Transition of municipal sludge anaerobic digestion from mesophilic to thermophilic and long-term performance evaluation

Bioresource Technology 170 (2014) 385–394

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Transition of municipal sludge anaerobic digestion from mesophilic to thermophilic and long-term performance evaluation Ulas Tezel 1, Madan Tandukar 2, Malek G. Hajaya 3, Spyros G. Pavlostathis ⇑ School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0512, USA

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Transition from 36 to 53.3 °C

digestion at a rate of 3 °C/day was successful, stable.  Operation at 60 °C led to relatively stable gas production but high levels of VFAs.  Methane production at 60 °C was lower than at mesophilic conditions (36 °C).  For high performance of municipal CSTR digesters, temperature should be below 60 °C.

a r t i c l e

i n f o

Article history: Received 3 June 2014 Received in revised form 30 July 2014 Accepted 2 August 2014 Available online 8 August 2014 Keywords: Acidogenesis Anaerobic digestion Methanogenesis Municipal sludge Temperature transition

a b s t r a c t Strategies for the transition of municipal sludge anaerobic digestion from mesophilic to thermophilic were assessed and the long-term stability and performance of thermophilic digesters operated at a solids retention time of 30 days were evaluated. Transition from 36 °C to 53.3 °C at a rate of 3 °C/day resulted in fluctuation of the daily gas and volatile fatty acids (VFAs) production. Steady-state was reached within 35 days from the onset of temperature increase. Transitions from either 36 or 53.3 °C to 60 °C resulted in relatively stable daily gas production, but VFAs remained at very high levels (in excess of 5000 mg COD/L) and methane production was lower than that of the mesophilic reactor. It was concluded that in order to achieve high VS and COD destruction and methane production, the temperature of continuous-flow, suspended growth digesters fed with mixed municipal sludge should be kept below 60 °C. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Anaerobic digestion has long been used as the main sludge stabilization process in municipal treatment plants (Tezel et al., 2011; ⇑ Corresponding author. Address: School of Civil and Environmental Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0512, USA. Tel.: +1 404 894 9367; fax: +1 404 894 8266. E-mail address: [email protected] (S.G. Pavlostathis). 1 Present address: The Institute of Environmental Sciences, Bogazici University, Istanbul 34342, Turkey. 2 Present address: North American Höganäs, Johnstown, PA 15902-2904, USA. 3 Present address: Civil Engineering Department, Tafila Technical University, Tafila 66110, Jordan. http://dx.doi.org/10.1016/j.biortech.2014.08.007 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Tchobanoglous et al., 2014). Most municipal digesters operate at mesophilic temperatures (35 to 37 °C). However, thermophilic digestion above 50 °C has been gaining popularity, primarily because it achieves a higher extent of pathogen reduction, resulting in Class A biosolids, and secondarily because it exhibits faster kinetics. The latter results in higher solids destruction and biogas production compared to mesophilic digestion for the same solids retention time (SRT), or the flexibility to achieve a desired extent of solids destruction while operating at a lower SRT value. Possible disadvantages of thermophilic digestion may include odor, reduced dewaterability of the digested sludge, and higher heating requirement, which may not be compensated for by higher biogas production as compared to mesophilic digestion.

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Conversion from mesophilic to thermophilic digestion has been practiced at both laboratory- and full-scale levels. Two strategies for the transition from mesophilic to thermophilic digestion have been tested: (a) one-step temperature increase in laboratory scale upflow anaerobic sludge blanket (UASB) reactors (Fang and Lau, 1996; Syutsubo et al., 1997; van Lier et al., 1992) and in continuous-flow stirred tank reactors (CSTR) (Bouskova et al., 2005); (b) step-wise temperature increase in CSTR (Zabranska et al., 2002; Bouskova et al., 2005; Palatsi et al., 2009). The results of the Bouskova et al. (2005) study, which used a SRT of 20 days and an organic loading rate of 1.38 g VS/L-day while the reactors were fed during the temperature transition period, showed that the one-step temperature increase from 37 to 55 °C resulted in stable operation in 30 days, as opposed to 70 days for the step-wise temperature increase (37, 42, 47, 51, and 55 °C). Palatsi et al. (2009) evaluated two strategies for the transition from mesophilic to thermophilic of a mixture of primary and secondary municipal sludge in CSTR laboratory reactors with a SRT and organic loading rate ranging from 19.6 to 23.4 days and from 1.29 to 1.73 g VS/Lday, respectively. A single temperature change from 35 to 55 °C required about 20 days for the reactor efficiency to fully recover, but resulted in higher transient VFAs production, especially propionic acid, compared to a step-wise temperature increase (35, 43, 50, and 55 °C), which required a longer time to complete the temperature transition. These researchers pointed out that the temperature range between 43 and 50 °C was critical in switching methanogenic activity from mesophilic to thermophilic. Peces et al. (2013) evaluated the response of a mesophilic anaerobic digester fed with municipal sludge to short- and long-term temperature fluctuations. Transition from mesophilic to thermophilic conditions of a laboratory CSTR and exposure at 55 °C for 24 h resulted in an increase in VFAs and a decrease in gas production; reactor recovery was achieved in 22 days and required a non-feeding period. A one-step temperature increase is not feasible at full-scale digesters because of heating capacity limits. Full-scale testing for the conversion of a mesophilic to thermophilic municipal sludge digester was assessed by Iranpour et al. (2002) using continuous heating at an average temperature rise of 3 °C/day, increasing the digester temperature from approximately 33 to 55 °C, while using a variable sludge feeding rate. This study achieved stable digester operation in less than 30 days. Further operation of the thermophilic digester achieved Class A biosolids and increased VS destruction and gas production. Information relative to the transition from mesophilic to thermophilic digestion for municipal sludge stabilization is very limited, especially for CSTR digesters, under real, full-scale conditions and constraints. In addition, the potential impact of thermophilic digestion on digestate quality has not been sufficiently assessed. The objective of the work presented here was to assess different conversion strategies for the transition of municipal sludge anaerobic digestion from mesophilic to thermophilic operation, and evaluate long-term stability and performance of thermophilic digesters in terms of solids destruction, gas production/composition, and digestate quality.

anaerobic digestion for sludge stabilization and biogas production, which is converted to electricity through combined heat and power technology. Primary and thickened waste activated sludge (TWAS) samples were collected at the F. Wayne Hill WRC. The primary sludge was sequentially passed through a 5  5 mm square mesh screen, a 2-mm sieve (US Standard No. 10), and finally a 1.4-mm sieve (US Standard No. 14). The TWAS was not further processed. Both sludge samples were stored under refrigeration (4 °C). The following analyses were performed for both sludge samples: pH, total and volatile solids (TS, VS), total and soluble chemical oxygen demand (tCOD and sCOD), VFAs, and ammonia. The water content of the two sludge samples was not affected by screening. 2.2. Ultimate sludge biodegradability The test was performed using 160-mL glass serum bottles (120 mL liquid volume), sealed with rubber stoppers and flushed with helium gas following previously described procedures (Tezel et al., 2006). Sludge obtained from a F. Wayne Hill WRC mesophilic anaerobic digester was anaerobically incubated in the laboratory and severed as inoculum (seed). Then, an aliquot of 75 mL of pre-digested sludge was anaerobically transferred to each bottle and 18 mL of media were then added. The media contained (in g/L): K2HPO4, 0.9; KH2PO4, 0.5; NH4Cl, 0.5; CaCl2
2H2O, 0.10; MgCl2
6H2O, 0.20; FeCl2
4H2O, 0.10; NaHCO3, 6.7. Also, 10 ml/L each of vitamin and trace metal stock solutions were added to the media (Beydilli and Pavlostathis, 2005). A total of five series in triplicate (5  3 = 15 bottles in total) were set up as follows. One series did not receive any sludge and served as the seed blank. Another series was amended with a mix of dextrin/peptone (800/ 400 mg/L) and served as a check of seed activity (reference series). Three more series were prepared with primary sludge, TWAS, and a mixture of primary sludge/TWAS, respectively. Primary sludge and TWAS were tested at a sample VS loading equal to 3 g/L. Combined primary and TWAS were tested at a total VS loading of 3 g/L and a primary/TWAS TS ratio of 20/80% as practiced at the F. Wayne Hill WRC. Incubation was carried out in the dark at 35 °C and the bottles were shaken manually once a day. Throughout the incubation period, total gas volume and composition (CH4 and CO2) were measured frequently. At the end of the incubation, pH, TS, VS, tCOD, sCOD, VFAs and ammonia were measured. The biodegradability test was carried out for 121 days, at which time all gas production had leveled off. 2.3. Digesters set up and operation All digesters used in this study were made of wide-mouth Pyrex reactors with a water jacket, and their temperature was controlled with water recirculation using heated water-circulating baths. The digesters were housed in a 22–24 °C room and their contents were mechanically mixed at 90 rpm using a shaft magnetically coupled to an external, variable-speed electric drive. Gas produced was collected in graduated burettes by displacement of an acidified brine solution (10% NaCl w/v and 2% H2SO4 v/v) and measured after equilibration to atmospheric pressure. Gas data reported here are either at 22 °C and 1 atm or at standard temperature and pressure (STP; 0 °C and 1 atm).

2. Methods 2.1. Sludge samples The study was designed and conducted by taking into account conditions and constraints at the F. Wayne Hill Water Resources Center (WRC), Gwinnett County, GA, USA. This municipal wastewater treatment plant uses activated sludge technology, achieving both N and P removal. It uses mesophilic (98 °F or 36.7 °C)

2.3.1. Mesophilic operation (36 °C) Two digesters (R1 and R2) were set up and operated at 36 °C and a SRT of 30 days (close to the SRT at the plant). The total digester volume was equal to 4 L with a liquid working volume equal to 3 L. Both digesters were started with 3 L mixed liquor obtained from a F. Wayne Hill WRC mesophilic anaerobic digester. The feed for both digesters was primary/TWAS sludge mixture (20/80% on TS basis), which was kept under refrigeration (4 °C). The combined

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sludge (30 mL) was fed to each digester once a day manually using a plastic syringe. Throughout this phase (98 days), pH, total gas volume and composition (CH4, CO2), and VFAs were measured frequently. At the end of this phase, the digesters mixed liquor was analyzed for pH, TS, VS, tCOD, sCOD, VFAs, ammonia, and phosphate. 2.3.2. Transition to moderate thermophilic operation (53 °C) One digester (R1) was maintained at 36 °C and operated as described above. The temperature of digester R2 was gradually increased at a rate of 3 °C/day and then maintained at 53.3 °C (128 °F) (transition 1). For this temperature transition, the following considerations were taken into account: a) the heating capacity at the F. Wayne Hill WRC is such that the digesters can be heated from 36.7 °C (98 °F) to 53.3 °C (128 °F) in about 5 days (i.e., at a temperature increase of about 3.3 °C/day); b) previous assessment of possible strategies for the transition from mesophilic to thermophilic digestion favored fast temperature increases while the digesters were fed. Based on the above, and to be conservative, for digester R2 the temperature increase from 36 °C to 53.3 °C occurred in 6 days (i.e., a temperature increase of about 3 °C/day). During the period of temperature increase of digester R2, sludge wasting and feeding was the same in both digesters (i.e., both R1 and R2 digesters received the same sludge loading as described above and were maintained at a SRT of 30 days). Throughout the temperature transition of digester R2 and until its performance was stable, pH, total gas volume and composition (CH4, CO2), and VFAs were measured daily. At the end of this phase, the digesters mixed liquor was analyzed for pH, TS, VS, tCOD, sCOD, VFAs, ammonia, and phosphate. 2.3.3. Transition to high thermophilic operation (60 °C) In order to evaluate the possibility of operating the thermophilic digester at 60 °C, three strategies were applied while the SRT was kept at 30 days: (a) transition from 36 to 60 °C at 3 °C/day (transition 2); (b) transition from 53.3 to 60 °C at 3 °C/day (transition 3); and (c) transition from 53.3 to 60 °C in four steps (55, 57, 58.5, and 60 °C) (transition 4). Corresponding reactors used for the three temperature transitions to 60 °C are referred to as R3, R4, and R5, respectively. Throughout this phase of the study, pH, total gas volume and composition (CH4, CO2), and VFAs were measured frequently. The mesophilic (36 °C) reactor was operated at 30 days and was used as control. At the end of this phase, the digesters mixed liquor was analyzed for pH, TS, VS, tCOD, sCOD, VFAs, ammonia, and phosphate. 2.4. Microbial community analysis Microbial community analysis related to temperature transition 2, digester R3, was conducted to quantitatively assess the change in bacterial and archeal communities associated with the change in the reactor’s temperature from 36 °C to 60 °C and its performance after was maintained at 60 °C for a relatively long period (about 10 SRTs). Coprothermobacter spp., Archaea, and the aceticlastic methanogenic families Methanosarcinaceae and Methanosaetaceae were selected as targets. Both Methanosarcinaceae and Methanosaetaceae belong to the order Methanosarcinales. Methanosaetaceae are strict aceticlastic methanogens, whereas Methanosarcinaceae utilize both acetate and H2/CO2 for methanogenesis. Coprothermobacter spp. is an important bacterial group in the anaerobic digestion of municipal sludge, as is proteolytic and regulates the release of amino acids and ammonium. Quantitative PCR analysis was performed to detect and quantify the 16S rRNA genes of the above mentioned target microorganisms. Three duplicate biomass samples were collected from R3 at the following day of operation and temperature: sample A at day 0, 36 °C; sample B

at day 8 when the temperature had just reached 60 °C during the temperature transition; and sample C at day 286 after the reactor had been maintained at 60 °C for 10 months and its performance was relatively stable in terms of biogas production and residual VFAs (see Section 3.3.1, below). Details on the procedures followed for DNA extraction, preparation of qPCR standards, and qPCR assays are included in Supplementary data (Text S1). 2.5. Analytical methods TS, VS, pH, COD, and ammonia measurements were conducted according to procedures described in Standard Methods (APHA, 2012). Total phosphorus in the sludge feed and digester effluent was measured following the molybdovanadate/acid persulfate digestion method (HACH procedure 10127; HACH, Loveland, CO, USA). Phosphate measurements were conducted using ion chromatography/conductivity detection with samples filtered through a 0.2 lm syringe filters (Tugtas and Pavlostathis, 2007). Total gas production was measured by displacement of an acidified brine solution (10% NaCl w/v and 2% H2SO4 v/v) in graduated burettes. The gas composition and VFAs were determined by gas chromatography with thermal conductivity and flame ionization detection, respectively, as previously reported (Okutman Tas and Pavlostathis, 2005; Misiti et al., 2013). 3. Results and discussion 3.1. Sludge characteristics and ultimate anaerobic biodegradability The results of the analysis of the primary sludge and TWAS samples are shown in Table 1. Both samples were acidic and the VS/TS ratio was higher for the primary sludge. Significant levels of soluble COD and VFAs were found in both samples, especially in the primary sludge, indicating that a degree of sludge solubilization and preacidification had taken place. Acetate and propionate were the major VFAs. The anaerobic biodegradability test was carried out for 121 days. At the end of the incubation, all samples tested had pH values between 7.04 and 7.15 and VFAs were not detected. Gas production in all five series started without any lag and the primary sludge sample had the highest gas production rate (Fig. S1; Supplementary data). After about 30 days of incubation, the gas production rate was relatively similar in all five series indicating that the gas production from this point forward was the result of

Table 1 Results of primary sludge and TWAS sample analysis.

a b

Parameter

Primary sludge

TWAS

pH TS, g/kg wet sample VS, g/kg wet sample VS/TS,% Total COD, mg/L Soluble COD, mg/L Ammonia, mg N/L Total VFAs, mg COD/L Acetic Propionic iso-Butyric n-Butyric iso-Valeric n-Valeric iso-Caproic n-Caproic Heptanoic

4.70 30.3 ± 0.1a 23.5 ± 0.1 77.6 41970 ± 1370 3450 ± 170 84 ± 5 2300 ± 43 1400 ± 17 622 ± 7 18 ± 10 203 ± 1 NDb 62 ± 1 ND 7±5 ND

6.13 58.8 ± 0.2 41.2 ± 0.2 70.1 59750 ± 1260 2350 ± 15 224 ± 5 1080 ± 24 567 ± 7 297 ± 5 34 ± 1 81 ± 2 75 ± 4 28 ± 7 ND ND ND

Mean ± standard deviation (n P 3). ND, not detected.

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slow seed and/or sludge destruction. Table 2 summarizes the results of the batch test for the two individual samples, as well as the combined primary/TWAS sample. A good COD balance was achieved indicating that methanogenesis was the main terminal electron transfer process. The anaerobic biodegradability of TWAS was significantly lower than the primary sludge. Based on the ultimate biodegradability (i.e., VS destruction) of each component (i.e., primary sludge and TWAS), and taking into account the VS concentration of each component in the combined primary/TWAS sample tested, the calculated VS destruction in the combined sample was equal to 43.6%, which is comparable to the measured VS destruction of 40.0% (Table 2). 3.2. Mesophilic operation and transition to moderate thermophilic operation Both digesters R1 and R2 were operated identically for 25 days at a SRT of 30 days and an organic loading rate of 1.1 g VS/L-day. The performance of both R1 and R2 based on gas production, pH and VFAs concentration was identical (Fig. 1). On day 25, the temperature of R2 was increased at a rate of 3 °C/day and reached 53.3 °C on day 31 (transition 1), whereas that of R1 remained at 36 °C. The daily gas production of R2 increased almost linearly with the increase in temperature, reached a high of 2100 mL/day on day 29 and then decreased precipitously and remained between 340 and 450 mL/day for about 8 days (Fig. 1). It is noteworthy that, while the reactor temperature was increased, a precipitous decrease in gas production occurred when the R2 temperature was 48 °C. Similar to our results, Palatsi et al. (2009) observed a drastic decrease in methane production when the reactor temperature was between 43 and 50 °C and considered this temperature range as the most critical for the transition from mesophilic to thermophilic digestion of municipal sludge. The decrease in gas production was accompanied by a sharp increase in VFAs concentration (Fig. 1). The performance of R2 was stabilized at day 60, i.e., after 35 days of operation past the initiation of temperature increase. The digesters characteristics and steady-state data are shown in Table 3. Although the mean feed pH was 5.8, the R1 and R2 mean pH values were 7.1 and 7.4, respectively, and the reactors were operated without any alkalinity addition even during the temperature transition of R2 when the VFAs concentration increased over 3 g COD/L. The soluble COD, VFAs, ammonia, and phosphate in R2 were 5.1-, 5.4-, 1.3-, and 1.2-fold higher compared to R1. The gas production was 335 and 344 mL at STP/g VS added for R1 and R2, respectively. The COD destruction in both digesters was also similar. In contrast to comparable total gas production and COD destruction, VS destruction and soluble COD in R2 were higher than in R1. Thermophilic conditions in R2 enhanced sludge

disintegration and hydrolysis but products were partially recalcitrant and thus not fully processed to methane. The thermophilic R2 reactor achieved only 5% higher methane production compared to R1. The specific methane production was 305 and 313 mL at STP/g COD destroyed in R1 and R2, respectively, which are below the theoretical value of 350 mL methane at STP/g COD destroyed. Reduction of alternative electron acceptors such as nitrate and sulfate results in a lower specific methane production due to electron channeling away from methane formation. However, neither nitrate nor sulfate was detected in the feed used in this study. The overall COD balance for R1 and R2 was 4.9 and 4.1, respectively (Table 3). The major VFAs in the feed, expressed as COD, were acetic (52%), propionic (23%), and n-butyric (12%) acids. Acetic acid was the major VFA in both the R1 and R2 reactors (54 and 79%, respectively) just before the temperature transition in R2. When the VFAs concentration was the highest in R2 after its temperature had reached 53.3 °C (day 36), the major VFAs component was acetic acid (72%), followed by propionic (13%). When the performance of R2 stabilized (day 60), acetic acid was the major VFAs component (82%), followed by propionic (9%) and heptanoic acid (9%). Based on these results, although the total VFAs concentration was higher in R2 compared to R1, acetic acid was the predominant VFA in both reactors. As mentioned above, although the soluble COD concentration in R2 was more than 5-fold higher compared to R1, the VFAs in both reactors accounted for about 7% of the soluble COD. 3.3. Transition and operation at 60 °C 3.3.1. Transition 2 The second temperature transition was from 36 to 60 °C at a rate of 3 °C/day while the digester, designated as R3, was operated with a SRT of 30 days. R3 was started with 1.5 L of mixed liquor from the mesophilic reactor R1 (36 °C, 30 days SRT) gradually collected from the daily waste over 15 days, kept unfed. Then, R3 was wasted and fed while maintained at 36 °C for 14 days before the temperature transition to 60 °C. Fig. 2 shows the performance of R3 before and during the temperature increase, as well as while it was maintained at 60 °C. As soon as the reactor temperature increased, the gas production increased from a mean value of 690 mL to a maximum of 1085 mL on day 17 when the reactor temperature was at 45 °C and then precipitously decreased to 370 mL by day 22 when the reactor temperature had reached 60 °C. From day 35 to 62, the gas production stabilized to about 200 mL, which is about one third of the gas produced by the mesophilic digester (R1, 36 °C) for the same reactor volume (1.5 L). The VFAs concentration in R3 increased linearly from 77 to 3700 mg COD/L on day 30 when the reactor temperature had been at

Table 2 Results of the batch anaerobic ultimate biodegradability test.

a b c

Parameter

Primary sludge (PS)

TWAS

Primary sludge + TWASa

Initial primary sludge, g VS/L Initial TWAS, g VS/L Initial total VS, g/L VS destruction,b % COD destruction,b % Methane,% of total gas Total gas produced,b mL @ STP/g VS added Methane, mL @ STP/g VS added Methane, mL @ STP/g VS destroyed COD balancec

3 – 3 56.7 68.2 67.3 598.3 399.8 705.6 5.4

– 3 3 40.0 46.4 67.7 363.7 244.7 611.8 0.5

0.65 2.35 3 40.0 40.7 68.6 404.8 278.5 696.2 3.8

PS/TWAS mix, 20/80% TS basis. Seed-corrected, corresponding to the individual component or mix of components. (CODinitial CODfinal CODmethane)  100/CODinitial.

389

pH

VFAs (mg COD/L)

GAS (mL/d)

o

TEMP. ( C)

U. Tezel et al. / Bioresource Technology 170 (2014) 385–394

60

A

50

Mesophilic (R1) Meso/Thermophilic (R2)

40

30 3000 2500 2000 1500 1000 500 0 4000

B Mesophilic (R1) Meso/Thermophilic (R2)

C

3000 2000

Meso/Thermophilic (R2)

1000 0 8.0 7.5 7.0 6.5 6.0 5.5

D Mesophilic (R1) Meso/Thermophilic (R2)

Feed

0

10

20

30

40

50

60

70

80

90

100

TIME (Days) Fig. 1. Temperature (A), daily gas production at 22 °C and 1 atm (B), VFAs (C) and pH (D) in the two digesters operated at 30 days SRT. On day 25, the R2 digester temperature was gradually increased to 53.3 °C at 3 °C/day (transition 1), whereas that of R1 remained at 36 °C. VFAs in the mesophilic digester R1 were below 25 mg COD/L at all times (error bars represent mean values ± one standard deviation; n = 3).

Table 3 Feed, effluent characteristics, and performance of digesters R1 and R2 (SRT 30 d; reactor volume 3 L)a.

a b c

Parameter

Feed

Mesophilic (R1)

Meso/thermophilic (R2)

Temperature pH TS, g/L VS, g/L Total COD, g/L Soluble COD, g/L VFAs, mg COD/L Ammonia, mg N/L Phosphate, mg P/L Total gas, mL at 22 °C/day Methane,% Methane, mL at 22 °C/day VS destruction,% COD destruction,% Total gas, mL at STP/g VS added Methane, mL at STP/g VS destroyed Methane, mL at STP/g COD destroyed COD balance, %c

– 5.75 ± 0.08b 46.9 ± 0.1 33.0 ± 0.1 59.7 ± 0.7 4.6 ± 0.2 2466 ± 49 336 ± 5 438 ± 5 – – – – – – – – –

36 °C 7.09 ± 0.01 36.4 ± 0.1 22.3 ± 0.1 37.0 ± 1.5 0.62 ± 0.03 43 ± 18 980 ± 5 413 ± 5 1196 ± 41 62.5 ± 0.7 748 ± 29 32.4 38.0 335 647 305 4.9

36–53.3 °C 7.38 ± 0.02 35.4 ± 0.1 21.3 ± 01 36.5 ± 1.9 3.19 ± 0.10 231 ± 40 1232 ± 5 505 ± 5 1226 ± 40 64.1 ± 1.3 786 ± 52 35.5 38.9 344 622 313 4.1

Steady-state data (day 70 to 91). Mean ± standard deviation (n P 5). COD balance = (CODin CODout CODCH4)  100/CODin.

60 °C for 8 days, and remained at this level for another 6 days before it increased again and reached 6610 mg COD/L on day 62 (Fig. 2). Due to low gas production and VFAs accumulation at a high concentration, R3 was kept at 60 °C unfed for a period of 44 days and then was intermittently fed while its volume was also increased first to 2 and then to 3 L. Frequent, daily feeding resumed on day 146 and continued till the end of this phase of the study (309 days of operation). During the latter period of operation when the reactor volume was 3 L, the mean gas production was 1125 mL/ day, but varied from a low 780 to a high 1450 mL/day. Although the mean gas production was close to that of the mesophilic reactor (R1, 36 °C, 30 days SRT) for the same reactor volume (3 L), the

VFAs in R3 remained at relatively high levels. As shown in Fig. 2, the VFAs decreased to a low value of 3900 mg COD/L during the period that the reactor was kept unfed, and then during the latter phase when feeding was resumed, the VFAs concentration varied between 3000 and 5500 mg COD/L. During the entire period of operation of R3, its pH remained above 7 without any alkalinity addition with the exception of a single addition of 2 g NaHCO3 on day 86 when the pH was 6.7. Just before the reactor temperature was increased on day 14, acetic acid was the major VFA component (62% of total VFAs COD), followed by propionic (11%), and heptanoic acid (9%), and the remaining VFAs were below 6.6%. On day 62, when the daily wasting and feeding was stopped converting R3 to a batch reactor,

U. Tezel et al. / Bioresource Technology 170 (2014) 385–394

VOLUME (L)

390

3

A

2 1

pH

VFAs (mg COD/L)

GAS (mL)

o

TEMP. ( C)

0 75 60 45 30 15 0 1600

B

C

Daily feeding Batch

1200 800 400 0 8000

D

6000 4000 2000 0 8.5 8.0 7.5 7.0 6.5 6.0 5.5

E

0

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320

TIME (Days) Fig. 2. Reactor volume (A), temperature profile (B), gas production at 22 °C and 1 atm (C), VFAs (D), and pH (E) in digester R3 operated at 30 days SRT and transitioned from 36 to 60 °C at 3 °C/day (transition 2) (error bars represent mean values ± one standard deviation; n = 3).

the major VFAs components were acetic (45%), propionic (22%), and iso-valeric acid (13%). On day 124, when intermittent wasting and feeding resumed, the total VFAs concentration decreased from 6610 to about 3900 mg COD/L. The major VFAs components were acetic (29%), propionic (44%), followed by iso-valeric acid (17%). Comparing the distribution of VFAs between day 62 and 124 shows that during the batch operation of the R3 reactor, the acetic and iso-valeric acid concentration decreased, whereas the concentration of propionic acid increased. Throughout this study, this was the first time that the propionic acid concentration exceeded that of the acetic acid. By the end of this phase of the study, the acetic acid concentration had decreased while that of the propionic and iso-valeric acid had increased, each representing 12%, 46%, and 21% of total VFAs COD, respectively. At the end of this phase of the study, the R3 effluent had the following characteristics: VS 23.2 ± 1.7 g/L, soluble COD 12330 ± 180 mg/L, total VFAs 4903 ± 472 mg COD/L, ammonia 1650 ± 7 mg N/L, and phosphate 325 ± 10 mg P/L. The soluble COD was about 26% of the total COD. The VS destruction was 38.7%, which is higher than that achieved by the mesophilic (36 °C; 32.4%) and thermophilic (53.3 °C; 35.5%) digesters, all operated at a SRT of 30 days (Table 3). It is noteworthy that Gray (Gabb) et al. (2006) reported a significant decrease in VS destruction with an increase in digestion temperature from 49 to 52 and 62 °C, a finding that has been observed in other previous studies according to these authors. As a result of high VS destruction and taking into account that the digester sludge feed was high in TWAS, a total ammonia concentration of 2184 mg N/L was recorded at 220 d of operation. Based on the reactor conditions (60 °C and pH 7.8), the un-ionized ammonia concentration was calculated as 540 mg N/L, which is well above 100 mg ammonia-N/L, a concentration that may be inhibitory to methanogens (Rittmann and McCarty, 2001), though ammonia

inhibition is a complex process affected by the total ammonia concentration, pH, temperature, and C/N ratio (Angelidaki and Ahring, 1994; Rajagopal et al., 2013; Borowski and Weatherley, 2013). The high un-ionized ammonia concentration may be responsible for the observed periodic increase and decrease of gas production towards the latter part of this phase of the study (Fig. 2). If the reactor pH was lowered to 7, for the same total ammonia concentration the un-ionized ammonia concentration would have decreased to 108 mg N/L and could have resulted in a more stable reactor operation. Given the unstable gas production and accumulation of VFAs at a high concentration, this reactor was abandoned. The inability to establish reactor operation at 60 °C with a relatively low VFAs level was attributed to lack of microbial acclimation to this temperature in spite the relatively long SRT value used and a relatively long period of batch operation (Fig. 2), as well as to possible ammonia inhibition. 3.3.2. Transition 3 The third temperature transition was from 53.3 to 60 °C at a rate of 3 °C/day while the digester, designated as R4, was operated with a SRT of 30 days. R4 was started with 2 L of mixed liquor collected from the thermophilic digester R2 (53.3 °C, 30 days SRT). Fig. 3 shows the performance of R4 before and during the temperature increase, as well as at 60 °C over a relatively long time. The gas production, after an initial small increase during the temperature increase, precipitously decreased to 270 mL by day 8 when the reactor temperature had reached 60 °C, increased again reaching the highest value of 720 mL by day 21, and then decreased and fluctuated between 230 and 440 mL/day (mean gas production, 325 mL/day) (Fig. 3C). The R4 mean gas production was about 40% of that produced by the mesophilic digester (R1, 36 °C) for the same reactor volume (2 L). The VFAs in R4 increased

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linearly from about 340 to 3800 mg COD/L on day 18 when the reactor temperature had been at 60 °C for 14 days. Then, the VFAs decreased, coinciding with the observed increase in gas production on day 21. Past day 24, the VFAs increased gradually and then fluctuated between 5500 and 7900 mg COD/L till the end of this phase of the study (Fig. 3B). During the entire period of operation of R4, its pH remained above 7 without any alkalinity addition with the exception of a single addition of 2 g NaHCO3 on day 140 when the pH had dropped to 6.9. Before the reactor temperature was increased on day 4, both acetic and propionic acid were the major VFA components (43 and 46% of total VFAs COD, respectively), followed by heptanoic acid (8%), and the remaining VFAs were all very low. However, on day 8, which marked the lowest gas production after the reactor temperature had reached 60 °C, the acetic and propionic acid represented 40% and 35% of the total VFAs COD, respectively. For the remaining operation period of R4, acetic acid increased at a higher rate and was always higher than propionic acid. On day 62, when the highest VFAs concentration was observed (7910 mg COD/L), the acetic and propionic acid represented 53% and 26% of the total VFAs COD, showing a disproportional increase of acetic acid concentration over that of propionic acid. By the end of this phase of the study, both the acetic and propionic acid concentration had decreased, while that of the iso-valeric acid had increased, each representing 38%, 27%, and 15% of total VFAs COD, respectively. Compared to the performance of reactor R3, which was developed at the same SRT of 30 days, but starting at 36 °C, the performance of R4 was more stable, i.e., its gas production and VFAs levels fluctuated less, but its COD destruction and gas production was much lower as discussed above. 3.3.3. Transition 4 In view of the fact that none of the previous temperature transitions was successful in establishing reactor operation at 60 °C with a relatively low VFAs level, a fourth temperature transition from 53.3 to 60 °C was tested, but instead of a continuous temperature rise at a rate of 3 °C/day used in all previous temperature transitions, the temperature rise in this case was done in four steps (55,

57, 58.5, and 60 °C), while the reactor was operated with a SRT of 30 days. The digester, designated as R5, was started with 2 L of mixed liquor collected from the thermophilic reactor R2 (53.3 °C, 30 days SRT). Fig. 4 shows the performance of R5 throughout its operation. R5 was operated at 53.3 °C for 7 days before the first temperature transition to 55 °C and achieved a mean gas production of 1040 mL/day during this period (Fig. 4D). With the increase of temperature to 55 and then to 57 °C, the gas production increased initially and then decreased and for 50 days (day 7 to day 57) fluctuated between 670 and 1060 mL/day. With further increase of the temperature to 58.5 °C, a gradual decrease of gas production was observed which continued after the reactor temperature was increased to 60 °C. After the reactor liquid volume had reached 3 L, the gas production stabilized first at about 285 mL/day and towards the latter part of operation at 415 mL/ day (Fig. 4C). The R5 mean gas production towards the end of its operation was about 35% of that produced by the mesophilic digester (R1, 36 °C) for the same reactor volume (3 L). With the exception of a few sudden increases and decreases, the VFAs concentration in R5 increased linearly from 415 to 3900 mg COD/L corresponding to reactor temperature 53.3 and 58.5 °C, respectively. Further increase of the reactor temperature to 60 °C resulted in an initial small decrease followed by an increase of the VFAs concentration which fluctuated between 3900 and 8150 mg COD/L, stabilizing to about 7000 mg COD/L towards the latter part of the R5 operation (Fig. 4D). During the entire period of operation of R5, its pH remained above 7 without any alkalinity addition with the exception of two additions of 3 g NaHCO3 on day 140 and 145 when the pH had dropped to 6.7. Fig. S2 shows the trend in total VFAs as well as that of the predominant VFAs (i.e., acetic, propionic, and iso-valeric) as a function of reactor R5 temperature. On day 21 when the reactor temperature had been at 55 °C for 14 days, the observed increase in the total VFAs was predominantly due to an increase in propionic acid, which accounted for 72% of the total VFAs COD. As the reactor temperature increased to 57 and then to 58.5 °C, the total VFAs increased by 6.3- and 9.4-fold, respectively, compared to the VFAs concentration at 53.3 °C. The acetic acid concentration at 57 and

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58.5 °C remained practically unchanged, but a significant increase of the propionic acid concentration was observed, which accounted for between 60% and 64% of the total VFAs COD (Fig. S2). Similar to our results, Wilson et al. (2008) and Palatsi et al. (2009) observed a higher propionic acid concentration compared to acetic acid as the digestion temperature increased to and above 50 °C. In the present study, transition and operation at 60 °C resulted in 16.5-fold increase in the total VFAs concentration compared to that at 53.3 °C. At 60 °C, a dramatic change in the VFAs distribution was observed, marked by a very high increase in the concentration of all acids, except iso-caproic and n-caproic, which remained practically at the levels observed at 58.5 °C, and propionic acid, which decreased by 36% compared to 58.5 °C. The predominant VFAs at 60 °C were acetic (40%), propionic (25%), and iso-valeric (14%) acid. The performance of R5 towards the end of this phase of the study was relatively stable, i.e., its gas production and VFAs levels had a lower fluctuation, but its COD destruction and gas production were low. 3.4. Comparison of performance at 60 °C As mentioned above, three reactors were transitioned to and operated at 60 °C using three different strategies, while the reactors were operated with a SRT of 30 days. In terms of specific gas production rate, the performance of the three reactors operated at 60 °C is compared to that of the mesophilic (36 °C) reactor operated with the same SRT and identical feed sludge and the results are shown in Fig. S3A. The thermophilic reactor R3 (transition 2) had about the same specific gas production rate as the mesophilic reactor (Fig. S3A), but as discussed above, its gas production fluctuated

over time. The specific gas production rate of reactors R4 and R5 (transition 3 and 4, respectively) was significantly lower than that of the mesophilic (36 °C) by 39% and 35%, respectively, but their gas production fluctuated less than that of reactor R3. Similar to our results, Wilson et al. (2008) reported 31–54% lower methane production in anaerobic digesters operated at 57.5 °C compared to a mesophilic (37 °C) digester and the methane production at 57.5 °C was about 23% of that achieved at 53 °C. Ahring et al. (2001) reported 18% decrease in the specific methane production from the anaerobic digestion of cattle manure at SRT of 15 days at 65 °C as compared to digestion at 55 °C. Digestion at 65 °C also resulted in a decrease of VS destruction to 22% from 28% observed at 55 °C and an increase of VFAs from below 0.3 g/L to as high as 2.6 g COD/L, mostly as acetate and propionate. Degradation of propionate was completely inhibited at 65 °C (Ahring et al., 2001). All three thermophilic digesters maintained at 60 °C had high levels of VFAs (between 4900 and 7600 mg COD/L) (Fig. S1B). It is noteworthy that the steady-state total VFAs concentration in the mesophilic (36 °C) and moderate thermophilic (53.3 °C) reactors, both operated at a SRT value of 30 days, was 43 ± 18 and 231 ± 40 mg COD/L, respectively (Table 3). In terms of VFAs distribution, reactors R4 and R5 (transition 3 and 4, respectively) had similar relative VFA components as follows (%): acetic (37.6–41.2), propionic (25.1–27.4), butyric (13.9–15.4), valeric (16.6–18.9), caproic (1.1–1.5), and heptanoic (0–0.8) acid. In contrast, reactor R3 (transition 2) had a relatively lower acetic acid (12.1%) and a higher propionic (46%) and valeric (23.1%) acid fraction (Fig. S3C). Aceticlastic methanogens are more sensitive than hydrogenotrophic methanogens at temperatures encountered in thermophilic

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digestion (Ahring et al., 2001), which may explain the relatively high acetate levels observed in all digesters operated at 60 °C. However, enrichment of syntrophic acetate-oxidizing, non-methanogenic thermophilic bacteria under the dual stress of high acetate and relatively high ammonia levels (Lee and Zinder, 1988; Hao et al., 2011, 2013; Fotidis et al., 2013; Lü et al., 2013; Ho et al., 2014) in R3 over its long-term operation, as well as acclimation and proliferation of aceticlastic methanogens at 60 °C while R3 was maintained for about 70 days in batch mode, may have contributed to the observed lower levels of acetic acid in R3 compared to all other digesters maintained at 60 °C with daily wasting/feeding, operated for relatively shorter times.

3.5. Microbial community response to temperature increase The gene numbers of the microbial community in the three samples collected from R3 operated at temperature values from 36 to 60 °C (transition 2) are presented in Fig. 5. The PCR and gel electrophoresis results revealed that the methanogenic archaea associated with the family Methanosaetaceae were not detected in any of the three samples analyzed, agreeing with the qPCR data which shows that the gene copy numbers of the family Methanosarcinaceae were very close to that of the domain Archaea. These observations suggest that the aceticlastic archeal community was dominated by Methanosarcinaceae. Mladenovska and Ahring (2000) and Demirel and Scherer (2008) have reported the dominance of Methanosarcinaceae in thermophilic digesters and unstable anaerobic digesters with high VFA concentrations, respectively. A previous study reported that Methanosarcinaceae dominate in high-rate anaerobic thermophilic digesters fed with waste activated sludge (Ho et al., 2013). Fig. 5 shows the changes in 16S rRNA gene concentrations of Coprothermobacter, Archaea, and Methanosarcinaceae in the three samples collected at different operational temperatures and duration of digester R3. At 36 °C, when the reactor performance was stable in terms of gas production and had negligible residual VFAs (sample A), the 16S rRNA gene concentrations of Coprothermobacter spp., Archaea, and Methanosarcinaceae, were 1.6 ± 0.46  106,

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SAMPLE Fig. 5. 16S rRNA gene concentrations of Coprothermobacter spp., Archaea, and Methanosarcinaceae in three samples collected from digester R3 transitioned from 36 to 60 °C (sample A and B; transition 2) and maintained at 60 °C for over 10 months (sample C) (Error bars represent mean values ± one standard deviation; n = 3).

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8.4 ± 0.46  105, and 1.3 ± 0.0017  105 copies/mL of extracted DNA, respectively. When the digester R3 temperature was transitioned from 36 °C and had just reached 60 °C (sample B), the 16S rRNA gene concentration of both Coprothermobacter spp. and Methanosarcinaceae decreased significantly. In contrast, the archeal 16S rRNA gene concentrations remained almost unchanged (Fig. 5). This observation indicates that hydrolytic bacteria and aceticlastic Methanosarcinaceae were negatively affected by the temperature increase. Zinder et al. (1984) reported that an anaerobic digester, fed with the organic fraction of municipal refuse at 10 days retention time, maintained at 58 °C, when was dominated by Methanosarcina sp., aceticlastic methanogenesis was maximal at 58 °C and completely inhibited at 65 °C. Ho et al. (2014) reported the dominance of Methanosarcinaceae in anaerobic digesters at 55–60 °C, but increase of temperature to 65 °C resulted in loss of Methanosarcinaceae, accumulation of VFAs, and a decrease in methane production. The change in bacterial and archeal populations observed in the present study, as shown by the 16S rRNA gene copies, could be due to a dynamic population shift to other bacterial species and methanogenic archaea, such as hydrogenotrophic methanogens, as well as an increase in non-methanogenic archaea (e.g., Crenarchaeota). Zinder et al. (1984) reported that methanogenesis from CO2 with a culture from an anaerobic digester maintained at 58 °C was optimal at 65 °C. Ahring et al. (2001) reported that the hydrogenotrophic methanogens were more tolerant and predominated in anaerobic CSTRs treating cattle manure when the operational temperature was changed from 55 to 65 °C. At relatively high pH (i.e., at or above 8.0), free ammonia could become inhibitory, particularly to Methanosaeta (Demirel and Scherer, 2008). In the latter phase of the thermophilic operation when R3 was maintained at 60 °C for 10 months (sample C), even lower numbers of Coprothermobacter spp., Archaea and Methanosarcinaceae were observed (Fig. 5). Although the gas production during this latter phase was stable, the reactor had a relatively high VFAs and ammonia concentration (see Sections 3.3.1 and 3.6, respectively). At this phase, the archeal community was vastly dominated by Methanosarcinaceae. Based on the qPCR data, it appears that the hydrogenotrophic methanogenic population also declined drastically at 60 °C. Thus, the increase in the reactor temperature had a significant effect on the microbial community and population dynamics, which eventually affected performance. 3.6. Digestate quality The phosphorus and ammonia concentrations in sludge filtrates from mesophilic and thermophilic digesters, all operated at SRT of 30 days, are shown in Table S2. The orthophosphate phosphorus (PO34 -P) concentration in filtered digesters effluent samples ranged from 285 to 505 mg P/L, which corresponds to 15.6 and 26.9% of the total sludge P, and increased as the reactor temperature increased from 36 to 53.3 °C. The total ammonia concentration in the digesters filtered effluent ranged from 980 to 2184 mg N/L and the highest concentration was observed at 60 °C, which is consistent with previous observations according to which the ammonia concentration increases with increased SRT and temperature (Bivins and Novak, 2001; Gray (Gabb) et al., 2006). The un-ionized free ammonia concentration calculated by taking into account the digester pH and temperature is also shown in Table S2. With the exception of digester R3, the un-ionized ammonia concentration values are well below 100 mg ammoniaN/L, a concentration that may be inhibitory to methanogens (Rittmann and McCarty, 2001), particularly to Methanosaeta spp. (Demirel and Scherer, 2008). In contrast, R3, operated at 60 °C and pH 7.8, exhibited high VS destruction which resulted in the highest total ammonia level, but also in a high un-ionized

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ammonia concentration (data at 220 days of operation), which may have contributed to the observed periodic increase and decrease of gas production towards the latter part of operation of R3 (Fig. 2). 4. Conclusions Transition of mixed municipal sludge anaerobic digestion from mesophilic to thermophilic conditions is feasible with continuous heating at a constant rate and sludge feeding of suspended growth, well mixed digesters. The strategy by which final temperature was achieved did not make any significant difference. However, in order to achieve stable, high performance (i.e., high VS and COD destruction and methane production), the digester temperature should be kept below 60 °C. Taking into account full-scale municipal plant constraints (e.g., heating capacity), gradual transition from mesophilic to thermophilic conditions can result in the successful conversion to a stable thermophilic sludge digestion process. Acknowledgements This work was supported by a contract from the Gwinnett County, Department of Water Resources, Lawrenceville, GA, USA administered through Hazen and Sawyer, P.C., Atlanta, GA, USA. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.08. 007. References Ahring, B.K., Ibrahim, A.A., Mladenovska, Z., 2001. Effect of temperature increase from 55 to 65 °C on performance and microbial population dynamics of an anaerobic reactor treating cattle manure. Water Res. 35 (10), 2446–2452. American Public Health Association (APHA), 2012. Standard Methods for the Examination of Water and Wastewater, 22nd ed. American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USA. Angelidaki, I., Ahring, B.K., 1994. Anaerobic thermophilic digestion of manure at different ammonia loads: effect of temperature. Water Res. 28 (3), 727–731. Beydilli, M.I., Pavlostathis, S.G., 2005. Decolorization kinetics of the azo dye Reactive Red 2 under methanogenic conditions: effect of long-term culture acclimation. Biodegradation 16 (2), 135–146. Bivins, J.L., Novak, J.T., 2001. Changes in dewatering properties between the thermophilic and mesophilic stages in temperature-phased anaerobic digestion systems. Water Environ. Res. 73 (4), 444–449. Borowski, S., Weatherley, L., 2013. Co-digestion of solid poultry manure with municipal sewage sludge. Biores. Technol. 142, 345–352. Bouskova, A., Dohanyos, M., Schmidt, J.E., Angelidaki, I., 2005. Strategies for changing temperature from mesophilic to thermophilic conditions in anaerobic CSTR reactors treating sewage sludge. Water Res. 39 (8), 1481–1488. Demirel, B., Scherer, P., 2008. The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review. Rev. Environ. Sci. Biotechnol. 7 (2), 173–190. Fang, H.H.P., Lau, I.W.C., 1996. Startup of thermophilic (55 °C) UASB reactors using different mesophilic seed sludges. Water Sci. Technol. 34 (5–6), 445–452. Fotidis, I.A., Karakashev, D., Kotsopoulos, T.A., Martzopoulos, G.G., Angelidaki, I., 2013. Effect of ammonium and acetate on methanogenic pathway and methanogenic community composition. FEMS Microbiol. Ecol. 83 (1), 38–48.

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