter. b Sum of propanol, butanol, formic, butyric, valeric, hexanoic, octanoic, nanoic and decanoic acid.

3. Results and discussion 3.1. Substrate composition The main characteristics of the SCV samples used for the semicontinuous experiment are presented in Table 2. The TS and COD values were similar (TS: 1.24–1.94% and COD: 22.72–23.07 g L1) to ones from a recent study analyzing samples along an operating

season from a distillery plant (A. Leite et al., 2015; A.F. Leite et al., 2015). However, they are considerably lower than the values found for annexed plants (combined sugar and bioethanol production), possibly due to the different feedstocks used for bioethanol production (mixture of molasses/sugarcane juice or only sugarcane juice) (Janke et al., 2015b).

Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110

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Fig. 2. Main parameters monitored during the evaluation of process stability. (A) Volatile organic acids (VOA); (B) ratio of volatile organic acids with total inorganic carbonate to calcium carbonate (VOA/TIC); (C) total inorganic carbonate to calcium carbonate (TIC); (D) pH value. Note: After day 166 the parameters VOA, VOA/TIC and TIC could not be measured in reactor R2 due to the pH value lower than 5.

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In both samples the C:N ratio (11–15:1) was found below the optimum range for AD (20–40:1), which could lead to ammonia inhibition if the surplus nitrogen is converted to FAN. For sample 1 the C:P ratio was also unfavorable (307:1) in comparison to the recommended optimum ratio (120:1), increasing the risk of process malfunctioning since phosphorus is an important element for the formation of energy carrier ATP, essential for microbial growth. Sulfur concentration was found critical for both SCV samples, around 7–8 times higher than the optimum value suggested by FNR (2010). Besides leading to the contamination of biogas with H2S, the high sulfur content could also negatively affect the availability of some important TE (through metal-sulfide precipitation), since iron, nickel, cobalt, molybdenum, manganese, copper and zinc were found below or close the lower limit recommended by Oechsner et al. (2008) and Kayhanian and Rich (1995).

3.2. Process stability 3.2.1. Phase I For the experiment’s phase I (days 0–65) the OLR was set to 1.5 g COD L1 d1 and gradually increased to 6.1 g COD L1 d1, while the HRT was decreased from 16.3 to 3.6 days. During the initial 35 days none of the reactors showed any sign of process unbalance with average VOA concentration of 0.18 g L1 and VOA/TIC ratio of 0.07, in spite of the decrease in the digestate’s pH due to the wash-out of residual NaHCO3 previously added to the reactors (Fig. 2). The Fig. 3 shows that around day 35 the urea supplementation in the reactor R1 has considerably increased the TAN concentration

(0.9 g L1) in comparison to the reactor R2 (TAN of 0.1 g L1). This supplementation strategy has compensated the loss of alkalinity previously supplied by the NaHCO3, since the results from Spearman’s rank analysis (P < 0.05) evidenced a strong positive correlation of 0.96 between HRT and TIC for reactor R2, while such correlation was not observed in reactor R1. Indeed, the TIC values in reactor R2 were continuously reduced to 1.2 g L1 until the end of the phase I, while in the reactor R1 a higher average value was observed (3.2 g L1). Additionally, the urea supplementation has also contributed to the maintenance of a constant pH of the digestate even under the influence of a VOA accumulation observed after day 35 (peak of 1.9 g L1 at day 62). Considering the unfavorable C:P ratio of the SCV Sample 1 for AD, a phosphate supplementation based on KH2PO4 was formulated and added to the reactor R1 from day 35. In contrary to other study (Scherer et al., 2009), the process has not benefitted from phosphate supplementation and even an increase of VOA from 0.15 to 1.9 g L1 (VOA/TIC of 0.58) was observed at day 62, while the VOA concentration in reactor R2 was kept below 0.15 g L1 (VOA/TIC of 0.07). In this case, the additional potassium supplied by the KH2PO4 could has directly affected the acetate-utilizing microorganisms, since acetic acid reached the concentration of 1.3 g L1 (85% of total VFA) (Fig. 4), or even intensified the negative effects of sulfide by removing essential micronutrients from the active sludge in the UASB reactor (Chen et al., 2008).

3.2.2. Phase II At day 65th of the experiment, when the OLR and HRT were constant at, respectively, 6.2 g COD L1 d1 and 3.6 days, a sudden increase of organic acids was observed in both reactors, which is

Fig. 3. Nitrogen profile monitored during the evaluation of process stability. (A) Total ammonium-nitrogen concentration (TAN); (B) free ammonia-nitrogen concentration (FAN).

Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110

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Fig. 4. Volatile fatty acids (VFA) profile. (A) Reactor R1; (B) reactor R2.

also reflected in the pH values of their digestates. The higher TIC concentration provided by urea supplementation reduced the negative effects of organic acids on the pH value, but did not completely prevent the process imbalance. In reactor R1 the VOA of 4.7 g L1 resulted in a pH of 6.5 at day 71, while a lower VOA concentration (3.3 g L1) caused a stronger drop of pH to 5.1 in the reactor R2 during the same period. Previous studies on AD of sulfur-rich substrate (Schmidt et al., 2013) suggested that the primary cause of this process imbalance is possibly due to deficiency of TE causing inhibition of propionate oxidizing bacteria and acetate-utilizing methanogens under high OLR and low HRT applied at that period, because the predominant accumulated VFA were acetic (50%) and propionic acids (30%) in both reactors. Thus, in order to recover the AD process, a TE solution was supplemented between days 71 and 75, keeping the same applied OLR and HRT. As a result a transient reduction of the VOA concentration from 4.7 to 2.9 g L1 between days 71 and 76 was observed in reactor R1 and VOA stopped to increase during the same period in reactor R2. However, the recovery strategy based only on TE addition was not able to completely stabilize the AD process, since acetic and propionic acids reached a peak concentration of 4.4 and 2.3 g L1 in reactor R1, and of 5.5 and 3.0 g L1 in reactor R2, respectively. Therefore, at day 76th of the experiment a drastic intervention was performed by reducing the OLR to 1.4 g COD L1 d1 and increasing the HRT to 16.3 days, together with the addition of 1.0 g of NaHCO3 per g COD in both reactors until day 105. Such strategy was able to reactivate the organic acids conversion by providing a more favorable pH for methanogenesis, resulting in a reduction of the VOA concentration from 7.4 to 2.3 g L1 (VOA/

TIC from 2.2 to 0.24) in reactor R1 and from 9.4 to 1.7 g L1 (VOA/TIC 4.1–0.21) in reactor R2. The positive effects of this bicarbonate buffer addition are also demonstrated by the Spearman rank analysis on TIC and VOA/TIC (q: <0.71) in both reactors, and subsequent correlation between the pH of the digestates and the decrease of OLR (q: 0.67), and the increase of HRT (q: 0.67). Furthermore, it is noteworthy that the combined urea and NaHCO3 supplementation in reactor R1 has considerable increased the FAN to a maximum concentration of 0.8 g L1 at day 118, which could have led to inhibition of the AD process by ammonia toxicity based on previous studies using continuous stirred-tank reactors (CSTR) under mesophilic condition (Nie et al., 2015). 3.2.3. Phase III After the process recovery, urea as a buffer agent was investigated in reactor R1 under the influence of TE supplementation (reactor R2 received only TE) by gradually increasing the OLR from 1.4 to 5.9 g COD L1 d1 and decreasing the HRT from 16.3 to 4.1 days during the period of 118–163 days. Despite of the strong correlation between OLR-TIC (q: <0.82) and HRT-TIC (q: >0.82) observed in both reactors due to the wash-out of the NaHCO3 added during the phase II of the experiment. The reactor R1 was able to keep a constant TIC of 4.0 g L1 after day 140 due to the higher NH4-N provided by urea supplementation (TAN of 1.0 g L1), while the TIC values of the reactor R2 kept decreasing to below 2.0 g L1 during the same period (TAN of 0.17 g L1). Such difference on alkalinity values is the main reason for the second accumulation of organic acids observed in reactor R2 after day 165. Despite of the supplementation of TE the reactor R2 was not able to maintain a stable AD process, since at a low TIC concentration small amounts of accumulated organic acids have a major

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effect on the reactor’s pH, causing inhibition to the AD process (Appels et al., 2008). In fact, the second VOA accumulation was responsible for the reduction of the pH of the digestate in the reactor R2 from a stable condition to less than 5 in a short period of 5 days. A maximum of total VFA of 4.6 g L1 (54% of acetic acid, 21% of n-butyric acid and 20% of propionic acid) was reached at day 169. In the meantime, the reactor R1 was not only able to keep a stable AD process under the same OLR and HRT at which the reactor R2 failed, but also a 62% higher OLR (9.6 g COD L1 d1) and a lower HRT of 2.5 days was reached without any major process unbalance, except a short VOA accumulation of 1.2 g L1 (VOA/ TIC 0.18) at day 183. According to Appels et al. (2008) for each mol of VFA are necessary at least 1.4 mol of bicarbonate for a stable and well buffered digestion system. Such fact supports the use of urea as a buffer agent, since based on the TIC concentration of 3.9 g L1 observed at that time, the reactor R1 could support around 2.8 g L1 of VFA without pH reduction below the optimum value for methanogenesis (>6.5). 3.3. Methane production and biogas composition 3.3.1. Phase I The methane production rate (MPR) and specific methane production (SMP) during the phase I of the experiment are shown in Fig. 5. Although the MPR strongly correlated with the OLR (q: >0.89) and the HRT (q: >0.89) in both reactors. The HRT reduction from 16.3 to 3.6 days and increase of OLR from 1.4 to 6.1 g COD L1 d1 have led to the reduction of the SMP by 17% from 242 to 201 mL g COD1 in reactor R2, and even a larger decrease (30%) in reactor R1 (from 263 to 184 mL g COD1) was observed

due to process imbalance. Whereas, the highest MPR during the experiment’s phase I was observed between days 50 and 60 when the OLR was set to 6.1 g COD L1 d1, in which the reactor R2 achieved 9% higher MPR (1.3 L L1 d1) than the reactor R1 (1.2 L L1 d1). In the meantime, biogas composition was similar in both reactors. At the beginning of the experiment, methane concentration was higher (75% v/v) and decreased over time (58% v/v between days 48 and 62), possibly due to the wash-out of NaHCO3 previously added to the reactors. By reducing the bicarbonate buffer more CO2 was released to the gas phase causing a reduction of the CH4 concentration in the biogas. Additionally, H2S values were found in most part of this phase higher than the maximum detection limit of the gas analyzer device (5000 ppm), except for a few measurements lower values around of 4500 ppm were observed (Fig. 6).

3.3.2. Phase II As a consequence of the process imbalance observed in both reactors after the 65th day of the experiment, the SMP and MPR were drastically reduced to nearly zero even before the ORL reduction and HRT increase at day 76. Such fact clearly demonstrates that the system was not able to support the applied OLR and HRT causing an inhibition of methanogenesis, since the CH4 concentration in the biogas was reduced to 25% v/v. After the addition of NaHCO3, reduction of OLR to 1.4 g COD L1 d1 and increase of HRT to 16.3 days from day 76, the methane production was recovered in both reactors. However, an unstable gas production was observed until the end of phase II (day 117) due the simultaneous conversion of the new substrate

Fig. 5. Monitored methane production. (A) Methane production rate (MPR) and organic loading rate (OLR); (B) specific methane production (SMP) and hydraulic retention time (HRT).

Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110

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Fig. 6. Biogas composition profile. (A) methane (CH4), carbon dioxide (CO2) and oxygen (O2) concentration in reactor R1; (B) hydrogen sulfide (H2S) concentration in reactor R1; (C) methane (CH4), carbon dioxide (CO2) and oxygen (O2) concentration in reactor R2; (D) hydrogen sulfide (H2S) concentration in reactor R2. Note: After day 163 the biogas composition could not be measured in reactor R2 due to the low biogas production.

supply and the accumulated organic acids, resulting in a high variation of the SMP between 130 and 420 mL g COD1. During this period the effects of NaHCO3 on biogas composition were also pronounced. However, at this time the addition of bicarbonate buffer trapped CO2 in the liquid phase resulting in an increase of the CH4 concentration in the gas phase to the highest observed level during the entire experiment (80% v/v). Moreover, it seems that the combined addition of NaHCO3 and TE (including FeCl3) has influenced the H2S concentration in the biogas. According to Firer et al. (2008) the FeS precipitation is a pH dependent reaction, in which at a higher pH value (caused by NaHCO3 addition) more FeS is expected to be formed. In this case, a reduction of the H2S in the biogas was observed at the same time when the alkalinity and pH reached their peak values. 3.3.3. Phase III The MPR of both reactors behaved similar to phase I between days 118 and 163, achieving a maximum value of 1.4 L L1 d1 at an OLR of 5.9 g COD L1 d1 and a corresponding HRT of 4.1 days. However, after this period the reactors behaved differently. The MPR and SMP of reactor R2 have reduced drastically to nearly zero as a consequence of the methanogenesis inhibition, while the reactor R1 kept a constant MPR and SMP of 1.4 L L1 d1 and 236 mL g COD1, respectively. Such behavior is confirmed by the different Spearman rank values obtained for phase III. In reactor R1 a positive correlation of 0.77 was obtained for SMP-OLR and the value of 0.77 for SMP-HRT, while in reactor R2 the opposite was observed (q: 0.60 for SMP-OLR and 0.60 for SMP-HRT). Regarding the biogas composition, a reduction of CH4 concentration to 59% (v/v) under stable conditions was observed until day 163 while the NaHCO3 was being washed-out from the reactors. At the same time the H2S concentration surpassed the detection limit of 5000 ppm, in the same way as previously observed when the reactors where not strongly buffered (phase I).

After this period the CH4 concentration in reactor R2 reduced to lower than 50% (v/v) while the CO2 increased, followed by a negligible SMP. In the meantime, the SMP in reactor R1 not only was kept constant in 239 mL g COD1 (CH4 of 59% v/v), but also the MPR achieved the highest observed level during the entire experiment (2.3 L L1 d1) at an OLR of 9.6 g COD L1 d1 and a HRT of 2.5 of days. 3.4. Final remarks A new strategy to stabilize the AD of SCV was proposed by combined supplementation of urea and TE. In contrary to other alternative buffering agents, such as sodium bicarbonate, sodium hydroxide or calcium hydroxide, the proposed addition of urea does not immediately result in the increase of the pH value or the alkalinity of the substrate, since the degradation of urea to ammonia is a biochemical reaction catalyzed by bacteria present in the reactor. Besides the substrate acidity/alkalinity, other aspects also influencing the requirements of alkalinity supplementation for a stable AD process are the reactor configuration, degradation kinetics, feed in regime and recirculation rate. Based on these aspects it would be unrealistic to recommend a standard urea dosage for all possible AD configurations that uses SCV as substrate. However, considering the method reviewed by Appels et al. (2008), which suggests that at least 1.4 mol of bicarbonate buffer are necessary to counteract one mol of VFA without reducing the pH value. It is possible to assume that the alkalinity value found in R1 (3.9 g L1) during the phase III would be able to counteract a VFA concentration of up to 2.8 g L1. Additionally, the combined urea supplementation with TE is considered to be a key factor to keep the process stable under the applied experimental set-up. Stimulation of the methanogenic activity with essential micronutrients results in a better VFA conversion process into biogas, hence

Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110

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avoiding major accumulation of these intermediate products and therefore reducing the alkalinity requirements. In fact, when urea and TE were supplemented separately, the AD process failed in R1 (urea) and R2 (TE) at days 65 and 165, respectively. These findings reinforce the synergistic effect of urea and TE supplementation for a stable AD process found in R1 (urea and TE) between days 118 and 190. In contrast to other studies that reported the positive effects of TE addition on the AD of other sulfur-rich substrates (Espinosa et al., 1995; Gustavsson et al., 2013), the TE supplementation alone has not stabilized the process in our system due to the low alkalinity found in R2. The low TS concentration of the SCV samples used during this experiment seems to be the reason of the low TAN accumulation in R2 (non-urea supplemented rector), resulting in an insufficient alkalinity for a stable AD process (Schmidt et al., 2014b). In this case without increasing the alkalinity by urea addition the benefits of TE could not have been realized. Future studies on the optimization of TE dosage for a rational use of this supplement are recommended. However, in case of reducing the urea dosage, a direct reduction of alkalinity would occur, increasing the risk of process failure, especially if higher OLRs and lower HRTs would be applied. 4. Conclusions The present study showed the synergistic effect of urea and trace elements supplementation for a stable biogas production from sugarcane vinasse. The addition of urea (2 g L1) improved the reactor’s alkalinity, but the process failed with an OLR of 6.1 g COD L1 d1 and HRT of 3.6 days. However, by supplementing the same amount of urea in combination with trace elements, enough alkalinity was provided for a stable biogas production even at higher OLR (9.6 g COD L1 d1) and lower HRT (2.5 days), since the addition of essential micronutrients stimulated the methanogenic activity avoiding major VFA accumulation. Acknowledgements The authors would like to acknowledge the support of the Brazilian National Scientific Counsel (CNPq) under the Program Science without Borders for the financial support of the PhD students Leandro Janke (237938/2012-0) and Athaydes Leite (202024/2012-1). The present research was partially financed by the i-NOPA Project ‘‘Sustainable bioeconomy in Brazil: Bioenergy from biogas using various types of waste substrates from the Brazilian bioethanol industry”. References Appels, L., Baeyens, J., Degrève, J., Dewil, R., 2008. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energy Combust. Sci. 34, 755–781. http://dx.doi.org/10.1016/j.pecs.2008.06.002. Boncz, M.A., Formagini, E.L., da Santos, L.S., Marques, R.D., Paulo, P.L., 2012. Application of urea dosing for alkalinity supply during anaerobic digestion of vinasse. Water Sci. Technol. 66, 2453–2460. http://dx.doi.org/10.2166/ wst.2012.476. Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol. 99, 4044–4064. http://dx.doi.org/10.1016/j. biortech.2007.01.057. Christofoletti, C.A., Escher, J.P., Correia, J.E., Marinho, J.F.U., Fontanetti, C.S., 2013. Sugarcane vinasse: environmental implications of its use. Waste Manage. 33, 2752–2761. http://dx.doi.org/10.1016/j.wasman.2013.09.005. Cysneiros, D., Banks, C.J., Heaven, S., Karatzas, K.A.G., 2012. The role of phase separation and feed cycle length in leach beds coupled to methanogenic reactors for digestion of a solid substrate (Part 1): optimisation of reactors’ performance. Bioresour. Technol. 103, 56–63. http://dx.doi.org/10.1016/j. biortech.2011.09.094. De Oliveira, B.G., Carvalho, J.L.N., Cerri, C.E.P., Cerri, C.C., Feigl, B.J., 2013. Soil greenhouse gas fluxes from vinasse application in Brazilian sugarcane areas. Geoderma 200–201, 77–84. http://dx.doi.org/10.1016/j.geoderma.2013.02.005.

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Please cite this article in press as: Janke, L., et al. Enhancing biogas production from vinasse in sugarcane biorefineries: Effects of urea and trace elements supplementation on process performance and stability. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.01.110