Waste Management xxx (2014) xxx–xxx
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Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge Qian Li a,b, Wei Qiao c,e, Xiaochang Wang a, Kazuyuki Takayanagi b, Mohammad Shofie d, Yu-You Li b,d,⇑ a
Key Lab of Northwest Water Resource, Environment and Ecology, MOE, Xi’an University of Architecture and Technology, Xi’an 710055, China Dept. of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579, Japan c College of Engineering, China Agriculture University, Beijing 10081, China d Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan e Key Laboratory for Solid Waste Management and Environment Safety, Ministry of Education of China, Tsinghua University, Beijing 100084, China b
a r t i c l e
i n f o
Article history: Received 6 September 2014 Accepted 25 November 2014 Available online xxxx Keywords: Anaerobic digestion Rate-limiting step Thermophilic Mesophilic Propionic acid degradation
a b s t r a c t This study was conducted to characterize the kinetics of an anaerobic process (hydrolysis, acetogenesis, acidogenesis and methanogenesis) under thermophilic (55 °C) and mesophilic (35 °C) conditions with coffee grounds and waste activated sludge (WAS) as the substrates. Special focus was given to the kinetics of propionic acid degradation to elucidate the accumulation of VFAs. Under the thermophilic condition, the methane production rate of all substrates (WAS, ground coffee and raw coffee) was about 1.5 times higher than that under the mesophilic condition. However, the effects on methane production of each substrate under the thermophilic condition differed: WAS increased by 35.8–48.2%, raw coffee decreased by 76.3–64.5% and ground coffee decreased by 74.0–57.9%. Based on the maximum reaction rate (Rmax) of each anaerobic stage obtained from the modified Gompertz model, acetogenesis was found to be the ratelimiting step for coffee grounds and WAS. This can be explained by the kinetics of propionate degradation under thermophilic condition in which a long lag-phase (more than 18 days) was observed, although the propionate concentration was only 500 mg/L. Under the mesophilic condition, acidogenesis and hydrolysis were found to be the rate-limiting step for coffee grounds and WAS, respectively. Even though reducing the particle size accelerated the methane production rate of coffee grounds, but did not change the rate-limiting step: acetogenesis in thermophilic and acidogenesis in mesophilic. Ó 2014 Published by Elsevier Ltd.
1. Introduction Anaerobic digestion is a complex microbiological process which converts organic materials into energy in the form of methane. During this process, different kinds of microorganisms cooperate to produce methane via hydrolysis, acidogenesis, acetogenesis and methanogenesis (Batston et al., 2002). The stability of the anaerobic system is a primary issue that needs to be considered when designing and operating an anaerobic digester. For a long time, the theory of the three stages fermentation was widely accepted and used to describe the anaerobic digestion of solid waste (Kaspar and Wuhrmann, 1978; Chiu et al., 1997). However, in fact, the digestion of many kinds of solid substrates cannot be reasonably expressed by the three stages model. In addition, the reaction rate in biochemical system was found to increase with ⇑ Corresponding author at: Dept. of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579, Japan. E-mail address: [email protected] (Y.-Y. Li).
increasing temperature. Although the thermophilic process is widely regarded as an effective way to overcome the rate-limiting step in anaerobic digestion, this has not been investigated in any depth as yet. According to a very recent study, among the many factors which determine the balance of the serial bio-chemical processes and the stability of a digester, temperature and the nature of the substrates were found to be the most important (Labatut et al., 2014). Thermophilic anaerobic digestion (55–60 °C) achieves a better pathogen inactivation and yields more biogas than mesophilic digestion (35–40 °C), and was considered a highly-efficiency system. It has, however, been reported in numerous studies that the thermophilic system is particularly susceptible to the accumulation of VFAs (especially to propionic acid), which inhibits the activity of methanogens and potentially decreases the pH-buffer system (Souza et al., 1992; Lier et al., 1993; Dinsdale et al., 1997; Nges and Liu, 2010; Qiao et al., 2013). In general, if the reaction rate of the VFAs production exceeds the degradation rate, VFAs accumulation is observed. Acetogenesis was the key step in the degradation of
http://dx.doi.org/10.1016/j.wasman.2014.11.016 0956-053X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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other VFAs, since all of the C3–C6 VFAs needs to be converted to acetate before being converted to methane (McCarty and Mosey, 1991). Until recently, a significant amount of attention has been paid to the hydrolysis step, since it was always regarded as the rate-limiting step in the anaerobic digestion of complex organic solids (Ghosh et al., 1975; Ge et al., 2011; Jeihanipour et al., 2011). However, due to the contradiction between two traditional views – that hydrolysis is the rate-limiting step in anaerobic digestion and that hydrolysis is responsible for the accumulation of VFAs under thermophilic condition – it is necessary to investigate the different rate-limiting steps in a specific condition. Attention also needs to be paid to the intermediate processes of anaerobic digestion, such as acidogenesis and acetogenesis. It should be noted that in just one of the processes (either thermophilic or mesophilic), different substrates exhibited distinct degradation kinetics due to the different reaction pathways and microbial communities involved. Carbohydrates, protein and lipids are the main components of organic waste. The first order hydrolysis rate constant k of carbohydrates, proteins and lipids was 0.025– 0.2, 0.015–0.075 and 0.005–0.010 day1 (Christ et al., 2000), respectively. This indicates that substrates which contain more carbohydrates are easily hydrolyzed. However, the effects of different substrate compositions on the rate-limiting step are still not clear, and this is especially the case for the thermophilic condition. Therefore, the kinetic characteristics of the thermophilic and mesophilic conditions should be discussed separately, rather than conclude a certain step as the rate-limiting step of the anaerobic digestion process. The kinetics of anaerobic digestion using different substrates and temperature levels has not been directly compared under the same experimental conditions. Four questions need to be considered: (1) whether high temperature enhances the hydrolysis process and stops it from being the rate-limiting step, (2) whether the accumulation of propionic acid implies that acetogenesis is the rate-limiting step, (3) whether the propionic acid accumulation under the thermophilic condition is related to the lower activity of acetogens, (4) whether different substrates exhibit different kinetic characteristics which determine the ratelimiting step. The aims of this paper were to discuss the effects of temperature and substrates on complex organic waste degradation and methanogenic activity by fitting the experimental data of the batch experiments using a modified Gompertz model. 2. Materials and methods 2.1. Seed sludge and substrates The seed sludge used in this batch experiment was taken from thermophilic (55 °C) and mesophilic (35 °C) anaerobic CSRT reactors in our lab, respectively, after more than 100 day’s acclimation. The seed sludge was washed by the nutrient solution (Table 1) twice before use in order to remove the excess VFAs and soluble COD. The characteristics of washed seed sludge are shown in Table 2. The substrates used in this study were coffee grounds and dewatered waste activated sludge (WAS). All the substrates were provided by Tokyo Gas Co Ltd. The raw coffee grounds were ground by a small crusher (Labo Milser LM-PLUS) for 10 min to reduce the particle size from 3–4 mm to 1–2 mm for use as ground coffee. These substrates were stored at 4 °C before use. The characteristics of the three substrates are shown in Table 3. 2.2. Batch experiment In this study, a batch experiment was carried out in duplicate using 120 mL serum bottles with 100 mL seed sludge. The raw
Table 1 The composition of nutrient solution. Component
Concentration
Macro-nutrients solution MgCl2 6H2O CaCl2 2H2O NH4Cl K2HPO4 KH2PO4 Cystein HCl 9H2O
1.00 g/L 0.375 g/L 1.25 g/L 2.18 g/L 1.70 g/L 0.5 g/L
Micro-nutrients solution FeCl2 4H2O CoCl2 6H2O ZnCl2 H3BO3 MnCl2 2H2O NiCl2 6H2O CuCl2 2H2O NaMoO4 2H2O EDTA
2.00 mg/L 0.17 mg/L 0.07 mg/L 0.06 mg/L 0.50 mg/L 0.04 mg/L 0.027 mg/L 0.025 mg/L 5.00 mg/L
Table 2 Characteristics of seed sludge.
TS (%) VS (%) SS (%) VSS (%) pH COD (g/L) Acetate (g-COD/L) Propionate (g-COD/L) Butyrate (g-COD/L) Valeric (g-COD/L) Total VFA (g-COD/L)
Thermophilic seed sludge
Mesophilic seed sludge
2.69 ± 0.08 1.81 ± 0.08 1.94 ± 0.04 0.83 ± 0.01 7.62 3.60 ± 0.08 N.d. 0.09 N.d. 0.06 0.15
2.19 ± 0.01 1.17 ± 0.02 1.72 ± 0.03 0.62 ± 0.01 7.63 0.15 ± 0.03 N.d. N.d. N.d. N.d. N.d.
‘‘N.d.’’: not detected.
Table 3 Characteristics of substrates.
TS (%) VS (%) VS/TS (%) COD/TS (g/g) Carbohydrate (g/g-TS) Protein (g/g-TS) Lipid (g/g-TS) Tannins (g/g-TS) C (%) H (%) O (%) H (%) S (%) C:N
Raw coffee
Ground coffee
Sludge
25.1 ± 0.24 24.7 ± 0.23 98.5 1.60 0.59 0.24 0.24 4.35 55.2 ± 3.71 7.07 ± 0.39 34.4 ± 4.54 2.33 ± 0.24 0.30 ± 0.19 23.7
23.7 ± 0.21 23.4 ± 0.17 98.4 1.60 0.59 0.24 0.24 4.35 55.2 ± 3.71 7.07 ± 0.39 34.4 ± 4.54 2.33 ± 0.24 0.30 ± 0.19 23.7
10.8 ± 0.05 8.80 ± 0.07 81.4 0.98 0.31 0.69 0.02 – 34.0 ± 0.22 5.47 ± 0.07 25.7 ± 0.42 5.93 ± 0.09 0.70 ± 0.02 5.8
coffee, ground coffee and sludge were added into bottles at concentrations of 8 g COD/L, 8 g COD/L and 5 g COD/L, respectively. The concentration of each substrate was determined by the theoretical gas production and the actual organic loading rate (OLR) in the reactors from which the seed sludge was taken. Fig. 1 showed the main procedure of the batch experiment, before the seed sludge was used: note that pretreatment was needed to remove excess VFAs and soluble COD. After the seed sludge was mixed with a nutrient solution (Table 1), the supernatant was removed after settling for 30 min. This step was repeated twice. The washed seed sludge was then transferred into a 1 L flask and put in a water bath for 2–3 days to allow the microorganisms to adapt. The treated seed
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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Nutrient solution
Recovered Seed Sludge Substrate
Water removal
Seed sludge
Nitrogen gas
Recovery
Bottling and inoculation
Seed sludge preparation
Gas monitor
Batch experiment Fig. 1. Procedure of batch experiment.
sludge was transferred into a 120 mL serum bottle with different substrates and purged with nitrogen gas for 2 min to remove the oxygen in the bottle (assuring the anaerobic condition). The bottles were subsequently incubated in a water-bath shaker at temperatures of 55 °C and 35 °C for thermophilic and mesophilic digestion, respectively. After each of the bottles reached the set temperature, the headspace was vented using a syringe to release the pressure caused by the thermal expansion. Two blank bottles without any substrates added were used as the control for both the thermophilic and mesophilic conditions. The biogas produced from inoculum bottles was corrected by the biogas produced from the blank bottles. A 1 mL sample of sludge was taken from each bottle on sampling days and stored at 4 °C after filtration through a 0.45 lm membrane filter. 2.3. Methanogenic activity test The accumulation of VFAs indicates a process imbalance due to low or inhibited methanogenic activity, especially under the presence of high concentration of propionic acid in the thermophilic condition. In order to ascertain the relationship between propionic acid degradation and methanogenic activity, an activity test was conducted under both the mesophilic and thermophilic conditions using sodium acetate and sodium propionate as substrates. The seed sludge and experiment procedure used for this activity test was the same as that used in Section 2.2. Methanogenic activity was determined by the methane production, with higher methane production rate indicates a higher methanogenic activity. 2.4. Analysis methods 2.4.1. Biogas measurement The biogas produced from each bottle was analyzed every 0.5– 4 days according to the volume of produced biogas. The bottles were shaken for 2–3 s in the water bath before measuring the bio-
gas production. The volume of biogas was measured using a syringe, while the composition of biogas (CO2 and CH4) was analyzed using a gas chromatograph (Shimadzu GC-8A). The volume of methane production was calculated as follows:
V CH4 ¼ V H DC CH4 þ V S C CH4
ð1Þ
where V CH4 is the volume of methane volume (mL), VH is the head space volume (mL), VS is the biogas volume in syringe, DC CH4 is the variation concentration of CH4 (%). 2.4.2. Other analysis COD was measured according to the Japan Standard Testing Methods for wastewater (JSWA , 1997). VFAs was analyzed by an Agilent-6890 gas chromatograph. All the data showed in the figures are the net value subtracted the blank sample. The four different steps reaction of anaerobic digestion, hydrolysis (Hyd), acidogenesis (Acid), acetogenesis (Acet) and methanogenesis (Meth), were expressed by cumulative hydrolyzed COD, cumulative acidified COD, cumulative acetified COD and methane COD (Neves et al., 2006), and were calculated as follows:
Hyd ¼ ðV CH4 =350 þ C SCOD VÞ COD1 add
ð2Þ
Acid ¼ ðV CH4 =350 þ C TVFA VÞ COD1 add
ð3Þ
Acet ¼ ðV CH4 =350 þ C acetate VÞ COD1 add
ð4Þ
Meth ¼ V CH4 =350 COD1 add
ð5Þ
where Hyd, Acid, Acet and Meth were cumulative hydrolyzed SCOD (%), cumulative acidified COD (%), cumulative acetified COD (%) and methane COD (%), respectively. V CH4 is the volume of methane volume (mL), V is the volume of seed sludge (L), CCODs is the concentration of soluble COD (g/L), CTVFA is the concentration of TVFA (g-COD/ L), Cacetate is the concentration of acetate acid (g-COD/L), CODadd is
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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Table 4 n h io e ðt0 tÞ þ 1 . The kinetic parameters of each step in anaerobic digestion obtained from Modified Gompertz model P ¼ P 0 exp exp Rmax P0 Thermophilic
Mesophilic
Hydrolysis
Acidogenesis
Acetogenesis
Methanogenesis
Hydrolysis
Acidogenesis
Acetogenesis
Methanogenesis
Raw coffee
P0 Rmax R2
0.813 ± 0.019 0.091 ± 0.011 0.978
0.715 ± 0.009 0.072 ± 0.004 0.992
0.645 ± 0.008 0.067 ± 0.003 0.994
0.645 ± 0.007 0.068 ± 0.003 0.996
0.838 ± 0.006 0.048 ± 0.001 0.999
0.763 ± 0.004 0.046 ± 0.001 0.999
0.763 ± 0.004 0.046 ± 0.001 0.999
0.763 ± 0.004 0.046 ± 0.001 0.999
Ground coffee
P0 Rmax R2
0.760 ± 0.165 0.125 ± 0.015 0.98
0.650 ± 0.004 0.099 ± 0.004 0.997
0.578 ± 0.004 0.096 ± 0.004 0.997
0.579 ± 0.003 0.097 ± 0.002 0.999
0.804 ± 0.005 0.069 ± 0.002 0.999
0.740 ± 0.003 0.062 ± 0.001 0.999
0.740 ± 0.003 0.062 ± 0.001 0.999
0.740 ± 0.003 0.062 ± 0.001 0.999
Sludge
P0 Rmax R2
0.490 ± 0.012 0.066 ± 0.009 0.962
0.458 ± 0.017 0.047 ± 0.009 0.925
0.482 ± 0.020 0.031 ± 0.004 0.95
0.482 ± 0.020 0.031 ± 0.004 0.951
0.361 ± 0.012 0.021 ± 0.003 0.955
0.358 ± 0.010 0.020 ± 0.002 0.972
0.358 ± 0.010 0.020 ± 0.002 0.972
0.358 ± 0.010 0.020 ± 0.002 0.972
(a')
Raw coffee (Thermophilic) 1 0.8 0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
0.2
Acid. Meth. Simulated Acid. Simulated Meth.
Raw coffee (Mesophilic) 1 0.8
g-COD/g-added COD
g-COD/g-added COD
(a)
0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
0.2
0
0 0
5
10
15
20
25
30
35
0
40
5
10
15
0.8 0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
Acid. Meth. Simulated Acid. Simulated Meth.
g-COD/g-added COD
g-COD/g-added COD
(b')
Ground coffee (Thermophilic) 1
0.2
30
35
40
Ground coffee (Mesophilic) 1
0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
0.2
Acid. Meth. Simulated Acid. Simulated Meth.
0 0
5
10
15
20
25
30
35
0
40
5
10
Sludge (Thermophilic)
(c')
1 Acid. Meth. Simulated Acid. Simulated Meth.
g-COD/g-added COD
Hyd. Acet. Simulated Hyd. Simulated Acet.
0.8
15
20
25
30
35
40
Time (days)
Time (days)
g-COD/g-added COD
25
0.8
0
(c)
20
Time (days)
Time (days)
(b)
Acid. Meth. Simulated Acid. Simulated Meth.
0.6 0.4 0.2 0
Sludge (Mesophilic) 1 Hyd. Acet. Simulated Hyd. Simulated Meth.
0.8 0.6
Acid. Meth. Simulated Acid. Simulated Acet.
0.4 0.2 0
0
5
10
15
20
25
Time (days)
30
35
40
0
5
10
15
20
25
30
35
40
Time (days)
e Fig. 2. Description of the 4 stages in anaerobic digestion by modified Gompertz model ðP ¼ P 0 exp exp Rmax ðk tÞ þ 1 Þ under thermophilic condition and mesophilic P condition. Symbols refer to the experimental data and lines to the simulation of the modified Gompertz model.
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Q. Li et al. / Waste Management xxx (2014) xxx–xxx
Concentration (mg-COD/L)
(a)
where P is the conversion ratio of substrates (gCOD/g-added COD), P0 is the conversion potential (gCOD/g-added COD), Rmax is the maximum conversion rate (gCOD/g-added COD d), t0 is the lag time (days) and e = 2.718281828. The constants P0, k, and t0 were estimated by a non-linear fitting program using the Original 8.5. This model was used by Lay et al. (1998) to describe the bacterial growth in a batch culture. In this study, simulations of both the batch experiment and methanogenic activity test were made using this model to obtain the kinetic parameters.
Raw coffee
600
Acetate Propionate Butyrate Valeric
500
5
400
300
200
100
3. Results and discussion 0 0
5
10
15
20
25
3.1. Effect of temperature on different steps in anaerobic digestion
30
Time (days)
Concentration (mg-COD/L)
(b)
Ground coffee
600
Acetate Propionate Butyrate Valeric
500
400
300
200
100
0 0
5
10
15
20
25
30
Time (days)
(c)
Sludge
600
Acetate Propionate Butyrate Valeric
Concentration (mg-COD/L)
500
400
300
200
100
0 0
5
10
15
20
25
30
Time (days) Fig. 3. Variation of VFA in anaerobic digestion under thermophilic condition.
the COD of substrates added into the bottles (g), 1 g COD equaled 350 mL CH4. Methanogenic activity was determined by the following equation:
Activ ity ¼ Rmax =S
ð6Þ
where Activity is the methanogenic activity (gCH4-COD/gVSS d), Rmax is the maximum methane production rate (gCH4-COD/d), and S is the amount of sludge (gVSS). 2.4.3. Kinetic models The experimental data of each step in the batch experiment and activity test was simulated by the modified Gompertz equation:
Rmax e P ¼ P0 exp exp ðt0 tÞ þ 1 P0
ð7Þ
The kinetic characteristics of anaerobic digestion were not the same under thermophilic and mesophilic conditions due to the different metabolism pathways and bacterial communities. Due to the contradiction between the traditional theory that hydrolysis is the rate-limiting step of anaerobic digestion and also the reason for the accumulation of VFAs under the thermophilic condition, the four steps of anaerobic digestion are discussed separately in order to determine the effects of temperature on the each step. According to the comparison between the maximum reaction rates (Rmax) and production potential (P0) obtained from the modified Gompertz model (Table 4 and Fig. 2), the effects of temperature on each step in the digestion of raw coffee and ground coffee were similar and irrelevant to the particle size of the substrates. Hydrolysis is the first step in anaerobic digestion. During hydrolysis, complex organic waste is hydrolyzed to monomers such as amino acids, sugars and fatty acids. Since hydrolysis potential directly affects the degradability of solid waste, it is always considered as the rate-limiting step in the anaerobic digestion of complex organic waste. As shown in Table 4, the hydrolysis rate of coffee grounds in the thermophilic condition was almost double what it was under the mesophilic condition. The higher hydrolysis potential of coffee grounds in both the mesophilic and thermophilic conditions may be attributable to the higher degradability of carbohydrates, which is the main component of coffee grounds. Even under the mesophilic condition, the degradation efficiency of coffee grounds was around 80%. For WAS, thermophilic digestion not only accelerated the reaction rates, but also increased the hydrolysis potential from 37.0% to 49.2%. In the acidogenesis stage, the hydrolysate (Soluble COD) was converted to VFA by acidogenic bacteria. The maximum acidification rate of coffee grounds under thermophilic condition was higher than that of the mesophilic condition by a factor of 1.56, but no significant increase in acidification potential was observed. Meanwhile the maximum acidification rate of WAS increased from 0.020 g-COD/g-added COD d1 to 0.047 g-COD/g-added COD d1, more than double. In addition to this, the acidification potential also increased from 35.8% to 45.8%. In the acetogenesis stage, all the C3–C6 VFAs needs to be converted to acetate before converting to methane. If the production rate exceeds the degradation rate, VFA will accumulate. Under the mesophilic condition, Rmax(ace) = Rmax(aci), indicating that all the VFAs was properly converted to acetate by acetogens, and was subsequently converted to methane by methanogens, leaving no VFAs. However, because Rmax(ace) < Rmax(aci) under the thermophilic condition, VFAs accumulation was observed, and propionic acid was found to be the main component of the VFAs, as shown in Fig. 3. This result agrees with the findings of Fernandez and Forster (1993) and Dinsdale et al. (1997), who reported that the metabolism of propionic acid by acetogens is lower under the thermophilic condition. Although the acetogenesis rate is accelerated in the thermophilic condition, the acetogenesis potential was
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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1000mg/L 2000mg/L 3000mg/L 5000mg/L 8000mg/L 10000mg/L
150
Mesophilic (acetate)
(a') 250
Thermophilic (acetate)
200
Methane production (mL)
Methane production (mL)
(a)
100
50
0
1000mg/L 3000mg/L 5000mg/L 8000mg/L 10000mg/L 20000mg/L
200 150 100 50 0
0
1
2
3
4
5
0
6
2
4
Time (days)
90
8
10
Mesophilic (propionate)
(b') 200
Thermophilic (propionate) 500mg/L 1000mg/L 1500mg/L 2000mg/L 3000mg/L 5000mg/L
Methane production (mL)
Methane production (mL)
(b) 120
6
Time (days)
60
30
0
500mg/L 1000mg/L 1500mg/L 2000mg/L 3000mg/L 5000mg/L
160 120 80 40 0
0
5
10
15
20
25
30
35
40
Time (days)
0
2
4
6
8
10
12
14
16
Time (days)
Fig. 4. Methane production of different acetate and propionate concentration under thermophilic and mesophilic condition.
Table 5 Kinetic parameters of methanogenic activity test obtained from modified Gompertz model. Concentration (mg/L)
Thermophilic
Mesophilic
Rmax (mL/d)
t0 (d)
Rmax (mL/d)
t0 (d)
Acetic acid 1000 2000 3000 5000 8000 10,000 20,000
23.6 38.2 44.6 55.3 57.4 55.1 –
0.112 0.180 0.232 0.312 0.358 0.359 –
36.7 – 38.5 43.3 41.8 36.0 2.50
0.613 – 0.470 0.633 0.883 0.878 0.846
Propionic acid 500 1000 1500 2000 3000 5000
3.01 5.89 8.04 11.6 17.91 –
18.0 20.1 20.5 24.1 26.6 –
8.02 10.7 14.5 18.9 24.7 25.7
1.32 2.59 2.57 3.34 4.22 5.91
found to decrease due to the accumulation in C3–C6 VFAs, which could not be degraded until the end of batch experiment in the case of coffee grounds digestion. For WAS, the thermophilic condition was found to increase the acetogenesis potential, since the accumulated VFA was degraded effectively in the latter half of anaerobic digestion, as shown in Fig. 3c. In the methanogenesis stage, acetate was completely converted to methane in both the thermophilic and mesophilic conditions. Therefore, the methane production potential depend on the part of organic wastes which could be converted to acetate. For both raw coffee and ground coffee, the methane production potential decreased by more than 15% under thermophilic condition due to VFAs accumulation, whereas it increased from 35.8% to 48.2% when using a WAS substrate.
A comparison of the Rmax and P0 in each stage revealed that one of the benefits of the thermophilic condition was an increased reaction rate, a notably higher hydrolysis potential, but that the methane production potential depended on the physicochemical properties of the substrates.
3.2. Effects of substrates on reaction rate The kinetic characteristics of organic waste degradation not only depended on temperature, but were also affected by the physicochemical properties of the substrates. The order of Rmax under both the thermophilic and mesophilic conditions in each stage was as follows: Rmax (ground coffee) > Rmax (raw coffee) > Rmax (WAS). For raw coffee and ground coffee, reducing the particle size increased the Rmax of hydrolysis from 0.048 to 0.069 g-COD/g-added COD d1 under the mesophilic condition, and from 0.091 to 0.125 g-COD/g-added COD d1 under the thermophilic condition. Smaller particle sizes increase the available specific surface, which leads to a higher reaction rate in anaerobic digestion. By reducing the particle size, the reaction rate at each stage of the anaerobic process was increased by more than 35%. The main component of coffee grounds and WAS are carbohydrates and protein, respectively. Generally, carbohydrates hydrolyze under the anaerobic condition at a faster rate than protein (Pavlostathis and Giraldo-Gomez, 1991). Due to the inherently low degradability of sludge, the methane production potential was still lower than 50% even under thermophilic condition. Because the main component of coffee grounds is carbohydrates, the reaction rate was higher than for WAS, which has protein as its main component. Still, the accumulation of VFAs (notably propionic acid) in thermophilic anaerobic digestion resulted in a lower methane production potential.
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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Thermophilic 0.15
(a') Methanogenic activity (gCH4-COD/gVSS·d)
Methanogenic activity (gCH4-COD/gVSS·d)
(a)
0.12 0.09 0.06 0.03
0.15 0.12 0.09 0.06 0.03 0
0
Acetate concentration (g/L)
0.1
Thermophilic
(b') Methanogenic activity (gCH4-COD/gVSS·d)
Methanogenic activity (gCH4-COD/gVSS·d)
(b)
Acetate concentration (g/L)
0.08 0.06 0.04 0.02 0
0.1
Mesophilic
0.08 0.06 0.04 0.02 0
Propionate concentration (g/L)
Propionate concentration (g/L)
Fig. 5. Variation of methanogenic activity with acetate/propionate concentration under thermophilic and mesophilic condition.
3.3. VFAs degradation and methanogenic activity In order to elucidate the effects of temperature on the degradation of propionic acid, and acetic acid, a methanogenic activity test was conducted using acetate and propionate as the substrates, respectively. The main reason for only investigating the methanogenic activity of acetate and propionate are as follows: (1) acetate is the final product of acetogenesis, and all the VFAs needs to be converted to acetate so that it can be converted to methane; (2) propionic acid is the main component of VFAs in this study and has a higher inhibitory effect on methanogenic bacteria than other types of VFAs. The curve shown in Fig. 4 was obtained from experimental data simulation by the modified Gompertz model, and the kinetic parameters are given in Table 5. Under both the thermophilic and mesophilic conditions, the lag time of acetate was less than one day. However, under the thermophilic condition, the lag time for propionate increased along with the increase in the propionate concentration and was longer than 17 days even when the propionate concentration was only 500 mg/L. It was clear that propionate degradation was strongly inhibited by high temperature, resulting in a longer lag time and a lower degradation rate. The main reason might be the lower ability of acetogens which could convert propionic acid to acetic acid. The variations of methanogenic activity with acetate/propionate concentration are shown in Fig. 5 and Table 6. Methanogenic activity under the mesophilic condition was a little higher than under the thermophilic condition when the concentration of acetic acid was lower than 3000 mg/L. This is consistent with the findings of a previous study by Isa et al., 1993, who also reported higher methanogenic activity under the mesophilic condition. For propionic acid, methanogenic activity was significantly lower under the thermophilic condition and completely inhibited when the concentration of propionate was higher than 5000 mg/L. The lower methanogenic activity and longer lag time of propionic acid degradation under thermophilic condition may be sufficient to explain the ease with which propionic acid accumulated in the thermophilic condition. In this study, despite the tendency for propionic acid to accumulate, it could be degraded when the concentration was not too high, as shown in Fig. 4. The degradation of propionic acid differed
Table 6 Variation of methanogenic activity with substrate concentration under thermophilic and mesophilic condition. Concentration (mg/L)
Methanogenic activity (gCH4-COD/gVSS d) Thermophilic
Mesophilic
Acetate 1000 2000 3000 5000 8000 10,000 20,000
0.058 0.094 0.109 0.135 0.140 0.135 –
0.102 – 0.108 0.120 0.115 0.099 0.007
Propionate 500 1000 1500 2000 3000 5000
0.007 0.014 0.020 0.028 0.044 0
0.022 0.030 0.040 0.052 0.068 0.071
according to the substrate. As shown in Fig. 3c, the concentration of propionic acid in WAS anaerobic digestion increased to the maximum within the first 9 days, then decreased sharply. However, a different phenomenon was observed in the thermophilic digestion of coffee grounds (Fig. 3a and b), where the propionic acid persisted during thermophilic digestion until the end of the batch experiment. The concentration of propionic acid was maintained below 500 mg-COD/L, which was a little higher than that in WAS digestion, but did not reach the inhibition threshold. The differences of propionic acid degradation kinetics between coffee grounds and WAS might be related to the different physicochemical properties of these substrates. The presence of inhibitory compounds contained in the coffee grounds was most likely the main reason for the inhibition of propionic acid degradation under the thermophilic condition, as has been reported in earlier studies. Fernandez and Forster (1993) reported that coffee waste contained some components which inhibited anaerobic digestion in the thermophilic temperature range. Neves et al. (2006) also found that the decreased methane yield was likely due to the intermediates formed during the hydrolysis step. The higher lipid content of cof-
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fee grounds (0.24 g/g-TS) also has an inhibitory effects in the anaerobic process due to the accumulation of long-chain fatty acids resulting from the hydrolysis of neutral lipids (Labatut et al., 2014). In this study, although there was no direct evidence indicating the inhibitory compounds, there was a clear and significant negative effect on propionic acid degradation and methane production during the thermophilic digestion of coffee. 3.4. Rate-limiting step determination Anaerobic digestion was considered a multi-step process, with the overall reaction rate depending on the slowest reaction, which is called the rate-limiting step (Hill and Barth, 1977). Theoretically, hydrolysis is considered the rate-limiting step in anaerobic digestion, but actually, the main problem in thermophilic digestion is the accumulation of VFAs. That is, it was concluded that hydrolysis cannot be considered the rate-limiting step in anaerobic digestion without first taking the effects of temperature and substrates into account. However, the focus of many researchers is still on the hydrolysis step, and how to enhance anaerobic digestion performance by improving the hydrolysis rate (Ristow et al., 2006; Ge et al., 2011). From this perspective, the effects of temperature on the determination of rate-limiting step in anaerobic digestion with different substrates need to be re-evaluated. In this study, the modified Gompertz model was used to simulate the four steps in anaerobic digestion using Origin 8.5, to determine the kinetic parameters shown in Table 4. The rate-limiting step was determined by comparing the Rmax obtained from modified Gompertz model at each stage in the digestion process. The rate-limiting step for the anaerobic digestion of raw coffee under the mesophilic condition was in this order: Rmax(hyd.) > Rmax(Acid.) = Rmax(Ace.) = Rmax(meth.), which meant that acidogenesis was the rate-limiting step. Under the thermophilic condition, the rate-limiting step was in this order: Rmax(hyd.) > Rmax(Acid.) > Rmax(Acet.) = Rmax(meth.). Because the accumulated VFAs could not be proportionally converted to acetate, methane production was affected, indicating that acetogenesis was the rate-limiting step. A similar conclusion was also obtained for ground coffee. Although reducing the particle size increased the reaction rate, it did not affect the rate-limiting step determination. For WAS, the thermophilic condition significantly enhanced the hydrolysis step and the methane yield. The order of the rate-limiting step for WAS under the thermophilic condition was as follows: Rmax(hyd.) > Rmax(Acid.) > Rmax(Acet.) = Rmax(meth.), indicating that acetogenesis was the rate-limiting step under the thermophilic condition. Under the mesophilic condition, due to the same reaction rate in each stage, the order of rate-limiting step was as follows: Rmax(hyd.) > Rmax(Acid.) > Rmax(Acet.) = Rmax(meth.). Hydrolysis was the rate-limiting step. The rate-limiting step was affected by both temperature and substrates. If hydrolysis is considered the rate-limiting step in all case of anaerobic digestion, it would result in problems. For coffee grounds, especially under thermophilic conditions, it was found that even though C3–C6 VFAs could not be normally converted to acetate, the VFAs concentration reached a limit and caused system failure when the hydrolysis rate was enhanced. In the case of WAS. However, a higher hydrolysis rate was found to be beneficial. Therefore, it is important to know the rate-limiting step of the anaerobic digestion process in order to optimize and control the process. 4. Conclusions 1. Thermophilic digestion was beneficial for increasing the methane production rate from WAS, coffee grounds and raw coffee grounds. However, under high temperature, methane produc-
tion potential when using coffee grounds decreased by around 15% compared to the mesophilic condition due to the accumulation of propionic acid. 2. Under the thermophilic condition, acetogenesis was the rate-limiting step for both coffee grounds and WAS. Under the mesophilic condition, acidogenesis and hydrolysis were the rate-limiting steps for coffee and sludge, respectively. 3. Although reducing the particle size of coffee grounds accelerated the reaction rate of acetogenesis and methanogenesis, the accumulation of propionate under thermophilic condition resulted in a lower methane production potential. 4. The lag time for propionate degradation under the thermophilic condition was almost 18-days longer under than mesophilic condition, but, the lag time was decreased by the thermophilic condition in the case of acetate degradation.
Acknowledgements This work was partially supported by Japan Society for the Promotion of Science (24-02053) and Key Laboratory for Solid Waste Management and Environment Safety, Ministry of Education of China (SWMES 2011-04). The overseas study conducted by the first author was finically supported by China Scholarship Council (CSC).
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Ristow, N.E., Sotemann, S.W., Wentzel, M.C., Loewental, R.E., Ekama, G.A., 2006. The effects of hydrolysis retention time and feed COD concentration on the rate of hydrolysis of primary sewage sludge under methanogenic condition. Water Sci. Technol. 54 (5), 91–100. Souza, M.E., Fuzaro, G., Polegato, A.R., 1992. Thermophilic anaerobic digestion of vinasse in pilot plant UASB reactor. Water Sci. Technol. 25 (7), 213–222.
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Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge Qian Li a,b, Wei Qiao c,e, Xiaochang Wang a, Kazuyuki Takayanagi b, Mohammad Shofie d, Yu-You Li b,d,⇑ a
Key Lab of Northwest Water Resource, Environment and Ecology, MOE, Xi’an University of Architecture and Technology, Xi’an 710055, China Dept. of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579, Japan c College of Engineering, China Agriculture University, Beijing 10081, China d Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan e Key Laboratory for Solid Waste Management and Environment Safety, Ministry of Education of China, Tsinghua University, Beijing 100084, China b
a r t i c l e
i n f o
Article history: Received 6 September 2014 Accepted 25 November 2014 Available online xxxx Keywords: Anaerobic digestion Rate-limiting step Thermophilic Mesophilic Propionic acid degradation
a b s t r a c t This study was conducted to characterize the kinetics of an anaerobic process (hydrolysis, acetogenesis, acidogenesis and methanogenesis) under thermophilic (55 °C) and mesophilic (35 °C) conditions with coffee grounds and waste activated sludge (WAS) as the substrates. Special focus was given to the kinetics of propionic acid degradation to elucidate the accumulation of VFAs. Under the thermophilic condition, the methane production rate of all substrates (WAS, ground coffee and raw coffee) was about 1.5 times higher than that under the mesophilic condition. However, the effects on methane production of each substrate under the thermophilic condition differed: WAS increased by 35.8–48.2%, raw coffee decreased by 76.3–64.5% and ground coffee decreased by 74.0–57.9%. Based on the maximum reaction rate (Rmax) of each anaerobic stage obtained from the modified Gompertz model, acetogenesis was found to be the ratelimiting step for coffee grounds and WAS. This can be explained by the kinetics of propionate degradation under thermophilic condition in which a long lag-phase (more than 18 days) was observed, although the propionate concentration was only 500 mg/L. Under the mesophilic condition, acidogenesis and hydrolysis were found to be the rate-limiting step for coffee grounds and WAS, respectively. Even though reducing the particle size accelerated the methane production rate of coffee grounds, but did not change the rate-limiting step: acetogenesis in thermophilic and acidogenesis in mesophilic. Ó 2014 Published by Elsevier Ltd.
1. Introduction Anaerobic digestion is a complex microbiological process which converts organic materials into energy in the form of methane. During this process, different kinds of microorganisms cooperate to produce methane via hydrolysis, acidogenesis, acetogenesis and methanogenesis (Batston et al., 2002). The stability of the anaerobic system is a primary issue that needs to be considered when designing and operating an anaerobic digester. For a long time, the theory of the three stages fermentation was widely accepted and used to describe the anaerobic digestion of solid waste (Kaspar and Wuhrmann, 1978; Chiu et al., 1997). However, in fact, the digestion of many kinds of solid substrates cannot be reasonably expressed by the three stages model. In addition, the reaction rate in biochemical system was found to increase with ⇑ Corresponding author at: Dept. of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579, Japan. E-mail address: [email protected] (Y.-Y. Li).
increasing temperature. Although the thermophilic process is widely regarded as an effective way to overcome the rate-limiting step in anaerobic digestion, this has not been investigated in any depth as yet. According to a very recent study, among the many factors which determine the balance of the serial bio-chemical processes and the stability of a digester, temperature and the nature of the substrates were found to be the most important (Labatut et al., 2014). Thermophilic anaerobic digestion (55–60 °C) achieves a better pathogen inactivation and yields more biogas than mesophilic digestion (35–40 °C), and was considered a highly-efficiency system. It has, however, been reported in numerous studies that the thermophilic system is particularly susceptible to the accumulation of VFAs (especially to propionic acid), which inhibits the activity of methanogens and potentially decreases the pH-buffer system (Souza et al., 1992; Lier et al., 1993; Dinsdale et al., 1997; Nges and Liu, 2010; Qiao et al., 2013). In general, if the reaction rate of the VFAs production exceeds the degradation rate, VFAs accumulation is observed. Acetogenesis was the key step in the degradation of
http://dx.doi.org/10.1016/j.wasman.2014.11.016 0956-053X/Ó 2014 Published by Elsevier Ltd.
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other VFAs, since all of the C3–C6 VFAs needs to be converted to acetate before being converted to methane (McCarty and Mosey, 1991). Until recently, a significant amount of attention has been paid to the hydrolysis step, since it was always regarded as the rate-limiting step in the anaerobic digestion of complex organic solids (Ghosh et al., 1975; Ge et al., 2011; Jeihanipour et al., 2011). However, due to the contradiction between two traditional views – that hydrolysis is the rate-limiting step in anaerobic digestion and that hydrolysis is responsible for the accumulation of VFAs under thermophilic condition – it is necessary to investigate the different rate-limiting steps in a specific condition. Attention also needs to be paid to the intermediate processes of anaerobic digestion, such as acidogenesis and acetogenesis. It should be noted that in just one of the processes (either thermophilic or mesophilic), different substrates exhibited distinct degradation kinetics due to the different reaction pathways and microbial communities involved. Carbohydrates, protein and lipids are the main components of organic waste. The first order hydrolysis rate constant k of carbohydrates, proteins and lipids was 0.025– 0.2, 0.015–0.075 and 0.005–0.010 day1 (Christ et al., 2000), respectively. This indicates that substrates which contain more carbohydrates are easily hydrolyzed. However, the effects of different substrate compositions on the rate-limiting step are still not clear, and this is especially the case for the thermophilic condition. Therefore, the kinetic characteristics of the thermophilic and mesophilic conditions should be discussed separately, rather than conclude a certain step as the rate-limiting step of the anaerobic digestion process. The kinetics of anaerobic digestion using different substrates and temperature levels has not been directly compared under the same experimental conditions. Four questions need to be considered: (1) whether high temperature enhances the hydrolysis process and stops it from being the rate-limiting step, (2) whether the accumulation of propionic acid implies that acetogenesis is the rate-limiting step, (3) whether the propionic acid accumulation under the thermophilic condition is related to the lower activity of acetogens, (4) whether different substrates exhibit different kinetic characteristics which determine the ratelimiting step. The aims of this paper were to discuss the effects of temperature and substrates on complex organic waste degradation and methanogenic activity by fitting the experimental data of the batch experiments using a modified Gompertz model. 2. Materials and methods 2.1. Seed sludge and substrates The seed sludge used in this batch experiment was taken from thermophilic (55 °C) and mesophilic (35 °C) anaerobic CSRT reactors in our lab, respectively, after more than 100 day’s acclimation. The seed sludge was washed by the nutrient solution (Table 1) twice before use in order to remove the excess VFAs and soluble COD. The characteristics of washed seed sludge are shown in Table 2. The substrates used in this study were coffee grounds and dewatered waste activated sludge (WAS). All the substrates were provided by Tokyo Gas Co Ltd. The raw coffee grounds were ground by a small crusher (Labo Milser LM-PLUS) for 10 min to reduce the particle size from 3–4 mm to 1–2 mm for use as ground coffee. These substrates were stored at 4 °C before use. The characteristics of the three substrates are shown in Table 3. 2.2. Batch experiment In this study, a batch experiment was carried out in duplicate using 120 mL serum bottles with 100 mL seed sludge. The raw
Table 1 The composition of nutrient solution. Component
Concentration
Macro-nutrients solution MgCl2 6H2O CaCl2 2H2O NH4Cl K2HPO4 KH2PO4 Cystein HCl 9H2O
1.00 g/L 0.375 g/L 1.25 g/L 2.18 g/L 1.70 g/L 0.5 g/L
Micro-nutrients solution FeCl2 4H2O CoCl2 6H2O ZnCl2 H3BO3 MnCl2 2H2O NiCl2 6H2O CuCl2 2H2O NaMoO4 2H2O EDTA
2.00 mg/L 0.17 mg/L 0.07 mg/L 0.06 mg/L 0.50 mg/L 0.04 mg/L 0.027 mg/L 0.025 mg/L 5.00 mg/L
Table 2 Characteristics of seed sludge.
TS (%) VS (%) SS (%) VSS (%) pH COD (g/L) Acetate (g-COD/L) Propionate (g-COD/L) Butyrate (g-COD/L) Valeric (g-COD/L) Total VFA (g-COD/L)
Thermophilic seed sludge
Mesophilic seed sludge
2.69 ± 0.08 1.81 ± 0.08 1.94 ± 0.04 0.83 ± 0.01 7.62 3.60 ± 0.08 N.d. 0.09 N.d. 0.06 0.15
2.19 ± 0.01 1.17 ± 0.02 1.72 ± 0.03 0.62 ± 0.01 7.63 0.15 ± 0.03 N.d. N.d. N.d. N.d. N.d.
‘‘N.d.’’: not detected.
Table 3 Characteristics of substrates.
TS (%) VS (%) VS/TS (%) COD/TS (g/g) Carbohydrate (g/g-TS) Protein (g/g-TS) Lipid (g/g-TS) Tannins (g/g-TS) C (%) H (%) O (%) H (%) S (%) C:N
Raw coffee
Ground coffee
Sludge
25.1 ± 0.24 24.7 ± 0.23 98.5 1.60 0.59 0.24 0.24 4.35 55.2 ± 3.71 7.07 ± 0.39 34.4 ± 4.54 2.33 ± 0.24 0.30 ± 0.19 23.7
23.7 ± 0.21 23.4 ± 0.17 98.4 1.60 0.59 0.24 0.24 4.35 55.2 ± 3.71 7.07 ± 0.39 34.4 ± 4.54 2.33 ± 0.24 0.30 ± 0.19 23.7
10.8 ± 0.05 8.80 ± 0.07 81.4 0.98 0.31 0.69 0.02 – 34.0 ± 0.22 5.47 ± 0.07 25.7 ± 0.42 5.93 ± 0.09 0.70 ± 0.02 5.8
coffee, ground coffee and sludge were added into bottles at concentrations of 8 g COD/L, 8 g COD/L and 5 g COD/L, respectively. The concentration of each substrate was determined by the theoretical gas production and the actual organic loading rate (OLR) in the reactors from which the seed sludge was taken. Fig. 1 showed the main procedure of the batch experiment, before the seed sludge was used: note that pretreatment was needed to remove excess VFAs and soluble COD. After the seed sludge was mixed with a nutrient solution (Table 1), the supernatant was removed after settling for 30 min. This step was repeated twice. The washed seed sludge was then transferred into a 1 L flask and put in a water bath for 2–3 days to allow the microorganisms to adapt. The treated seed
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Nutrient solution
Recovered Seed Sludge Substrate
Water removal
Seed sludge
Nitrogen gas
Recovery
Bottling and inoculation
Seed sludge preparation
Gas monitor
Batch experiment Fig. 1. Procedure of batch experiment.
sludge was transferred into a 120 mL serum bottle with different substrates and purged with nitrogen gas for 2 min to remove the oxygen in the bottle (assuring the anaerobic condition). The bottles were subsequently incubated in a water-bath shaker at temperatures of 55 °C and 35 °C for thermophilic and mesophilic digestion, respectively. After each of the bottles reached the set temperature, the headspace was vented using a syringe to release the pressure caused by the thermal expansion. Two blank bottles without any substrates added were used as the control for both the thermophilic and mesophilic conditions. The biogas produced from inoculum bottles was corrected by the biogas produced from the blank bottles. A 1 mL sample of sludge was taken from each bottle on sampling days and stored at 4 °C after filtration through a 0.45 lm membrane filter. 2.3. Methanogenic activity test The accumulation of VFAs indicates a process imbalance due to low or inhibited methanogenic activity, especially under the presence of high concentration of propionic acid in the thermophilic condition. In order to ascertain the relationship between propionic acid degradation and methanogenic activity, an activity test was conducted under both the mesophilic and thermophilic conditions using sodium acetate and sodium propionate as substrates. The seed sludge and experiment procedure used for this activity test was the same as that used in Section 2.2. Methanogenic activity was determined by the methane production, with higher methane production rate indicates a higher methanogenic activity. 2.4. Analysis methods 2.4.1. Biogas measurement The biogas produced from each bottle was analyzed every 0.5– 4 days according to the volume of produced biogas. The bottles were shaken for 2–3 s in the water bath before measuring the bio-
gas production. The volume of biogas was measured using a syringe, while the composition of biogas (CO2 and CH4) was analyzed using a gas chromatograph (Shimadzu GC-8A). The volume of methane production was calculated as follows:
V CH4 ¼ V H DC CH4 þ V S C CH4
ð1Þ
where V CH4 is the volume of methane volume (mL), VH is the head space volume (mL), VS is the biogas volume in syringe, DC CH4 is the variation concentration of CH4 (%). 2.4.2. Other analysis COD was measured according to the Japan Standard Testing Methods for wastewater (JSWA , 1997). VFAs was analyzed by an Agilent-6890 gas chromatograph. All the data showed in the figures are the net value subtracted the blank sample. The four different steps reaction of anaerobic digestion, hydrolysis (Hyd), acidogenesis (Acid), acetogenesis (Acet) and methanogenesis (Meth), were expressed by cumulative hydrolyzed COD, cumulative acidified COD, cumulative acetified COD and methane COD (Neves et al., 2006), and were calculated as follows:
Hyd ¼ ðV CH4 =350 þ C SCOD VÞ COD1 add
ð2Þ
Acid ¼ ðV CH4 =350 þ C TVFA VÞ COD1 add
ð3Þ
Acet ¼ ðV CH4 =350 þ C acetate VÞ COD1 add
ð4Þ
Meth ¼ V CH4 =350 COD1 add
ð5Þ
where Hyd, Acid, Acet and Meth were cumulative hydrolyzed SCOD (%), cumulative acidified COD (%), cumulative acetified COD (%) and methane COD (%), respectively. V CH4 is the volume of methane volume (mL), V is the volume of seed sludge (L), CCODs is the concentration of soluble COD (g/L), CTVFA is the concentration of TVFA (g-COD/ L), Cacetate is the concentration of acetate acid (g-COD/L), CODadd is
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Table 4 n h io e ðt0 tÞ þ 1 . The kinetic parameters of each step in anaerobic digestion obtained from Modified Gompertz model P ¼ P 0 exp exp Rmax P0 Thermophilic
Mesophilic
Hydrolysis
Acidogenesis
Acetogenesis
Methanogenesis
Hydrolysis
Acidogenesis
Acetogenesis
Methanogenesis
Raw coffee
P0 Rmax R2
0.813 ± 0.019 0.091 ± 0.011 0.978
0.715 ± 0.009 0.072 ± 0.004 0.992
0.645 ± 0.008 0.067 ± 0.003 0.994
0.645 ± 0.007 0.068 ± 0.003 0.996
0.838 ± 0.006 0.048 ± 0.001 0.999
0.763 ± 0.004 0.046 ± 0.001 0.999
0.763 ± 0.004 0.046 ± 0.001 0.999
0.763 ± 0.004 0.046 ± 0.001 0.999
Ground coffee
P0 Rmax R2
0.760 ± 0.165 0.125 ± 0.015 0.98
0.650 ± 0.004 0.099 ± 0.004 0.997
0.578 ± 0.004 0.096 ± 0.004 0.997
0.579 ± 0.003 0.097 ± 0.002 0.999
0.804 ± 0.005 0.069 ± 0.002 0.999
0.740 ± 0.003 0.062 ± 0.001 0.999
0.740 ± 0.003 0.062 ± 0.001 0.999
0.740 ± 0.003 0.062 ± 0.001 0.999
Sludge
P0 Rmax R2
0.490 ± 0.012 0.066 ± 0.009 0.962
0.458 ± 0.017 0.047 ± 0.009 0.925
0.482 ± 0.020 0.031 ± 0.004 0.95
0.482 ± 0.020 0.031 ± 0.004 0.951
0.361 ± 0.012 0.021 ± 0.003 0.955
0.358 ± 0.010 0.020 ± 0.002 0.972
0.358 ± 0.010 0.020 ± 0.002 0.972
0.358 ± 0.010 0.020 ± 0.002 0.972
(a')
Raw coffee (Thermophilic) 1 0.8 0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
0.2
Acid. Meth. Simulated Acid. Simulated Meth.
Raw coffee (Mesophilic) 1 0.8
g-COD/g-added COD
g-COD/g-added COD
(a)
0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
0.2
0
0 0
5
10
15
20
25
30
35
0
40
5
10
15
0.8 0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
Acid. Meth. Simulated Acid. Simulated Meth.
g-COD/g-added COD
g-COD/g-added COD
(b')
Ground coffee (Thermophilic) 1
0.2
30
35
40
Ground coffee (Mesophilic) 1
0.6 0.4 Hyd. Acet. Simulated Hyd. Simulated Acet.
0.2
Acid. Meth. Simulated Acid. Simulated Meth.
0 0
5
10
15
20
25
30
35
0
40
5
10
Sludge (Thermophilic)
(c')
1 Acid. Meth. Simulated Acid. Simulated Meth.
g-COD/g-added COD
Hyd. Acet. Simulated Hyd. Simulated Acet.
0.8
15
20
25
30
35
40
Time (days)
Time (days)
g-COD/g-added COD
25
0.8
0
(c)
20
Time (days)
Time (days)
(b)
Acid. Meth. Simulated Acid. Simulated Meth.
0.6 0.4 0.2 0
Sludge (Mesophilic) 1 Hyd. Acet. Simulated Hyd. Simulated Meth.
0.8 0.6
Acid. Meth. Simulated Acid. Simulated Acet.
0.4 0.2 0
0
5
10
15
20
25
Time (days)
30
35
40
0
5
10
15
20
25
30
35
40
Time (days)
e Fig. 2. Description of the 4 stages in anaerobic digestion by modified Gompertz model ðP ¼ P 0 exp exp Rmax ðk tÞ þ 1 Þ under thermophilic condition and mesophilic P condition. Symbols refer to the experimental data and lines to the simulation of the modified Gompertz model.
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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Concentration (mg-COD/L)
(a)
where P is the conversion ratio of substrates (gCOD/g-added COD), P0 is the conversion potential (gCOD/g-added COD), Rmax is the maximum conversion rate (gCOD/g-added COD d), t0 is the lag time (days) and e = 2.718281828. The constants P0, k, and t0 were estimated by a non-linear fitting program using the Original 8.5. This model was used by Lay et al. (1998) to describe the bacterial growth in a batch culture. In this study, simulations of both the batch experiment and methanogenic activity test were made using this model to obtain the kinetic parameters.
Raw coffee
600
Acetate Propionate Butyrate Valeric
500
5
400
300
200
100
3. Results and discussion 0 0
5
10
15
20
25
3.1. Effect of temperature on different steps in anaerobic digestion
30
Time (days)
Concentration (mg-COD/L)
(b)
Ground coffee
600
Acetate Propionate Butyrate Valeric
500
400
300
200
100
0 0
5
10
15
20
25
30
Time (days)
(c)
Sludge
600
Acetate Propionate Butyrate Valeric
Concentration (mg-COD/L)
500
400
300
200
100
0 0
5
10
15
20
25
30
Time (days) Fig. 3. Variation of VFA in anaerobic digestion under thermophilic condition.
the COD of substrates added into the bottles (g), 1 g COD equaled 350 mL CH4. Methanogenic activity was determined by the following equation:
Activ ity ¼ Rmax =S
ð6Þ
where Activity is the methanogenic activity (gCH4-COD/gVSS d), Rmax is the maximum methane production rate (gCH4-COD/d), and S is the amount of sludge (gVSS). 2.4.3. Kinetic models The experimental data of each step in the batch experiment and activity test was simulated by the modified Gompertz equation:
Rmax e P ¼ P0 exp exp ðt0 tÞ þ 1 P0
ð7Þ
The kinetic characteristics of anaerobic digestion were not the same under thermophilic and mesophilic conditions due to the different metabolism pathways and bacterial communities. Due to the contradiction between the traditional theory that hydrolysis is the rate-limiting step of anaerobic digestion and also the reason for the accumulation of VFAs under the thermophilic condition, the four steps of anaerobic digestion are discussed separately in order to determine the effects of temperature on the each step. According to the comparison between the maximum reaction rates (Rmax) and production potential (P0) obtained from the modified Gompertz model (Table 4 and Fig. 2), the effects of temperature on each step in the digestion of raw coffee and ground coffee were similar and irrelevant to the particle size of the substrates. Hydrolysis is the first step in anaerobic digestion. During hydrolysis, complex organic waste is hydrolyzed to monomers such as amino acids, sugars and fatty acids. Since hydrolysis potential directly affects the degradability of solid waste, it is always considered as the rate-limiting step in the anaerobic digestion of complex organic waste. As shown in Table 4, the hydrolysis rate of coffee grounds in the thermophilic condition was almost double what it was under the mesophilic condition. The higher hydrolysis potential of coffee grounds in both the mesophilic and thermophilic conditions may be attributable to the higher degradability of carbohydrates, which is the main component of coffee grounds. Even under the mesophilic condition, the degradation efficiency of coffee grounds was around 80%. For WAS, thermophilic digestion not only accelerated the reaction rates, but also increased the hydrolysis potential from 37.0% to 49.2%. In the acidogenesis stage, the hydrolysate (Soluble COD) was converted to VFA by acidogenic bacteria. The maximum acidification rate of coffee grounds under thermophilic condition was higher than that of the mesophilic condition by a factor of 1.56, but no significant increase in acidification potential was observed. Meanwhile the maximum acidification rate of WAS increased from 0.020 g-COD/g-added COD d1 to 0.047 g-COD/g-added COD d1, more than double. In addition to this, the acidification potential also increased from 35.8% to 45.8%. In the acetogenesis stage, all the C3–C6 VFAs needs to be converted to acetate before converting to methane. If the production rate exceeds the degradation rate, VFA will accumulate. Under the mesophilic condition, Rmax(ace) = Rmax(aci), indicating that all the VFAs was properly converted to acetate by acetogens, and was subsequently converted to methane by methanogens, leaving no VFAs. However, because Rmax(ace) < Rmax(aci) under the thermophilic condition, VFAs accumulation was observed, and propionic acid was found to be the main component of the VFAs, as shown in Fig. 3. This result agrees with the findings of Fernandez and Forster (1993) and Dinsdale et al. (1997), who reported that the metabolism of propionic acid by acetogens is lower under the thermophilic condition. Although the acetogenesis rate is accelerated in the thermophilic condition, the acetogenesis potential was
Please cite this article in press as: Li, Q., et al. Kinetic characterization of thermophilic and mesophilic anaerobic digestion for coffee grounds and waste activated sludge. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2014.11.016
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1000mg/L 2000mg/L 3000mg/L 5000mg/L 8000mg/L 10000mg/L
150
Mesophilic (acetate)
(a') 250
Thermophilic (acetate)
200
Methane production (mL)
Methane production (mL)
(a)
100
50
0
1000mg/L 3000mg/L 5000mg/L 8000mg/L 10000mg/L 20000mg/L
200 150 100 50 0
0
1
2
3
4
5
0
6
2
4
Time (days)
90
8
10
Mesophilic (propionate)
(b') 200
Thermophilic (propionate) 500mg/L 1000mg/L 1500mg/L 2000mg/L 3000mg/L 5000mg/L
Methane production (mL)
Methane production (mL)
(b) 120
6
Time (days)
60
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0
500mg/L 1000mg/L 1500mg/L 2000mg/L 3000mg/L 5000mg/L
160 120 80 40 0
0
5
10
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40
Time (days)
0
2
4
6
8
10
12
14
16
Time (days)
Fig. 4. Methane production of different acetate and propionate concentration under thermophilic and mesophilic condition.
Table 5 Kinetic parameters of methanogenic activity test obtained from modified Gompertz model. Concentration (mg/L)
Thermophilic
Mesophilic
Rmax (mL/d)
t0 (d)
Rmax (mL/d)
t0 (d)
Acetic acid 1000 2000 3000 5000 8000 10,000 20,000
23.6 38.2 44.6 55.3 57.4 55.1 –
0.112 0.180 0.232 0.312 0.358 0.359 –
36.7 – 38.5 43.3 41.8 36.0 2.50
0.613 – 0.470 0.633 0.883 0.878 0.846
Propionic acid 500 1000 1500 2000 3000 5000
3.01 5.89 8.04 11.6 17.91 –
18.0 20.1 20.5 24.1 26.6 –
8.02 10.7 14.5 18.9 24.7 25.7
1.32 2.59 2.57 3.34 4.22 5.91
found to decrease due to the accumulation in C3–C6 VFAs, which could not be degraded until the end of batch experiment in the case of coffee grounds digestion. For WAS, the thermophilic condition was found to increase the acetogenesis potential, since the accumulated VFA was degraded effectively in the latter half of anaerobic digestion, as shown in Fig. 3c. In the methanogenesis stage, acetate was completely converted to methane in both the thermophilic and mesophilic conditions. Therefore, the methane production potential depend on the part of organic wastes which could be converted to acetate. For both raw coffee and ground coffee, the methane production potential decreased by more than 15% under thermophilic condition due to VFAs accumulation, whereas it increased from 35.8% to 48.2% when using a WAS substrate.
A comparison of the Rmax and P0 in each stage revealed that one of the benefits of the thermophilic condition was an increased reaction rate, a notably higher hydrolysis potential, but that the methane production potential depended on the physicochemical properties of the substrates.
3.2. Effects of substrates on reaction rate The kinetic characteristics of organic waste degradation not only depended on temperature, but were also affected by the physicochemical properties of the substrates. The order of Rmax under both the thermophilic and mesophilic conditions in each stage was as follows: Rmax (ground coffee) > Rmax (raw coffee) > Rmax (WAS). For raw coffee and ground coffee, reducing the particle size increased the Rmax of hydrolysis from 0.048 to 0.069 g-COD/g-added COD d1 under the mesophilic condition, and from 0.091 to 0.125 g-COD/g-added COD d1 under the thermophilic condition. Smaller particle sizes increase the available specific surface, which leads to a higher reaction rate in anaerobic digestion. By reducing the particle size, the reaction rate at each stage of the anaerobic process was increased by more than 35%. The main component of coffee grounds and WAS are carbohydrates and protein, respectively. Generally, carbohydrates hydrolyze under the anaerobic condition at a faster rate than protein (Pavlostathis and Giraldo-Gomez, 1991). Due to the inherently low degradability of sludge, the methane production potential was still lower than 50% even under thermophilic condition. Because the main component of coffee grounds is carbohydrates, the reaction rate was higher than for WAS, which has protein as its main component. Still, the accumulation of VFAs (notably propionic acid) in thermophilic anaerobic digestion resulted in a lower methane production potential.
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Thermophilic 0.15
(a') Methanogenic activity (gCH4-COD/gVSS·d)
Methanogenic activity (gCH4-COD/gVSS·d)
(a)
0.12 0.09 0.06 0.03
0.15 0.12 0.09 0.06 0.03 0
0
Acetate concentration (g/L)
0.1
Thermophilic
(b') Methanogenic activity (gCH4-COD/gVSS·d)
Methanogenic activity (gCH4-COD/gVSS·d)
(b)
Acetate concentration (g/L)
0.08 0.06 0.04 0.02 0
0.1
Mesophilic
0.08 0.06 0.04 0.02 0
Propionate concentration (g/L)
Propionate concentration (g/L)
Fig. 5. Variation of methanogenic activity with acetate/propionate concentration under thermophilic and mesophilic condition.
3.3. VFAs degradation and methanogenic activity In order to elucidate the effects of temperature on the degradation of propionic acid, and acetic acid, a methanogenic activity test was conducted using acetate and propionate as the substrates, respectively. The main reason for only investigating the methanogenic activity of acetate and propionate are as follows: (1) acetate is the final product of acetogenesis, and all the VFAs needs to be converted to acetate so that it can be converted to methane; (2) propionic acid is the main component of VFAs in this study and has a higher inhibitory effect on methanogenic bacteria than other types of VFAs. The curve shown in Fig. 4 was obtained from experimental data simulation by the modified Gompertz model, and the kinetic parameters are given in Table 5. Under both the thermophilic and mesophilic conditions, the lag time of acetate was less than one day. However, under the thermophilic condition, the lag time for propionate increased along with the increase in the propionate concentration and was longer than 17 days even when the propionate concentration was only 500 mg/L. It was clear that propionate degradation was strongly inhibited by high temperature, resulting in a longer lag time and a lower degradation rate. The main reason might be the lower ability of acetogens which could convert propionic acid to acetic acid. The variations of methanogenic activity with acetate/propionate concentration are shown in Fig. 5 and Table 6. Methanogenic activity under the mesophilic condition was a little higher than under the thermophilic condition when the concentration of acetic acid was lower than 3000 mg/L. This is consistent with the findings of a previous study by Isa et al., 1993, who also reported higher methanogenic activity under the mesophilic condition. For propionic acid, methanogenic activity was significantly lower under the thermophilic condition and completely inhibited when the concentration of propionate was higher than 5000 mg/L. The lower methanogenic activity and longer lag time of propionic acid degradation under thermophilic condition may be sufficient to explain the ease with which propionic acid accumulated in the thermophilic condition. In this study, despite the tendency for propionic acid to accumulate, it could be degraded when the concentration was not too high, as shown in Fig. 4. The degradation of propionic acid differed
Table 6 Variation of methanogenic activity with substrate concentration under thermophilic and mesophilic condition. Concentration (mg/L)
Methanogenic activity (gCH4-COD/gVSS d) Thermophilic
Mesophilic
Acetate 1000 2000 3000 5000 8000 10,000 20,000
0.058 0.094 0.109 0.135 0.140 0.135 –
0.102 – 0.108 0.120 0.115 0.099 0.007
Propionate 500 1000 1500 2000 3000 5000
0.007 0.014 0.020 0.028 0.044 0
0.022 0.030 0.040 0.052 0.068 0.071
according to the substrate. As shown in Fig. 3c, the concentration of propionic acid in WAS anaerobic digestion increased to the maximum within the first 9 days, then decreased sharply. However, a different phenomenon was observed in the thermophilic digestion of coffee grounds (Fig. 3a and b), where the propionic acid persisted during thermophilic digestion until the end of the batch experiment. The concentration of propionic acid was maintained below 500 mg-COD/L, which was a little higher than that in WAS digestion, but did not reach the inhibition threshold. The differences of propionic acid degradation kinetics between coffee grounds and WAS might be related to the different physicochemical properties of these substrates. The presence of inhibitory compounds contained in the coffee grounds was most likely the main reason for the inhibition of propionic acid degradation under the thermophilic condition, as has been reported in earlier studies. Fernandez and Forster (1993) reported that coffee waste contained some components which inhibited anaerobic digestion in the thermophilic temperature range. Neves et al. (2006) also found that the decreased methane yield was likely due to the intermediates formed during the hydrolysis step. The higher lipid content of cof-
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fee grounds (0.24 g/g-TS) also has an inhibitory effects in the anaerobic process due to the accumulation of long-chain fatty acids resulting from the hydrolysis of neutral lipids (Labatut et al., 2014). In this study, although there was no direct evidence indicating the inhibitory compounds, there was a clear and significant negative effect on propionic acid degradation and methane production during the thermophilic digestion of coffee. 3.4. Rate-limiting step determination Anaerobic digestion was considered a multi-step process, with the overall reaction rate depending on the slowest reaction, which is called the rate-limiting step (Hill and Barth, 1977). Theoretically, hydrolysis is considered the rate-limiting step in anaerobic digestion, but actually, the main problem in thermophilic digestion is the accumulation of VFAs. That is, it was concluded that hydrolysis cannot be considered the rate-limiting step in anaerobic digestion without first taking the effects of temperature and substrates into account. However, the focus of many researchers is still on the hydrolysis step, and how to enhance anaerobic digestion performance by improving the hydrolysis rate (Ristow et al., 2006; Ge et al., 2011). From this perspective, the effects of temperature on the determination of rate-limiting step in anaerobic digestion with different substrates need to be re-evaluated. In this study, the modified Gompertz model was used to simulate the four steps in anaerobic digestion using Origin 8.5, to determine the kinetic parameters shown in Table 4. The rate-limiting step was determined by comparing the Rmax obtained from modified Gompertz model at each stage in the digestion process. The rate-limiting step for the anaerobic digestion of raw coffee under the mesophilic condition was in this order: Rmax(hyd.) > Rmax(Acid.) = Rmax(Ace.) = Rmax(meth.), which meant that acidogenesis was the rate-limiting step. Under the thermophilic condition, the rate-limiting step was in this order: Rmax(hyd.) > Rmax(Acid.) > Rmax(Acet.) = Rmax(meth.). Because the accumulated VFAs could not be proportionally converted to acetate, methane production was affected, indicating that acetogenesis was the rate-limiting step. A similar conclusion was also obtained for ground coffee. Although reducing the particle size increased the reaction rate, it did not affect the rate-limiting step determination. For WAS, the thermophilic condition significantly enhanced the hydrolysis step and the methane yield. The order of the rate-limiting step for WAS under the thermophilic condition was as follows: Rmax(hyd.) > Rmax(Acid.) > Rmax(Acet.) = Rmax(meth.), indicating that acetogenesis was the rate-limiting step under the thermophilic condition. Under the mesophilic condition, due to the same reaction rate in each stage, the order of rate-limiting step was as follows: Rmax(hyd.) > Rmax(Acid.) > Rmax(Acet.) = Rmax(meth.). Hydrolysis was the rate-limiting step. The rate-limiting step was affected by both temperature and substrates. If hydrolysis is considered the rate-limiting step in all case of anaerobic digestion, it would result in problems. For coffee grounds, especially under thermophilic conditions, it was found that even though C3–C6 VFAs could not be normally converted to acetate, the VFAs concentration reached a limit and caused system failure when the hydrolysis rate was enhanced. In the case of WAS. However, a higher hydrolysis rate was found to be beneficial. Therefore, it is important to know the rate-limiting step of the anaerobic digestion process in order to optimize and control the process. 4. Conclusions 1. Thermophilic digestion was beneficial for increasing the methane production rate from WAS, coffee grounds and raw coffee grounds. However, under high temperature, methane produc-
tion potential when using coffee grounds decreased by around 15% compared to the mesophilic condition due to the accumulation of propionic acid. 2. Under the thermophilic condition, acetogenesis was the rate-limiting step for both coffee grounds and WAS. Under the mesophilic condition, acidogenesis and hydrolysis were the rate-limiting steps for coffee and sludge, respectively. 3. Although reducing the particle size of coffee grounds accelerated the reaction rate of acetogenesis and methanogenesis, the accumulation of propionate under thermophilic condition resulted in a lower methane production potential. 4. The lag time for propionate degradation under the thermophilic condition was almost 18-days longer under than mesophilic condition, but, the lag time was decreased by the thermophilic condition in the case of acetate degradation.
Acknowledgements This work was partially supported by Japan Society for the Promotion of Science (24-02053) and Key Laboratory for Solid Waste Management and Environment Safety, Ministry of Education of China (SWMES 2011-04). The overseas study conducted by the first author was finically supported by China Scholarship Council (CSC).
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