Appl Biochem Biotechnol DOI 10.1007/s12010-014-0941-z
Dry Anaerobic Co-digestion of Cow Dung with Pig Manure for Methane Production Jianzheng Li & Ajay Kumar Jha & Tri Ratna Bajracharya
Received: 8 February 2014 / Accepted: 21 April 2014 # Springer Science+Business Media New York 2014
Abstract The performance of dry anaerobic digestions of cow dung, pig manure, and their mixtures into different ratios were evaluated at 35±1 °C in single-stage batch reactors for 63 days. The specific methane yields were 0.33, 0.37, 0.40, 0.38, 0.36, and 0.35 LCH4/gVSr for cow dung to pig manure ratios of 1:0, 4:1, 3:2, 2:3, 1:4, and 0:1, respectively, while volatile solid (VS) and chemical oxygen demand (COD) removal efficiencies were 48.59, 50.79, 53.20, 47.73, 46.10, and 44.88 % and 55.44, 57.96, 60.32, 56.96, 53.32, and 50.86 %, respectively. The experimental results demonstrated that the co-digestions resulted in 5.10–18.01 % higher methane yields, 2.03– 12.95 % greater VS removals, 2.98–12.52 % greater COD degradation and so had positive synergism. The various mixtures of pig manure with cow dung might persuade a better nutrient balance and dilution of high ammonia concentration in pig manure and therefore enhanced digester performance efficiency and higher biogas yields. The dry co-digestion of 60 % cow dung and 40 % pig manure achieved the highest methane yield and the greatest organic materials removal efficiency than other mixtures and controls. Keywords Dry anaerobic digestion . Co-digestion . Manures . Biogas . Organic materials removal Introduction The continuously increasing demand for renewable energy sources such as methane renders anaerobic digestion to one of the most promising methods for stabilization of organic wastes and green energy production from biogas combustion [1, 2]. The anaerobic digestion technology produces less greenhouse gases than other waste treatment techniques like incineration [3], composting [4], and land filling [5]. The anaerobic digestion process follows hydrolysis, J. Li (*) : A. K. Jha State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, the People’s Republic of China e-mail: [email protected] A. K. Jha e-mail: [email protected] A. K. Jha : T. R. Bajracharya Pulchowk Campus, Institute of Engineering, Tribhuvan University, Lalitpur, Nepal T. R. Bajracharya e-mail: [email protected]
Appl Biochem Biotechnol
acidogenesis, acetogenesis, and methanogenesis [6]. In an oxygen-free environment, anaerobic microbes—such as methanogenic bacteria, acetogenic bacteria, and fermentative bacteria— digest biodegradable matter into biogas with 50–75 % methane as potential energy content, 25–40 % carbon dioxide, and other gases in small amount. De Baere [7], Jha et al. [8], and Pavan et al. [9] noted the following advantages of dry anaerobic digestion when compared to liquid anaerobic digestion: higher biogas yield per unit volume of digester, greater organic loading rate, lower energy requirements for heating, reduced nutrient run off during storage and distribution of residues, limited environmental consequences, and energetically effective performance. Mainly due to its reduced cost in digesters and easier slurry handling, the dry anaerobic digestion process has attracted increasing attention all around the world recently. However, the high-solid anaerobic digestion is known to suffer from many inhibition problems [10]. The major disadvantages of the dry anaerobic digestion are the requirement of larger amount of inocula and much longer retention time [11]. Jha et al. [12] have presented that the dry methane fermentation of cow manure took relatively longer retention time than wet fermentation to produce same amount of biogas while Fongsatitkul et al. [13] reported that the specific gas production from organic fraction of municipal waste steadily dropped with increase in total solid of the feedstocks. Furthermore, dry anaerobic digestion exhibits a poor start-up performance, while the conversion of acetate to methane is generally considered as rate limiting due to slow growth of methanogens [14]. Also, the accumulation of volatile fatty acids (VFAs) is known to restrict the biogas yield [15]. Moreover, complete mixing is difficult to achieve. Hence, this technology needs enhancement of reliability in operation to become more sustainable [16]. An option for significantly improving yields of anaerobic digestion of solid wastes is the codigestion of multiple substrates [17–19]. Co-digestion enhances the methane yield due to positive synergisms established in the digestion medium, bacterial diversities in different wastes, and the supply of missing nutrients by the co-substrates. Animal manures contain rumen microorganisms that assist to carry out anaerobic digestion process faster, and cattle manure-based biogas plants are successful in the rural area of many developing countries, but they are affected due to the continuous increasing scarcity of feedstocks. The co-digestion process can assist to solve the feedstock scarcity dilemma. Moreover, the addition of other biomass products increases biogas yield and makes the biogas plants economically profitable. However, although the cow dung and pig manure have good biogas potential, the biodegradation of pig manure as sole substrate is inhibited by presence of high ammonia concentration [20, 21]. The mixing of pig manure with cow dung reduces the ammonia concentration in the homogeneous mixture, and their simultaneous digestion might provide additional energy. The wet biomethanation process of the mixture of different wastes is relatively well understood and documented; however, limited research reports were found about the dry anaerobic co-digestion of organic wastes including the codigestion of cow dung with pig manure. The aim of this study was to assess the feasibility of dry anaerobic co-digestion of cow dung, pig manure, and their mixtures using single-stage batch digesters under mesophilic condition. Biogas and methane yields, chemical oxygen demand (COD) and volatile solid (VS) removal efficiency, and VFAs and ammonia-nitrogen accumulation and degradation are considered for comparisons.
Materials and Methods Experimental Setup and Procedure The experiments were carried out in single-stage batch lab reactors. Each bioreactor has 2.5-L effective volume with an internal diameter of 13 cm and height of 25 cm. The capped reactors
Appl Biochem Biotechnol
were kept in a water bath of operational temperature 35±1 °C, the optimum temperature for mesophilic range. Each reactor was fitted with four ports (Fig. 1). The two ports were fitted on the cover while other two ports were fitted on the side. One of the cover ports was used for measuring biogas production. The sample for analysis of biogas quality was also taken out from the same port. The other cover port was set aside as spare. One of the side ports was kept above 5 cm from the bottom. This port was used to take out the sample for the analysis of various parameters while pH meter was set up at the other side port. The samples were stored at −4 °C in a freezer before analysis. The analysis was generally performed within 1 week. In the fermentation process, the substrates were pretreated and fed into air-tight digester under specified environmental conditions for 63 days without dilution. Pretreatment means separation of manures from foreign materials like stones, woods, metals, and other inorganic materials and the addition of inoculants into the feedstocks. The visible straw and feathers were removed by hand. Each digester was purged with nitrogen for 15–20 min to create complete anaerobic environment. The contents of the reactors were slowly shaken once daily for 2–3 min to create homogeneous substrate preventing stratification and formation of a surface crust and distributing microorganisms throughout the digester. Characteristics of Feedstocks The average values of the physicochemical characteristics of the manures and inoculum are presented in Table 1. The manures were obtained from the livestock farms of Harbin, China. The digested slurry from the previous dry anaerobic digestion experiment of cow dung was utilized as inoculum to provide the initial population of methane bacteria for initiating digestion in the system [22]. The digesters, R1–R6, were filled with the cow dung and pig manure in the ratios of 1:0, 1:4, 3:2, 2:3, 4:1, and 0:1, respectively, on wet-weight basis along with inoculum (Table 2). No other nutrients, chemicals, or water was fed into the reactors. The experiments were performed in triplicate, and the average values were reported. Analytical Methods The physicochemical parameters analyzed were temperature, pH, total solid (TS), VS, COD, soluble chemical oxygen demand (SCOD), VFAs, total phosphorus (TP), total Kjeldahl nitrogen (TKN), ammonia nitrogen, and free ammonia. All the analytical determinations were performed according to the standard methods [23]. All the tests were conducted in triplicate, and mean values were reported. The pH of the mixtures was measured with a digital pH meter (Seven Multi SK40, Switzerland). The free ammonia was calculated using the previously Biogas
Reactor
Cylinder
Thermostat sensor
Biogas outlet
pH meter
Water bath
Fig. 1 Schematic diagram of the reactor
Acidified water
Appl Biochem Biotechnol
Table 1 Characteristics of substrates and inoculum
Parameters
Cow dung
Pig manure
Inoculum
pH
7.76
7.82
7.83
Total solids (%) Volatile solids (% of TS)
15.98±0.2 84.24±0.2
18.96±0.3 77.42±0.2
10.55 59.18
COD (g/L)
155.44±5
164.15±4
69.18
Soluble COD (g/L)
68.97±2
75.44±2
15.62
Total organic carbon (g/L)
40.32±2
43.13±2
11.72
Total phosphorus (g/L)
1.38±0.2
1.77±0.2
1.19
Total Kjeldahl nitrogen (g/L)
2.96±0.2
4.75±0.3
2.11
Ammonia concentration (g/L)
1.53±0.1
2.28±0.2
1.22
Free ammonia (g/L)
0.09
0.17
0.12
reported formula [24]. The yielded biogas was measured per day by downward water displacement method at atmospheric pressure using a calibrated 1- or 2-L cylindrical jar for each reactor. The constituents (CH4, CO2, and H2) of the biogas were determined using gas chromatography (SP-6800A, Shandong Lunan Instrument Factory, China) equipped with a thermal conductivity detector and a 2-m stainless column packed with Porapak TDS201 (60–80 mesh). Nitrogen was employed as the carrier gas at a flow rate of 40 mL/min. The operation temperatures for the injection port, oven, and detector were all 80 °C. The cumulative methane production for each test was determined by summing daily methane production, which was calculated by timing daily biogas production with corresponding methane content. The samples taken from the batch culture reactor were centrifuged at 6,000 rpm for 15 min and then acidified with hydrochloric acid and filtered through a 0.2-μm membrane for the analysis of VFAs and ethanol. The concentrations of the VFAs and ethanol were determined using an another gas chromatograph (SP6890, Shandong Lunan Instrument Factory, China) equipped with a flame ionization detector and a 2-m stainless (5 mm inside diameter) column packed with Porapak GDX-103 (60/80 mesh). The operational temperatures of the injection port, the column, and the detector were 220, 190, and 220 °C, respectively. Nitrogen was used as carrier gas at a flow rate of 50 mL/min. Microbial Community Analysis Genomic DNA of the sludge samples was extracted using a DNA extraction kit (MO Bio Laboratories, Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. Extracted DNA was dissolved in 60-μL 1×TE buffer solution. The V3 and V4 regions of 16S ribosomal Table 2 Composition and condition of the reactors Reactors
Cow dung (g)
Pig manure (g)
Inoculant (g)
pH
TS (%)
VS (% TS)
R1
1,000
0
200
7.77
15.11±0.2
81.28±0.2
R2
800
200
200
7.79
15.64±0.2
79.39±0.15
R3
600
400
200
7.80
16.17±0.3
78.18±0.2
R4
400
600
200
7.81
16.54±0.2
77.91±0.2
R5
200
800
200
7.80
17.13±0.3
76.02±0.2
R6
0
1,000
200
7.82
17.55±0.3
75.52±0.25
Appl Biochem Biotechnol
RNA (rRNA) were amplified by PCR using universal bacterial primers (341F, 5′-CCTACG GGAGGCAGCAG-3′ with a GC clamp and 907R, 5′-CCGTCAATTCMTTTGAGTTT-3′) and universal archaeal primers (344F, 5′-ACGGGGYGCAGCAGGCGCGA-3′ with a GC clamp and 915R, 5′-GTGCTCCCCCGCCAATTCCT-3′). The PCR amplification was conducted in a 50-μL system containing 5 μL 10×Ex Taq buffer, 4 μL dNTP mixture (2.50 mM), 1 μL forward primer (20 μM), 1 μL reverse primer (20 μM), 2.5 ng DNA template, and 0.15 U Ex Taq DNA polymerase (Takara, Dalian, China), using a thermal cycler (model 9700; ABI, Foster, CA, USA), started with an initial denaturation of DNA for 10 min at 94 °C, followed by 30 cycles for 1 min at 94 °C, 30 s at 55 °C (decreasing by 0.10 °C per cycle to 52 °C), and 1 min at 72 °C; final extension was 10 min at 72 °C. The PCR products were separated using the Dcode™ universal mutation detection system (Biorad Laboratories, Hercules, CA, USA). Polyacrylamide gels with 40–60 % vertical denaturing gradient were prepared. The 10-μL PCR products were loaded and electrophoresed at 120 V and 60 °C for 10 h. Gels were stained silver as described in the previous research [25]. All DGGE bands were excised and dissolved in 30 μL 1×TE at 40 °C for 3 h and then centrifuged at 12,000 rpm for 3 min. The 3-μL supernatant was used as the template and conducted PCR amplification under the conditions as described above using the same primers. The PCR products were pured by Gel Extraction Mini Kit (Watson Biotechnologies, Inc, China) and ligated into pMD18-T vector (Takara, Dalian, China), and then cloned into Escherichia coli DH5α. Some white clones from each sample were randomly selected for PCR detection, and positive clones were selected for sequencing by ABI3730, and partial 16S rRNA gene sequences were analyzed using the BLAST program in GenBank.
Results and Discussion Evaluation of pH, NH3-N, and VFAs Figure 2 illustrates the evolution of pH, NH3-N, and VFAs in average for each six types of the functional reactors during the digestion period. The pHs of cow dung and pig manure were initially around 7.76 and 7.82, respectively. The pH variation pattern was observed similar for all the tests. It was decreased to 6.74, 6.80, 6.78, 6.81, 6.75, and 6.78 in 14, 14, 14, 21, 21, and 21 days after the beginning of the digestion process for the reactor types R1–R6, respectively (Fig. 2a). It happened due to the increase in VFAs production by acidogenic bacteria during the start-up phase of each experiment. The easily digestible fraction of organic matter was hydrolyzed and converted to fatty acids rapidly. The pH value did not drop off much lower because the substrates were able to buffer themselves and prevent the acidification occurrence due to proper alkalinity of cow dung and pig manure to maintain optimal biological activity and stability of the anaerobic digestion system. The pH value for all the experiments began to rise gradually as the VFAs were consumed by methanogens and transferred to the methane. The biogas process becomes more sensitive towards increase of pH value because the concentration of free ammonia increases as pH value raises [26]. The ammonia concentration for cow dung was noted 1.53 and 2.28 g/L for pig manure. In this study, the accumulation of ammonia has been increased to some extent due to the hydrolysis of amino acids and proteins during the start-up period. Afterwards, the concentration of ammonia was decreased since it was used as nitrogen source for methanogen growth. It was again increased since the protein-containing hard biodegradable fraction began to hydrolyze after some days of the beginning of digestion. As a result, fluctuated ammonia variation patterns were observed for all the tests during the digestion period (Fig. 2b). It can be noted
Appl Biochem Biotechnol
a
7.90 7.70
pH
7.50 7.30 7.10
R1
R2
R3
R4
R5
R6
6.90 6.70
0
Ammonia nitrogen (g/L)
2.60
7
14
7
14
21
28
35
42
49
56
63
b
2.40 2.20 2.00 1.80 1.60 1.40 1.20
0
21
28
35
42
49
56
63
Time (d)
Fig. 2 Evolution of a pH and b ammonia nitrogen
that the biodegradation of pig manure and the mixtures was partially inhibited due to the presence of high ammonia concentration because the ammonia inhibition has been observed to commence at concentrations of 1.50–2.50 g/L [20]. In this study, average calculated free ammonia for bioreactor types R1–R6 were determined in the range of 0.02–0.55, 0.02–0.58, 0.02–0.66, 0.03–0.71, 0.03–0.82, and 0.03–0.87 g/L, respectively. Furthermore, Angelidaki and Ahring [27] reported that the existence of high content of the free ammonia is toxic for biogas processes because the specific growth rate of methanogenic bacteria is a function of the free ammonia concentration and decreases as the concentration of ammonia increases [26]. The growth of acetate-utilizing bacteria has been started to inhibit from 0.1 to 0.15 g/L [28] while H2-utilizing methanogenic bacteria are inhibited at free ammonia concentrations higher than 1.20 g/L [26]. It indicated that the process was partially inhibited for the acetate-utilizing bacteria. It was also noted that the inhibition was more in pig manure than cow dung and their mixtures. Volatile fatty acids are usually produced due to the degradation of the complex organic polymers during hydrolysis and acidogenic stages. The conversion of intermediate products— VFAs—has been treated as an indicator of the digestion efficiency, but the high concentration of VFAs results in decrease in pH, acidification, and destructions/demolish activity of methanogenic bacteria, leading to failure of digester ultimately. In this study, the average highest values of VFAs for the reactor types R1–R6 were determined 8.20, 7.89, 8.06, 7.50, 7.66, and 7.58 g/L, respectively. All the reactors showed high VFA concentrations in the start-up phase (Fig. 3) due to higher acidogenesis and lower methanogenic activities. The higher concentration of VFAs during the start-up period results in the increasing amount of ammonia within the limits having no significant influence on the hydrolysis and acidogenesis. The principal volatile acids formed were acetic, butyric, and propionic acids. Acetic acid was the dominant VFAs. Moreover, the acetic acid production rate was apparently higher than the acetic acid consumption rate during the start-up period. The share of propionic and butyric acids was
9 8 7 6
R1
5 4 3 2 1 0 7
14
21
28
9 8 7 6 5 4 3
35
42
49
56
9 8 7 6 5 4 3 2 1 0
R2
0
63
7
14
21
28
35
42
49
56
63
42
49
56
63
28 35 42 Time (d)
49
56
63
8 R3
VFAs & ethanol (g/L)
VFAs & ethanol (g/L)
0
2 1 0
7
R4
6 5 4 3 2 1 0
0
7
14
21
9 8 7 6 5 4 3 2 1 0
28
35
42
49
56
0
63
7
14
21
28
35
8 R5
VFAs & ethanol (g/L)
VFAs & ethanol (g/L)
Ethanol Acetic Propionic Butric Total
VFAs & ethanol (g/L)
VFAs & ethanol (g/L)
Appl Biochem Biotechnol
R6
7 6 5 4 3 2 1 0
0
7
14
21
28 35 42 Time (d)
49
56
63
0
7
14
21
Fig. 3 VFAs and ethanol variation pattern
observed low because of the sufficient propionate- and butyric-degrading syntrophs which could rapidly convert propionic acid and butyric acid into acetic acid [29]. The degradation of propionate and butyrate by syntrophic acetogenic bacteria (e.g., Syntropher wolinii, syntrophomonas wolfei) produced acetic acid that was subsequently degraded into methane and CO2 by acetoclastic methanogens [29]. During methanogenic stage, the methanogens were in exponential growth phase, and the acetic acid consumption rate was higher though hydrolysis and acidogenesis were still going on. The consumption rate of propionate was observed slower than that of butyrate because the oxidation of propionate to acetate is relatively more difficult. Therefore, as methanogenesis and methane gas yield have been increased, the VFA concentrations were decreased. The residual total VFA was observed highest in the reactor with pig manure followed by reactors with their mixtures and lowest in the reactor with cow dung. VFAs and alkalinity together are good indicators for evaluating the process stability of the anaerobic reactor. Figure 4 shows the variation in total VFA to alkalinity ratio. The ratio varied
Appl Biochem Biotechnol
VFAs/alkalinity
0.80 R1 R3 R5
0.60
R2 R4 R6
0.40 0.20 0.00
0
7
14
21
28 35 Time (d)
42
49
56
63
Fig. 4 VFAs to alkalinity ratio variation pattern
below 0.80, and the process seemed stable [30]. No accumulation of VFAs and no drastic fall in pH also support that the process was not inhibited extensively. Biogas Yield and Organic Material Removal Figure 5 presents the daily biogas yield, percentage methane content, and cumulative methane production in the operational reactors R1–R6 during the digestion period. In this study, similar trends of daily biogas and methane yields were observed for all the tests. The biogas generation was started after seeding, kept increasing until reaching the peak, and then began to decline. The initial biogas production was due to readily biodegradable organic matter in the substrates and presence of methanogens. The reactor with cow dung alone generated higher biogas and methane than those of co-digestion mixtures and pig manure during the start-up phase and obtained a peak value (2.13 L biogas with 1.29 L methane) promptly on day 15. The difference in obtaining peak values was occurred because the cow dung was not significantly inhibited due to existence of lower ammonium nitrogen compared to pig manure. However, the anaerobic digestion of pig manure was partially inhibited by the presence of ammonia; there was a steady production of biogas. Angelidaki and Ahring [27] explained that interaction among free ammonia, VFAs, and pH leads to an inhibited steady state, which is a condition where the process is running stable but with a lower methane yield. The daily biogas yield for pig manure alone reached a peak value of 2.03 L biogas with 1.19 L methane on day 26 and decreased slowly. The peak values for the mixtures in the reactors R2–R5 were obtained 2.23 L biogas with 1.33 L methane on the 17th day, 2.45 L biogas with 1.45 L methane on the 16th day, 2.20 L biogas with 1.33 L methane on the 21st day, and 2.11 L biogas with 1.25 L methane on the 24th day, respectively. The cumulative biogas generation of the reactors R1, R2, R3, R4, R5, and R6 measured were 56.38, 63.26, 69.04, 65.81, 62.19, and 61.35 L/kg with 31.66, 35.57, 38.98, 37.68, 35.34, and 35.04 L/kg methane contents, respectively. The biogas production rate and specific biogas production were statistically different for the treatments compared. According to the results, they were ranked as follows: R3>R4>R2>R5>R6>R1. The methane yield in the co-mixture of cow dung and pig manure increased linearly with higher ratios of pig manure up to 40 % proportion and then decreased linearly, showing partial inhibition signs due to higher free ammonia in the reactors with greater mass of pig manure. Though biogas generation was initially inferior in the reactor containing pig manure alone, higher cumulative biogas and methane were obtained in the digester R6 with 100 % pig manure than those in the reactor R1 with 100 % cow dung. The reason behind this was the pig manure had contained high volatile solids. This also concurs with Hobson et al.’s [31] findings that attributed the lower production to low biodegradable material in the cow dung. However,
Appl Biochem Biotechnol
15 10
0.50 0.00 0
7
0.50
0
0.00
25 20 15
1.00
10
0.50
5 0 7
2.50
14 21 28 35 42 49 56 63 R4
2.00 Daily biogas (L)
30
1.50
10
40 35
2.00
15
0
Cumu. methane (L)
2.50
20 1.00
15 10 5
0.00
0 0
40 R5
2.00
25 20
1.00
15 10
0.50
5 0.00
0 0
7
14 21 28 35 42 49 56 63 40
2.50 R6
35 30
1.50
0 0
7 14 21 28 35 42 49 56 63
2.50
35 25
5
0.00
40 30
1.50
0.50
2.00 Daily biogas (d)
Daily biogas (L)
20
5
45 R3
25
1.00
14 21 28 35 42 49 56 63
3.00
30
1.50
35 30 25
1.50
20 1.00
15 10
0.50
Cumu. methane (L)
1.00
20
35 Cumu. methane
Daily biogas Cumu. methane
1.50
2.00
Cumu. methane (L)
25
Daily biogas (L)
2.00
40 R2
Cumu. methane (L)
30
Cumu. methane (L)
Dialy biogas (L)
R1
Daily biogas (L)
2.50
35
2.50
5 0.00
7 14 21 28 35 42 49 56 63 Time (d)
0 0
7
14 21 28 35 42 49 56 63 Time (d)
Fig. 5 Daily biogas production and cumulative methane yield
Yeole and Ranande [32] attributed the higher biogas yield from the pig manure due to the presence of native microflora in the pig manure. It was also detected that the addition of cow dung into pig manure has prompted the start-up period with early generation of biogas and biodegradability as the co-digestion reduces the ammonium inhibition and provides balanced nutrients for microorganisms. The methane content was determined low during start-up period and increased swiftly in all the functional reactors. The average and highest methane content for the reactors R1–R6 were determined 56.15, 56.24, 56.58, 57.32, 56.82, and 57.10 % and 60.48, 61.24, 61.84, 62.32, 60.86, and 62.07 %, respectively. It was shown that the co-digestions could not improve the biogas quality as there were no significant variations in methane contents among different treatments. This result is consistent with the result of Li et al. [18] which stated that the dry anaerobic co-digestion of cow manure with solid sludge can improve biogas and methane yields but unable to increase methane content in the biogas. As previous studies [12, 18, 33, 34]
Appl Biochem Biotechnol
pointed, the quality of biogas was identical among solid-state anaerobic digestions and to the liquid anaerobic digestion process. The percentage of carbon dioxide for all the tests has increased and stabilized in between 25 and 40 %. Hydrogen gas was detected in very small percentage (
Dry Anaerobic Co-digestion of Cow Dung with Pig Manure for Methane Production Jianzheng Li & Ajay Kumar Jha & Tri Ratna Bajracharya
Received: 8 February 2014 / Accepted: 21 April 2014 # Springer Science+Business Media New York 2014
Abstract The performance of dry anaerobic digestions of cow dung, pig manure, and their mixtures into different ratios were evaluated at 35±1 °C in single-stage batch reactors for 63 days. The specific methane yields were 0.33, 0.37, 0.40, 0.38, 0.36, and 0.35 LCH4/gVSr for cow dung to pig manure ratios of 1:0, 4:1, 3:2, 2:3, 1:4, and 0:1, respectively, while volatile solid (VS) and chemical oxygen demand (COD) removal efficiencies were 48.59, 50.79, 53.20, 47.73, 46.10, and 44.88 % and 55.44, 57.96, 60.32, 56.96, 53.32, and 50.86 %, respectively. The experimental results demonstrated that the co-digestions resulted in 5.10–18.01 % higher methane yields, 2.03– 12.95 % greater VS removals, 2.98–12.52 % greater COD degradation and so had positive synergism. The various mixtures of pig manure with cow dung might persuade a better nutrient balance and dilution of high ammonia concentration in pig manure and therefore enhanced digester performance efficiency and higher biogas yields. The dry co-digestion of 60 % cow dung and 40 % pig manure achieved the highest methane yield and the greatest organic materials removal efficiency than other mixtures and controls. Keywords Dry anaerobic digestion . Co-digestion . Manures . Biogas . Organic materials removal Introduction The continuously increasing demand for renewable energy sources such as methane renders anaerobic digestion to one of the most promising methods for stabilization of organic wastes and green energy production from biogas combustion [1, 2]. The anaerobic digestion technology produces less greenhouse gases than other waste treatment techniques like incineration [3], composting [4], and land filling [5]. The anaerobic digestion process follows hydrolysis, J. Li (*) : A. K. Jha State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, the People’s Republic of China e-mail: [email protected] A. K. Jha e-mail: [email protected] A. K. Jha : T. R. Bajracharya Pulchowk Campus, Institute of Engineering, Tribhuvan University, Lalitpur, Nepal T. R. Bajracharya e-mail: [email protected]
Appl Biochem Biotechnol
acidogenesis, acetogenesis, and methanogenesis [6]. In an oxygen-free environment, anaerobic microbes—such as methanogenic bacteria, acetogenic bacteria, and fermentative bacteria— digest biodegradable matter into biogas with 50–75 % methane as potential energy content, 25–40 % carbon dioxide, and other gases in small amount. De Baere [7], Jha et al. [8], and Pavan et al. [9] noted the following advantages of dry anaerobic digestion when compared to liquid anaerobic digestion: higher biogas yield per unit volume of digester, greater organic loading rate, lower energy requirements for heating, reduced nutrient run off during storage and distribution of residues, limited environmental consequences, and energetically effective performance. Mainly due to its reduced cost in digesters and easier slurry handling, the dry anaerobic digestion process has attracted increasing attention all around the world recently. However, the high-solid anaerobic digestion is known to suffer from many inhibition problems [10]. The major disadvantages of the dry anaerobic digestion are the requirement of larger amount of inocula and much longer retention time [11]. Jha et al. [12] have presented that the dry methane fermentation of cow manure took relatively longer retention time than wet fermentation to produce same amount of biogas while Fongsatitkul et al. [13] reported that the specific gas production from organic fraction of municipal waste steadily dropped with increase in total solid of the feedstocks. Furthermore, dry anaerobic digestion exhibits a poor start-up performance, while the conversion of acetate to methane is generally considered as rate limiting due to slow growth of methanogens [14]. Also, the accumulation of volatile fatty acids (VFAs) is known to restrict the biogas yield [15]. Moreover, complete mixing is difficult to achieve. Hence, this technology needs enhancement of reliability in operation to become more sustainable [16]. An option for significantly improving yields of anaerobic digestion of solid wastes is the codigestion of multiple substrates [17–19]. Co-digestion enhances the methane yield due to positive synergisms established in the digestion medium, bacterial diversities in different wastes, and the supply of missing nutrients by the co-substrates. Animal manures contain rumen microorganisms that assist to carry out anaerobic digestion process faster, and cattle manure-based biogas plants are successful in the rural area of many developing countries, but they are affected due to the continuous increasing scarcity of feedstocks. The co-digestion process can assist to solve the feedstock scarcity dilemma. Moreover, the addition of other biomass products increases biogas yield and makes the biogas plants economically profitable. However, although the cow dung and pig manure have good biogas potential, the biodegradation of pig manure as sole substrate is inhibited by presence of high ammonia concentration [20, 21]. The mixing of pig manure with cow dung reduces the ammonia concentration in the homogeneous mixture, and their simultaneous digestion might provide additional energy. The wet biomethanation process of the mixture of different wastes is relatively well understood and documented; however, limited research reports were found about the dry anaerobic co-digestion of organic wastes including the codigestion of cow dung with pig manure. The aim of this study was to assess the feasibility of dry anaerobic co-digestion of cow dung, pig manure, and their mixtures using single-stage batch digesters under mesophilic condition. Biogas and methane yields, chemical oxygen demand (COD) and volatile solid (VS) removal efficiency, and VFAs and ammonia-nitrogen accumulation and degradation are considered for comparisons.
Materials and Methods Experimental Setup and Procedure The experiments were carried out in single-stage batch lab reactors. Each bioreactor has 2.5-L effective volume with an internal diameter of 13 cm and height of 25 cm. The capped reactors
Appl Biochem Biotechnol
were kept in a water bath of operational temperature 35±1 °C, the optimum temperature for mesophilic range. Each reactor was fitted with four ports (Fig. 1). The two ports were fitted on the cover while other two ports were fitted on the side. One of the cover ports was used for measuring biogas production. The sample for analysis of biogas quality was also taken out from the same port. The other cover port was set aside as spare. One of the side ports was kept above 5 cm from the bottom. This port was used to take out the sample for the analysis of various parameters while pH meter was set up at the other side port. The samples were stored at −4 °C in a freezer before analysis. The analysis was generally performed within 1 week. In the fermentation process, the substrates were pretreated and fed into air-tight digester under specified environmental conditions for 63 days without dilution. Pretreatment means separation of manures from foreign materials like stones, woods, metals, and other inorganic materials and the addition of inoculants into the feedstocks. The visible straw and feathers were removed by hand. Each digester was purged with nitrogen for 15–20 min to create complete anaerobic environment. The contents of the reactors were slowly shaken once daily for 2–3 min to create homogeneous substrate preventing stratification and formation of a surface crust and distributing microorganisms throughout the digester. Characteristics of Feedstocks The average values of the physicochemical characteristics of the manures and inoculum are presented in Table 1. The manures were obtained from the livestock farms of Harbin, China. The digested slurry from the previous dry anaerobic digestion experiment of cow dung was utilized as inoculum to provide the initial population of methane bacteria for initiating digestion in the system [22]. The digesters, R1–R6, were filled with the cow dung and pig manure in the ratios of 1:0, 1:4, 3:2, 2:3, 4:1, and 0:1, respectively, on wet-weight basis along with inoculum (Table 2). No other nutrients, chemicals, or water was fed into the reactors. The experiments were performed in triplicate, and the average values were reported. Analytical Methods The physicochemical parameters analyzed were temperature, pH, total solid (TS), VS, COD, soluble chemical oxygen demand (SCOD), VFAs, total phosphorus (TP), total Kjeldahl nitrogen (TKN), ammonia nitrogen, and free ammonia. All the analytical determinations were performed according to the standard methods [23]. All the tests were conducted in triplicate, and mean values were reported. The pH of the mixtures was measured with a digital pH meter (Seven Multi SK40, Switzerland). The free ammonia was calculated using the previously Biogas
Reactor
Cylinder
Thermostat sensor
Biogas outlet
pH meter
Water bath
Fig. 1 Schematic diagram of the reactor
Acidified water
Appl Biochem Biotechnol
Table 1 Characteristics of substrates and inoculum
Parameters
Cow dung
Pig manure
Inoculum
pH
7.76
7.82
7.83
Total solids (%) Volatile solids (% of TS)
15.98±0.2 84.24±0.2
18.96±0.3 77.42±0.2
10.55 59.18
COD (g/L)
155.44±5
164.15±4
69.18
Soluble COD (g/L)
68.97±2
75.44±2
15.62
Total organic carbon (g/L)
40.32±2
43.13±2
11.72
Total phosphorus (g/L)
1.38±0.2
1.77±0.2
1.19
Total Kjeldahl nitrogen (g/L)
2.96±0.2
4.75±0.3
2.11
Ammonia concentration (g/L)
1.53±0.1
2.28±0.2
1.22
Free ammonia (g/L)
0.09
0.17
0.12
reported formula [24]. The yielded biogas was measured per day by downward water displacement method at atmospheric pressure using a calibrated 1- or 2-L cylindrical jar for each reactor. The constituents (CH4, CO2, and H2) of the biogas were determined using gas chromatography (SP-6800A, Shandong Lunan Instrument Factory, China) equipped with a thermal conductivity detector and a 2-m stainless column packed with Porapak TDS201 (60–80 mesh). Nitrogen was employed as the carrier gas at a flow rate of 40 mL/min. The operation temperatures for the injection port, oven, and detector were all 80 °C. The cumulative methane production for each test was determined by summing daily methane production, which was calculated by timing daily biogas production with corresponding methane content. The samples taken from the batch culture reactor were centrifuged at 6,000 rpm for 15 min and then acidified with hydrochloric acid and filtered through a 0.2-μm membrane for the analysis of VFAs and ethanol. The concentrations of the VFAs and ethanol were determined using an another gas chromatograph (SP6890, Shandong Lunan Instrument Factory, China) equipped with a flame ionization detector and a 2-m stainless (5 mm inside diameter) column packed with Porapak GDX-103 (60/80 mesh). The operational temperatures of the injection port, the column, and the detector were 220, 190, and 220 °C, respectively. Nitrogen was used as carrier gas at a flow rate of 50 mL/min. Microbial Community Analysis Genomic DNA of the sludge samples was extracted using a DNA extraction kit (MO Bio Laboratories, Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. Extracted DNA was dissolved in 60-μL 1×TE buffer solution. The V3 and V4 regions of 16S ribosomal Table 2 Composition and condition of the reactors Reactors
Cow dung (g)
Pig manure (g)
Inoculant (g)
pH
TS (%)
VS (% TS)
R1
1,000
0
200
7.77
15.11±0.2
81.28±0.2
R2
800
200
200
7.79
15.64±0.2
79.39±0.15
R3
600
400
200
7.80
16.17±0.3
78.18±0.2
R4
400
600
200
7.81
16.54±0.2
77.91±0.2
R5
200
800
200
7.80
17.13±0.3
76.02±0.2
R6
0
1,000
200
7.82
17.55±0.3
75.52±0.25
Appl Biochem Biotechnol
RNA (rRNA) were amplified by PCR using universal bacterial primers (341F, 5′-CCTACG GGAGGCAGCAG-3′ with a GC clamp and 907R, 5′-CCGTCAATTCMTTTGAGTTT-3′) and universal archaeal primers (344F, 5′-ACGGGGYGCAGCAGGCGCGA-3′ with a GC clamp and 915R, 5′-GTGCTCCCCCGCCAATTCCT-3′). The PCR amplification was conducted in a 50-μL system containing 5 μL 10×Ex Taq buffer, 4 μL dNTP mixture (2.50 mM), 1 μL forward primer (20 μM), 1 μL reverse primer (20 μM), 2.5 ng DNA template, and 0.15 U Ex Taq DNA polymerase (Takara, Dalian, China), using a thermal cycler (model 9700; ABI, Foster, CA, USA), started with an initial denaturation of DNA for 10 min at 94 °C, followed by 30 cycles for 1 min at 94 °C, 30 s at 55 °C (decreasing by 0.10 °C per cycle to 52 °C), and 1 min at 72 °C; final extension was 10 min at 72 °C. The PCR products were separated using the Dcode™ universal mutation detection system (Biorad Laboratories, Hercules, CA, USA). Polyacrylamide gels with 40–60 % vertical denaturing gradient were prepared. The 10-μL PCR products were loaded and electrophoresed at 120 V and 60 °C for 10 h. Gels were stained silver as described in the previous research [25]. All DGGE bands were excised and dissolved in 30 μL 1×TE at 40 °C for 3 h and then centrifuged at 12,000 rpm for 3 min. The 3-μL supernatant was used as the template and conducted PCR amplification under the conditions as described above using the same primers. The PCR products were pured by Gel Extraction Mini Kit (Watson Biotechnologies, Inc, China) and ligated into pMD18-T vector (Takara, Dalian, China), and then cloned into Escherichia coli DH5α. Some white clones from each sample were randomly selected for PCR detection, and positive clones were selected for sequencing by ABI3730, and partial 16S rRNA gene sequences were analyzed using the BLAST program in GenBank.
Results and Discussion Evaluation of pH, NH3-N, and VFAs Figure 2 illustrates the evolution of pH, NH3-N, and VFAs in average for each six types of the functional reactors during the digestion period. The pHs of cow dung and pig manure were initially around 7.76 and 7.82, respectively. The pH variation pattern was observed similar for all the tests. It was decreased to 6.74, 6.80, 6.78, 6.81, 6.75, and 6.78 in 14, 14, 14, 21, 21, and 21 days after the beginning of the digestion process for the reactor types R1–R6, respectively (Fig. 2a). It happened due to the increase in VFAs production by acidogenic bacteria during the start-up phase of each experiment. The easily digestible fraction of organic matter was hydrolyzed and converted to fatty acids rapidly. The pH value did not drop off much lower because the substrates were able to buffer themselves and prevent the acidification occurrence due to proper alkalinity of cow dung and pig manure to maintain optimal biological activity and stability of the anaerobic digestion system. The pH value for all the experiments began to rise gradually as the VFAs were consumed by methanogens and transferred to the methane. The biogas process becomes more sensitive towards increase of pH value because the concentration of free ammonia increases as pH value raises [26]. The ammonia concentration for cow dung was noted 1.53 and 2.28 g/L for pig manure. In this study, the accumulation of ammonia has been increased to some extent due to the hydrolysis of amino acids and proteins during the start-up period. Afterwards, the concentration of ammonia was decreased since it was used as nitrogen source for methanogen growth. It was again increased since the protein-containing hard biodegradable fraction began to hydrolyze after some days of the beginning of digestion. As a result, fluctuated ammonia variation patterns were observed for all the tests during the digestion period (Fig. 2b). It can be noted
Appl Biochem Biotechnol
a
7.90 7.70
pH
7.50 7.30 7.10
R1
R2
R3
R4
R5
R6
6.90 6.70
0
Ammonia nitrogen (g/L)
2.60
7
14
7
14
21
28
35
42
49
56
63
b
2.40 2.20 2.00 1.80 1.60 1.40 1.20
0
21
28
35
42
49
56
63
Time (d)
Fig. 2 Evolution of a pH and b ammonia nitrogen
that the biodegradation of pig manure and the mixtures was partially inhibited due to the presence of high ammonia concentration because the ammonia inhibition has been observed to commence at concentrations of 1.50–2.50 g/L [20]. In this study, average calculated free ammonia for bioreactor types R1–R6 were determined in the range of 0.02–0.55, 0.02–0.58, 0.02–0.66, 0.03–0.71, 0.03–0.82, and 0.03–0.87 g/L, respectively. Furthermore, Angelidaki and Ahring [27] reported that the existence of high content of the free ammonia is toxic for biogas processes because the specific growth rate of methanogenic bacteria is a function of the free ammonia concentration and decreases as the concentration of ammonia increases [26]. The growth of acetate-utilizing bacteria has been started to inhibit from 0.1 to 0.15 g/L [28] while H2-utilizing methanogenic bacteria are inhibited at free ammonia concentrations higher than 1.20 g/L [26]. It indicated that the process was partially inhibited for the acetate-utilizing bacteria. It was also noted that the inhibition was more in pig manure than cow dung and their mixtures. Volatile fatty acids are usually produced due to the degradation of the complex organic polymers during hydrolysis and acidogenic stages. The conversion of intermediate products— VFAs—has been treated as an indicator of the digestion efficiency, but the high concentration of VFAs results in decrease in pH, acidification, and destructions/demolish activity of methanogenic bacteria, leading to failure of digester ultimately. In this study, the average highest values of VFAs for the reactor types R1–R6 were determined 8.20, 7.89, 8.06, 7.50, 7.66, and 7.58 g/L, respectively. All the reactors showed high VFA concentrations in the start-up phase (Fig. 3) due to higher acidogenesis and lower methanogenic activities. The higher concentration of VFAs during the start-up period results in the increasing amount of ammonia within the limits having no significant influence on the hydrolysis and acidogenesis. The principal volatile acids formed were acetic, butyric, and propionic acids. Acetic acid was the dominant VFAs. Moreover, the acetic acid production rate was apparently higher than the acetic acid consumption rate during the start-up period. The share of propionic and butyric acids was
9 8 7 6
R1
5 4 3 2 1 0 7
14
21
28
9 8 7 6 5 4 3
35
42
49
56
9 8 7 6 5 4 3 2 1 0
R2
0
63
7
14
21
28
35
42
49
56
63
42
49
56
63
28 35 42 Time (d)
49
56
63
8 R3
VFAs & ethanol (g/L)
VFAs & ethanol (g/L)
0
2 1 0
7
R4
6 5 4 3 2 1 0
0
7
14
21
9 8 7 6 5 4 3 2 1 0
28
35
42
49
56
0
63
7
14
21
28
35
8 R5
VFAs & ethanol (g/L)
VFAs & ethanol (g/L)
Ethanol Acetic Propionic Butric Total
VFAs & ethanol (g/L)
VFAs & ethanol (g/L)
Appl Biochem Biotechnol
R6
7 6 5 4 3 2 1 0
0
7
14
21
28 35 42 Time (d)
49
56
63
0
7
14
21
Fig. 3 VFAs and ethanol variation pattern
observed low because of the sufficient propionate- and butyric-degrading syntrophs which could rapidly convert propionic acid and butyric acid into acetic acid [29]. The degradation of propionate and butyrate by syntrophic acetogenic bacteria (e.g., Syntropher wolinii, syntrophomonas wolfei) produced acetic acid that was subsequently degraded into methane and CO2 by acetoclastic methanogens [29]. During methanogenic stage, the methanogens were in exponential growth phase, and the acetic acid consumption rate was higher though hydrolysis and acidogenesis were still going on. The consumption rate of propionate was observed slower than that of butyrate because the oxidation of propionate to acetate is relatively more difficult. Therefore, as methanogenesis and methane gas yield have been increased, the VFA concentrations were decreased. The residual total VFA was observed highest in the reactor with pig manure followed by reactors with their mixtures and lowest in the reactor with cow dung. VFAs and alkalinity together are good indicators for evaluating the process stability of the anaerobic reactor. Figure 4 shows the variation in total VFA to alkalinity ratio. The ratio varied
Appl Biochem Biotechnol
VFAs/alkalinity
0.80 R1 R3 R5
0.60
R2 R4 R6
0.40 0.20 0.00
0
7
14
21
28 35 Time (d)
42
49
56
63
Fig. 4 VFAs to alkalinity ratio variation pattern
below 0.80, and the process seemed stable [30]. No accumulation of VFAs and no drastic fall in pH also support that the process was not inhibited extensively. Biogas Yield and Organic Material Removal Figure 5 presents the daily biogas yield, percentage methane content, and cumulative methane production in the operational reactors R1–R6 during the digestion period. In this study, similar trends of daily biogas and methane yields were observed for all the tests. The biogas generation was started after seeding, kept increasing until reaching the peak, and then began to decline. The initial biogas production was due to readily biodegradable organic matter in the substrates and presence of methanogens. The reactor with cow dung alone generated higher biogas and methane than those of co-digestion mixtures and pig manure during the start-up phase and obtained a peak value (2.13 L biogas with 1.29 L methane) promptly on day 15. The difference in obtaining peak values was occurred because the cow dung was not significantly inhibited due to existence of lower ammonium nitrogen compared to pig manure. However, the anaerobic digestion of pig manure was partially inhibited by the presence of ammonia; there was a steady production of biogas. Angelidaki and Ahring [27] explained that interaction among free ammonia, VFAs, and pH leads to an inhibited steady state, which is a condition where the process is running stable but with a lower methane yield. The daily biogas yield for pig manure alone reached a peak value of 2.03 L biogas with 1.19 L methane on day 26 and decreased slowly. The peak values for the mixtures in the reactors R2–R5 were obtained 2.23 L biogas with 1.33 L methane on the 17th day, 2.45 L biogas with 1.45 L methane on the 16th day, 2.20 L biogas with 1.33 L methane on the 21st day, and 2.11 L biogas with 1.25 L methane on the 24th day, respectively. The cumulative biogas generation of the reactors R1, R2, R3, R4, R5, and R6 measured were 56.38, 63.26, 69.04, 65.81, 62.19, and 61.35 L/kg with 31.66, 35.57, 38.98, 37.68, 35.34, and 35.04 L/kg methane contents, respectively. The biogas production rate and specific biogas production were statistically different for the treatments compared. According to the results, they were ranked as follows: R3>R4>R2>R5>R6>R1. The methane yield in the co-mixture of cow dung and pig manure increased linearly with higher ratios of pig manure up to 40 % proportion and then decreased linearly, showing partial inhibition signs due to higher free ammonia in the reactors with greater mass of pig manure. Though biogas generation was initially inferior in the reactor containing pig manure alone, higher cumulative biogas and methane were obtained in the digester R6 with 100 % pig manure than those in the reactor R1 with 100 % cow dung. The reason behind this was the pig manure had contained high volatile solids. This also concurs with Hobson et al.’s [31] findings that attributed the lower production to low biodegradable material in the cow dung. However,
Appl Biochem Biotechnol
15 10
0.50 0.00 0
7
0.50
0
0.00
25 20 15
1.00
10
0.50
5 0 7
2.50
14 21 28 35 42 49 56 63 R4
2.00 Daily biogas (L)
30
1.50
10
40 35
2.00
15
0
Cumu. methane (L)
2.50
20 1.00
15 10 5
0.00
0 0
40 R5
2.00
25 20
1.00
15 10
0.50
5 0.00
0 0
7
14 21 28 35 42 49 56 63 40
2.50 R6
35 30
1.50
0 0
7 14 21 28 35 42 49 56 63
2.50
35 25
5
0.00
40 30
1.50
0.50
2.00 Daily biogas (d)
Daily biogas (L)
20
5
45 R3
25
1.00
14 21 28 35 42 49 56 63
3.00
30
1.50
35 30 25
1.50
20 1.00
15 10
0.50
Cumu. methane (L)
1.00
20
35 Cumu. methane
Daily biogas Cumu. methane
1.50
2.00
Cumu. methane (L)
25
Daily biogas (L)
2.00
40 R2
Cumu. methane (L)
30
Cumu. methane (L)
Dialy biogas (L)
R1
Daily biogas (L)
2.50
35
2.50
5 0.00
7 14 21 28 35 42 49 56 63 Time (d)
0 0
7
14 21 28 35 42 49 56 63 Time (d)
Fig. 5 Daily biogas production and cumulative methane yield
Yeole and Ranande [32] attributed the higher biogas yield from the pig manure due to the presence of native microflora in the pig manure. It was also detected that the addition of cow dung into pig manure has prompted the start-up period with early generation of biogas and biodegradability as the co-digestion reduces the ammonium inhibition and provides balanced nutrients for microorganisms. The methane content was determined low during start-up period and increased swiftly in all the functional reactors. The average and highest methane content for the reactors R1–R6 were determined 56.15, 56.24, 56.58, 57.32, 56.82, and 57.10 % and 60.48, 61.24, 61.84, 62.32, 60.86, and 62.07 %, respectively. It was shown that the co-digestions could not improve the biogas quality as there were no significant variations in methane contents among different treatments. This result is consistent with the result of Li et al. [18] which stated that the dry anaerobic co-digestion of cow manure with solid sludge can improve biogas and methane yields but unable to increase methane content in the biogas. As previous studies [12, 18, 33, 34]
Appl Biochem Biotechnol
pointed, the quality of biogas was identical among solid-state anaerobic digestions and to the liquid anaerobic digestion process. The percentage of carbon dioxide for all the tests has increased and stabilized in between 25 and 40 %. Hydrogen gas was detected in very small percentage (
