Waste Management 35 (2015) 119–126
Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
Influence of initial pH on thermophilic anaerobic co-digestion of swine manure and maize stalk Tong Zhang a,c, Chunlan Mao b,c, Ningning Zhai a,c, Xiaojiao Wang a,c, Gaihe Yang a,c,⇑ a
College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China c Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling, 712100, Shaanxi, China b
a r t i c l e
i n f o
Article history: Received 27 March 2014 Accepted 9 September 2014 Available online 31 October 2014 Keywords: Thermophilic anaerobic digestion Initial pH value Manure ratios Optimum reaction condition
a b s t r a c t The contradictions between the increasing energy demand and decreasing fossil fuels are making the use of renewable energy the key to the sustainable development of energy in the future. Biogas, a renewable clean energy, can be obtained by the anaerobic fermentation of manure waste and agricultural straw. This study examined the initial pH value had obvious effect on methane production and the process in the thermophilic anaerobic co-digestion. Five different initial pH levels with three different manure ratios were tested. All digesters in different initial pH showed a diverse methane production after 35 days. The VFA/alkalinity ratio of the optimum reaction condition for methanogens activity was in the range of 0.1–0.3 and the optimal condition that at the 70% dung ratio and initial pH 6.81, was expected to achieve maximum total biogas production (146.32 mL/g VS). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The contradictions between the increasing energy demand and decreasing fossil fuels are making the use of renewable energy the key to the sustainable development of energy in the future. Anaerobic digestion process, which is an effective way of nutrients recycling, offers a potential means of converting organic solid waste into fuel gas and fertilizer, thereby provides a supplemental and readily utilizable source of energy (Cooney and Wise, 1975; De Baere, 2000; Keshtkar et al., 2001; El-Mashad et al., 2004; Alvarez et al., 2006; Uludag-Demirer et al., 2008). Currently, using biogas fermentation technology to improve the utilization of crop straw and livestock manure has become popular in various areas (Shi et al., 2005). Many studies have shown that co-digestion is a well-accepted process which can compensate the balance of nutrients and pH buffer capacity, dilute inhibitory compounds, and alleviate toxicity from sole-substrate(Kim and Oh, 2011; Wan et al., 2011; Mata-Alvarez, 2003). In the past, priority was given to anaerobic fermentation under mesophilic condition. Recently, thermophilic anaerobic fermentation gained more and more attention (Sung and Liu, 2003) due to its advantages such as shorter startup time, faster process of methane gas production and stability (Aitken and Mullennix, 1992). ⇑ Corresponding author at: College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China. http://dx.doi.org/10.1016/j.wasman.2014.09.004 0956-053X/Ó 2014 Elsevier Ltd. All rights reserved.
Compared to mesophilic process, thermophilic anaerobic fermentation enhances hydrolysis of complex organic/biological materials and reduces foaming (Rimkus et al., 1982; Sung, 2001). Study showed that microbial consortia proximity can alleviate effluent quality in thermophilic non-mixed reactor, which showed higher biogas production potential and better volatile solids destruction (Kim et al., 2002). There are kinetic advantages in fermentation of livestock manure at thermophilic condition (Chen et al., 1980), such as reducing the hydraulic retention time and improving the dewaterability of the sludge (Buhr and Andrews, 1977). Thermophilic digestion can effectively remove organic matter, reduce the accumulation of pathogens, such as bacteria and viruses (Cooney and Wise, 1975), and weed seeds or insect eggs, thus enable effluent hygienization (Ferrer et al., 2008). However, there are some limiting parameters in thermophilic anaerobic fermentation. Thermophilic anaerobic fermentation of animal waste fertilizers such as swine (Hansen et al., 1999), cattle (Sung, 2001), and poultry waste is not ideal. Many experiments showed that fermentation of high nitrogen raw materials with low C/N, such as swine manure (SM), easily led to ammonium nitrogen accumulation, which caused material liquid ammonia poisoning, inhibited digestion (Hansen et al., 1998; Salminem et al., 2002) and reduced gas production rate. On the contrary, fermentation of materials with high C/N, such as straw, was liable to produce fermented acid accumulation phenomenon, which caused the inhibition of CH4 bacteria activity, the decrease of organic
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carbon conversion (Poliafico and Murphy, 2007) and the extension of processing time. Thermophilic fermentation appeared to be more sensitive to pH than mesophilic fermentation for both hydrolysis and acidogenesis (Kim et al., 2003). Performance of fermentation with different materials varies with different initial pH conditions. Sutaryo et al. (2012) demonstrated that digesters can run with stable biogas production and low VFA levels using 30% of acidified dairy cow manure (SFDCM) instead of dairy cow manure (DCM) in thermophilic anaerobic co-digestion. But as the concentration of SFDCM increased, the methane concentration was significantly lower. An increased in acetic, butyric, and total VFA contents was caused by the acidified pig slurry content was more than 40% (Moset et al., 2012). The culture pH shift can cause the change of the dominant microbial populations affecting the main organic acids products (Horiuchi et al., 2002). The maximum hydrolysis and acidogenesis occurred when the pH was 6.5 (Kim et al., 2003). Therefore, a suitable pH is an important guarantee of anaerobic fermentation. In view of the environmental factors that affect the process of the thermophilic anaerobic digestion, such as initial pH, temperature and toxic substances (Sung and Liu, 2003), and the lack of the comprehensive study on these factors, to explore the optimal reaction conditions and optimize environmental factors became the target of this study. In this research, we investigated the thermophilic anaerobic fermentation of mixed swine manure (SM) and maize stalks (MS) with different ratios. The optimal initial pH value and fermentation ratio were obtained for future intensification of rural biogas fermentation. 2. Material and methods 2.1. Substrates and inoculum Fresh SM was collected from a local livestock farm near Northwest Agriculture and Forestry University (NW A & F U), Yangling, Shaanxi, China. Wood, debris and other garbage were removed and the maggots should be separated out from SM before being put into digesters. Maize stalks (MS) of Luodan 9, harvested a year ago from the experimental field of NW A & F U, were crushed into particles with diameter of 1 mm after dry grinding before being put into digesters. Anaerobic sludge used as inoculum was obtained from an anaerobic digester in a local biogas demonstration village in Yangling, Shaanxi, China. Table 1 shows the chemical characteristics of each material used in this study.
inoculum had been run in order to extract the inoculum’s endogenous methane yield from the results and the inoculum had been depleted the residual biodegradable organic material. Three different total solid (TS) ratios of SM content to total material (30%, 50%, and 70%) were set and each ratio was conducted at five initial pH levels (6.0, 6.5, 7.0, 7.5, and 8.0) with sodium hydroxide and hydrochloric acid. Each treatment had three repetitions. The initial pH value of the mixed raw materials was measured by Gasboard3200P (Table 1). Meanwhile unadjusted mixtures (CK) were anaerobically digested as no-treatment control group for every ratio. After mixing the anaerobic sludge, full stir was conducted to make good contact of raw materials and inoculum. Mixtures of SM and MS were poured into the lab-scale anaerobic digesters fabricated 1 L Erlenmeyer flasks. The co-fermentations of SM and MS were detected by batch digesters (Zhang et al. 2014). Every digester contained 500 g mixture and 200 g inoculum. All treatments had the same total solid content of 8%. Table 2 shows the actual amount in each conical flask. The fermentation flasks were placed in a thermostatic water bath with temperature of 55 °C ± 2 °C. The reactors were gently shaken before measuring biogas volume. The period of the fermentation was 35 days. Samples of the substrates, which had the best performance of biogas yield, were collected every five days and related indicators were measured. 2.3. Measurement In this study, TS, volatile solids (VS), pH, volatile fatty acid (VFA), alkalinity, and total ammonium nitrogen (TAN) of the digested biomass material were measured in accordance with the Standard Methods for the Examination of Water and Wastewater of the American Public Health Association (1995). Total carbon (TC) was analyzed using the method described in Song et al. (2012) and Cuetos et al. (2011). Drainage method was used to measure the volume of biogas yield every day and fast CH4 analyzer (Model DLGA-1000, Infrared Analyzer; Dafang, Beijing, China) was used to measure the CH4 content of the produced biogas. 2.4. Analysis and calculations The significances of each treatment and the second-order polynomial coefficients were analyzed through the analysis of variance (ANOVA) using SAS version 8.12 (SAS Institute Inc., Cary, NC, USA) and Design-Expert8.0.6 software. 3. Results and discussion
2.2. Experimental design and set-up 3.1. Daily biogas production The experiment was conducted in the digesters of our own design, which have been operated stably for several years (Wang et al., 2012). Before mixing the materials, all the substrates and inoculum were homogenized individually. Blank bottles with only
The daily biogas production of the treatments with three ratios of SM contents at five different initial pH values are shown in Fig. 1. Significant biogas yield peak values of most treatments appeared in
Table 1 Chemical characteristics of substrates. Substrate c
SM MSd Ine
TSa content (%)
VSb content/% of TS
pH value
Total carbon
Total Kjeldahl nitrogen (g/kg VS)
53.58 ± 2.14 96.55 ± 0.37 6.59 ± 0.45
70.01 ± 3.44 88.99 ± 0.63 53.56 ± 1.05
7.2 ± 0.2 ND 7.9
26.66 ± 0.99 33.03 ± 1.11 NDf
1.21 ± 0.025 0.96 ± 0.07 ND
Result = mean ± standard deviation (SD). a TS: total solid. b VS: volatile solids. c SM: swine manure. d MS: maize stalks. e In: inoculum. f ND: not determined.
T. Zhang et al. / Waste Management 35 (2015) 119–126 Table 2 Experimental design of mix component batch co-digestion sets (WWa/g).
a b
SM ratio/TS%
SM
MS
DWb
In
30% 50% 70%
23.98 39.96 55.94
29.97 22.18 13.31
446.05 437.86 430.75
200 200 200
WW: wet weight. DW: deionized water.
the early fermentation (the first 10 days). From the first day after seeding, aerogenesis started and kept growing. Most treatments reached their peak values in 3–4 days. When the process went into hydrolytic acidification, the daily biogas production reduced gradually as the pH went down below 6.0. Biogas yield increased with
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the raise of pH little by little after acid phase and of different treatments reached the second or third peak value at different times except the group with 30% SM ratio. Only one biogas yield peak value appeared in the treatments with SM ratio of 30%. After 13 days of the fermentation, biogas yields of all treatments were less than 200 mL. Unlike any other treatments in 30% SM ratio group, the daily biogas production peak value of pH 6.0 appeared in the 11th day (Fig. 1a). In the group with SM ratio of 50%, pH 7.0 daily biogas productions were significant higher than other treatments due to the pH rose over 7.0 after 10 days and there were four evident biogas yield peak values on days 2, 12, 17, and 25. But because of the low pH, other treatments’ biogas yield declined under 100 mL/d after 15 days. The rest treatments of this group at the late digestion (from 25 to 35 days) reduced gradually without significant fluctuations (Fig. 1b). The group with SM ratio of 70% was significantly different from the two groups above. The
Fig. 1. Daily biogas production of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
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pH of every treatment had risen up after acid phase, so in the middle (from 11 to 24 days) and late reaction, the daily biogas production in 70% SM ratio group kept active. Each treatment had bigger yield peak values than that in the first 10 days except pH 8.0 and CK treatments, and the durations of the yield peak value were different (Fig. 1c). According to the performance of daily biogas production, it can be concluded that pH can affect the biogas yield effects, such as the number and the durations of the biogas yield peak values, in thermophilic anaerobic co-digestion under different manure ratio. 3.2. Change of ammonia nitrogen under different conditions Compared with MS, SM has higher nitrogen contents. Lots of studies reported ammonia inhibition occurred during the biogas production progress particularly when mono-digestion of manure under thermophilic conditions (Hansen et al., 1998; Bujoczek et al., 2000). Previous studies have shown that ammonia concentrations greater than 1500 mg/L inhibited digestion when pH was higher than 7.4 (Siegrist et al., 2002). It is shown that the higher SM content, the higher ammonia content in response (Fig. 2). The ammonia content was instable in the early process, which led to VFA accumulation lowering the pH (Angelidaki and Ahring, 1993; Yenigün and Demirel, 2013). Ammonia nitrogen contents of all the treatments in SM content 30% groups declined slightly during the acidification phase in the early stages. Subsequently, ammonia contents began to rise and remained stable between 600 and 850 mL/L in the middle and late periods (Fig. 2a). Different from the group of 30% SM, ammonia nitrogen contents of only the pH 6.5 treatments in 50% and 70% SM were declined in the first 10 days, and showed secular upheaval in the rest of treatments (Fig. 2b and c). In the group with SM ratio of 30% and 50%, the trends of ammonia nitrogen content were smooth in the medium and period (Fig. 2a and b). In the treatments with SM ratio of 70%, three cases (pH 7.0, 7.5, and 8.0) had relatively similar ammonia contents to single SM fermentation in the middle of the digestion (Fig. 2c). At the end of digestion, no treatment had higher ammonia content than mono-digestion of manure compared with all the treatments. At the last phase of the fermentation, every ammonia level of the treatments lay in the thresholds of the mono-fermentation with manure and straw (Fig. 2). Overall, as the co-digestion proceeded, ammonia nitrogen content increased gradually. Accumulated ammonia generated in the degradation of the protein in SM would cause a certain degree of ammonia inhibition (Sung and Liu, 2003). In this study, no obvious ammonia inhibition was observed, which is similar to the research of Wang et al. (2012). 3.3. pH trend during fermentation Horiuchi’s et al. study showed that the main products of organic acids were changed by shifting the culture pH in the reactor (Horiuchi et al., 2001). Therefore, the trend of the pH change affected the daily biogas production to a certain degree. With same initial pH values, the pH resilience in the late stage of fermentation improved significantly as SM ratio increased. In the treatments with SM ratio of 30%, pH value of each treatment was lower than 6.0 in the fifth day after inoculation, which seriously affected the activity of methane bacteria. Since methane bacteria cannot survive in pH lower than 6.0 environment, at the late fermentation, the buffering capacity of the substrate was imbalance showing high alkalinity/VFA ratio. In the treatments with SM ratio of 50%, because of the accumulation of organic acids, the pH of each treatment apart from the initial pH 7.0 went to lower than 6.0. Only that of the initial pH 7.0 treatment recovered to larger than 7.0 after 10 days (Fig. 3a), and the alkalinity/VFA ratio was below 0.2 during
Fig. 2. Change of ammonia nitrogen of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
the 35 days’ digestion, so the total biogas yield of pH 7.0 treatment in 50% SM ratio group was much higher than other treatments in the same SM ratio group. In treatments with SM ratio of 70%, pH value of each treatment gradually recovered to above 7.0. Recovery speed of pH 8.0 treatment was the fastest, followed by 6.0, 7.5, 6.5, and 7.0. At the same, the low alkalinity/VFA ratio showed there was no excessive accumulation of organic acids and the environment of the substrate was suitable for bacteria activity. It can be speculated that increasing manure content would help enhance
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Fig. 3. Change of pH of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
the buffering capacity of the substance, reducing the impact of organic acids accumulation of the fermentation process (Fig. 3b). The fermentation of 70% SM group had final pH values ranging from 7.4 to 8.2 (Fig. 3c). Thus, the biogas yield of treatments with 70% SM content was significantly better than other treatments with same pH and in the same SM group the biogas yield of initial pH 7.0 treatments was better than other treatments. 3.4. Effect of SM ratios on VFA/alkalinity Decrease of the buffering capacity caused by the VFAs’ accumulation is earlier than the pH decreases (Chen et al., 2014), so VFA/ alkalinity ratio were used in this research as a more reliable parameter for monitoring fermentation condition imbalance. Studies have shown that the ratio of volatile fatty acid (VFA) to alkalinity in a range of 0.3–0.5 was optimum for methanogens (Liew et al., 2011). The VFA/alkalinity ratios of all treatments in the first 25 days were lower than 0.4 (Fig. 4). The accumulation of organic acids caused the VFA/alkalinity ratios raised and declined the pH, so in the groups with SM ratios of 30% and 50% (except 50% pH 7.0 treatment), VFA/alkalinity ratios raised sharply after the 25th day (Fig. 4a and b). However, the VFA/alkalinity ratios of treatments with SM ratio of 70% remained in the appropriate range (Fig. 4c), which showed the pH of the substrate was suitable for anaerobic digestion. Combined with the daily biogas production, the optimum VFA/alkalinity ratio for co-digestion of SM/MS was 0.1–0.3. When the VFA/alkalinity ratio hovered from 0.3 to 0.8, the buffering capacity of co-digestion system was restricted to a certain extent. While when VFA/alkalinity ratio was larger than 0.8, the buffering capacity of the system was not enough for fermentation. SM could be used as cushioning material to improve the buffering capacity of the co-digestion system. But it is worth noting that ammonia inhibition is prone to accrue when the ratio of SM is too high.
The variance analysis on effects of SM content and initial pH on the total biogas yield was conducted using SAS software. Tests had proved that data followed a normal distribution. The results showed that the SM ratio and pH as well as their interaction had significant impacts on total gas yield. Based on the total biogas yield of each treatment (Table 3), twofactor analysis of variance for pH and manure ratio is showed in Table 4. From the data in Table 4, it is obvious that pH has significant effects on total biogas yield (Table 5) and the ratio and proportion and its interaction effects with pH reach highly significant. Shifting the initial pH in the digesters from 6.0 to 8.0 by step, the total biogas products showed a trend that decrease came after increase. At the same initial pH, the performances of 70% SM ratio were the best. The total biogas yield of 70% SM ratio was significantly higher than that of the other two ratios. It can be speculated that pH has a very significant effect on the thermophilic anaerobic co-digestion and optimal yields could be obtained by optimizing conditions of the manure ratio and pH. The ratio of SM and initial pH value were designed as factors influencing the total methane volume. Design-Expert software for response surface was used to analyze the relationship between these two factors and total biogas yield (Table 6). Correlation regression equation models were achieved, and 3D map was drawn accordingly. 3.6. Two factors correlation regression equation In order to optimize the studied parameters, response surface methodology (RSM) was used in this study. The methane yield per unit volume was dependent output responses. The following second-order polynomial model showing the estimating coefficients based on experimental data was used to describe the functional relationships between responses (Y) and the set of factors (A and B).
Y ¼ b0 þ b1 A þ b2 B þ b3 AB þ b4 A2 þ b5 B2
ð1Þ
3.5. Modeling and optimization of methane yield From Fig. 5, the accumulative biogas production of treatments with various SM content changed differently. Biogas yield increased with the pH rising when the SM content was 30%. When SM content rose to 50%, the biogas yield of pH 7.0 was significantly higher than others, followed by the 6.5, 7.5, CK, 8.0, and 6.0. S-shaped growth curves were appeared in the SM ratio of 70% different from other conditional control group. In addition to CK group, the rest of the treatments had a second high biogas yield after the 15th days. Through the regression analysis on the influences of the two factors on the biogas yield, the following equations were obtained.
where Y represents the predicted response, A is the ratio of SM; B is the pH; b0 is a constant; b1 and b2 are linear coefficients; b3 is interaction coefficients and b4 and b5 are quadratic coefficients. 3.7. Final equation in terms of coded factors
Total methane yield ¼ þ54:63 þ 44:34 A þ 2:50 B 25:72 A B þ 35:07 A2 333:91 B2 ð2Þ The model was implied significant by the F-value of 10.24 and the R-squared was 82.31%. Both interaction factor and quadratic
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Fig. 4. Change of VFA/ alkalinity of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
factors emerged a significant effect for the ratio of SM and the pH over the response among all the evaluated factors. Using Design-Expert software to optimize the design of the model, the optimal conditions for maximum methane potential (Fig. 6) were calculated as a SM ratio of 70% and a pH of 6.81. Accordingly, the maximum total methane yield was expected to reach 146.32 mL/g VS. Compared with the results that the highest methane yield of 145.98 mL/g VS was appeared in pH 7.0 treatment of SM 70% in this study, the optimal conditions for maximum
Fig. 5. Accumulative biogas production of treatments with different SM contents (a) 30% SM, (b) 50% SM, and (c) 70% SM.
Table 3 Total biogas yield of each treatment (mL/g VS). SM ratio/TS%
30% 50% 70%
pH 6.0
6.5
7.0
7.5
8.0
CK
62.53 22.19 240.41
88.27 129.11 285.59
84.58 215.01 307.68
92.38 92.49 296.28
87.33 58.75 203.40
89.77 84.90 273.61
T. Zhang et al. / Waste Management 35 (2015) 119–126 Table 4 The two-factor variance analysis of the total biogas yield. Source
df
Sum of squares
Mean square
F value
Model Error Corrected total
17 36 53
412441.2406 18179.8013 430621.0419
Source
df
pH Ratio pH * ratio
5 2 10
Pr > F
24261.2494 504.9945
48.04
Anova SS
Mean square
F value
Pr > F
59006.6033 319187.8381 34246.7992
11801.3207 159593.9190 3424.6799
23.37 316.03 6.78
Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
Influence of initial pH on thermophilic anaerobic co-digestion of swine manure and maize stalk Tong Zhang a,c, Chunlan Mao b,c, Ningning Zhai a,c, Xiaojiao Wang a,c, Gaihe Yang a,c,⇑ a
College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China c Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling, 712100, Shaanxi, China b
a r t i c l e
i n f o
Article history: Received 27 March 2014 Accepted 9 September 2014 Available online 31 October 2014 Keywords: Thermophilic anaerobic digestion Initial pH value Manure ratios Optimum reaction condition
a b s t r a c t The contradictions between the increasing energy demand and decreasing fossil fuels are making the use of renewable energy the key to the sustainable development of energy in the future. Biogas, a renewable clean energy, can be obtained by the anaerobic fermentation of manure waste and agricultural straw. This study examined the initial pH value had obvious effect on methane production and the process in the thermophilic anaerobic co-digestion. Five different initial pH levels with three different manure ratios were tested. All digesters in different initial pH showed a diverse methane production after 35 days. The VFA/alkalinity ratio of the optimum reaction condition for methanogens activity was in the range of 0.1–0.3 and the optimal condition that at the 70% dung ratio and initial pH 6.81, was expected to achieve maximum total biogas production (146.32 mL/g VS). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The contradictions between the increasing energy demand and decreasing fossil fuels are making the use of renewable energy the key to the sustainable development of energy in the future. Anaerobic digestion process, which is an effective way of nutrients recycling, offers a potential means of converting organic solid waste into fuel gas and fertilizer, thereby provides a supplemental and readily utilizable source of energy (Cooney and Wise, 1975; De Baere, 2000; Keshtkar et al., 2001; El-Mashad et al., 2004; Alvarez et al., 2006; Uludag-Demirer et al., 2008). Currently, using biogas fermentation technology to improve the utilization of crop straw and livestock manure has become popular in various areas (Shi et al., 2005). Many studies have shown that co-digestion is a well-accepted process which can compensate the balance of nutrients and pH buffer capacity, dilute inhibitory compounds, and alleviate toxicity from sole-substrate(Kim and Oh, 2011; Wan et al., 2011; Mata-Alvarez, 2003). In the past, priority was given to anaerobic fermentation under mesophilic condition. Recently, thermophilic anaerobic fermentation gained more and more attention (Sung and Liu, 2003) due to its advantages such as shorter startup time, faster process of methane gas production and stability (Aitken and Mullennix, 1992). ⇑ Corresponding author at: College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China. http://dx.doi.org/10.1016/j.wasman.2014.09.004 0956-053X/Ó 2014 Elsevier Ltd. All rights reserved.
Compared to mesophilic process, thermophilic anaerobic fermentation enhances hydrolysis of complex organic/biological materials and reduces foaming (Rimkus et al., 1982; Sung, 2001). Study showed that microbial consortia proximity can alleviate effluent quality in thermophilic non-mixed reactor, which showed higher biogas production potential and better volatile solids destruction (Kim et al., 2002). There are kinetic advantages in fermentation of livestock manure at thermophilic condition (Chen et al., 1980), such as reducing the hydraulic retention time and improving the dewaterability of the sludge (Buhr and Andrews, 1977). Thermophilic digestion can effectively remove organic matter, reduce the accumulation of pathogens, such as bacteria and viruses (Cooney and Wise, 1975), and weed seeds or insect eggs, thus enable effluent hygienization (Ferrer et al., 2008). However, there are some limiting parameters in thermophilic anaerobic fermentation. Thermophilic anaerobic fermentation of animal waste fertilizers such as swine (Hansen et al., 1999), cattle (Sung, 2001), and poultry waste is not ideal. Many experiments showed that fermentation of high nitrogen raw materials with low C/N, such as swine manure (SM), easily led to ammonium nitrogen accumulation, which caused material liquid ammonia poisoning, inhibited digestion (Hansen et al., 1998; Salminem et al., 2002) and reduced gas production rate. On the contrary, fermentation of materials with high C/N, such as straw, was liable to produce fermented acid accumulation phenomenon, which caused the inhibition of CH4 bacteria activity, the decrease of organic
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carbon conversion (Poliafico and Murphy, 2007) and the extension of processing time. Thermophilic fermentation appeared to be more sensitive to pH than mesophilic fermentation for both hydrolysis and acidogenesis (Kim et al., 2003). Performance of fermentation with different materials varies with different initial pH conditions. Sutaryo et al. (2012) demonstrated that digesters can run with stable biogas production and low VFA levels using 30% of acidified dairy cow manure (SFDCM) instead of dairy cow manure (DCM) in thermophilic anaerobic co-digestion. But as the concentration of SFDCM increased, the methane concentration was significantly lower. An increased in acetic, butyric, and total VFA contents was caused by the acidified pig slurry content was more than 40% (Moset et al., 2012). The culture pH shift can cause the change of the dominant microbial populations affecting the main organic acids products (Horiuchi et al., 2002). The maximum hydrolysis and acidogenesis occurred when the pH was 6.5 (Kim et al., 2003). Therefore, a suitable pH is an important guarantee of anaerobic fermentation. In view of the environmental factors that affect the process of the thermophilic anaerobic digestion, such as initial pH, temperature and toxic substances (Sung and Liu, 2003), and the lack of the comprehensive study on these factors, to explore the optimal reaction conditions and optimize environmental factors became the target of this study. In this research, we investigated the thermophilic anaerobic fermentation of mixed swine manure (SM) and maize stalks (MS) with different ratios. The optimal initial pH value and fermentation ratio were obtained for future intensification of rural biogas fermentation. 2. Material and methods 2.1. Substrates and inoculum Fresh SM was collected from a local livestock farm near Northwest Agriculture and Forestry University (NW A & F U), Yangling, Shaanxi, China. Wood, debris and other garbage were removed and the maggots should be separated out from SM before being put into digesters. Maize stalks (MS) of Luodan 9, harvested a year ago from the experimental field of NW A & F U, were crushed into particles with diameter of 1 mm after dry grinding before being put into digesters. Anaerobic sludge used as inoculum was obtained from an anaerobic digester in a local biogas demonstration village in Yangling, Shaanxi, China. Table 1 shows the chemical characteristics of each material used in this study.
inoculum had been run in order to extract the inoculum’s endogenous methane yield from the results and the inoculum had been depleted the residual biodegradable organic material. Three different total solid (TS) ratios of SM content to total material (30%, 50%, and 70%) were set and each ratio was conducted at five initial pH levels (6.0, 6.5, 7.0, 7.5, and 8.0) with sodium hydroxide and hydrochloric acid. Each treatment had three repetitions. The initial pH value of the mixed raw materials was measured by Gasboard3200P (Table 1). Meanwhile unadjusted mixtures (CK) were anaerobically digested as no-treatment control group for every ratio. After mixing the anaerobic sludge, full stir was conducted to make good contact of raw materials and inoculum. Mixtures of SM and MS were poured into the lab-scale anaerobic digesters fabricated 1 L Erlenmeyer flasks. The co-fermentations of SM and MS were detected by batch digesters (Zhang et al. 2014). Every digester contained 500 g mixture and 200 g inoculum. All treatments had the same total solid content of 8%. Table 2 shows the actual amount in each conical flask. The fermentation flasks were placed in a thermostatic water bath with temperature of 55 °C ± 2 °C. The reactors were gently shaken before measuring biogas volume. The period of the fermentation was 35 days. Samples of the substrates, which had the best performance of biogas yield, were collected every five days and related indicators were measured. 2.3. Measurement In this study, TS, volatile solids (VS), pH, volatile fatty acid (VFA), alkalinity, and total ammonium nitrogen (TAN) of the digested biomass material were measured in accordance with the Standard Methods for the Examination of Water and Wastewater of the American Public Health Association (1995). Total carbon (TC) was analyzed using the method described in Song et al. (2012) and Cuetos et al. (2011). Drainage method was used to measure the volume of biogas yield every day and fast CH4 analyzer (Model DLGA-1000, Infrared Analyzer; Dafang, Beijing, China) was used to measure the CH4 content of the produced biogas. 2.4. Analysis and calculations The significances of each treatment and the second-order polynomial coefficients were analyzed through the analysis of variance (ANOVA) using SAS version 8.12 (SAS Institute Inc., Cary, NC, USA) and Design-Expert8.0.6 software. 3. Results and discussion
2.2. Experimental design and set-up 3.1. Daily biogas production The experiment was conducted in the digesters of our own design, which have been operated stably for several years (Wang et al., 2012). Before mixing the materials, all the substrates and inoculum were homogenized individually. Blank bottles with only
The daily biogas production of the treatments with three ratios of SM contents at five different initial pH values are shown in Fig. 1. Significant biogas yield peak values of most treatments appeared in
Table 1 Chemical characteristics of substrates. Substrate c
SM MSd Ine
TSa content (%)
VSb content/% of TS
pH value
Total carbon
Total Kjeldahl nitrogen (g/kg VS)
53.58 ± 2.14 96.55 ± 0.37 6.59 ± 0.45
70.01 ± 3.44 88.99 ± 0.63 53.56 ± 1.05
7.2 ± 0.2 ND 7.9
26.66 ± 0.99 33.03 ± 1.11 NDf
1.21 ± 0.025 0.96 ± 0.07 ND
Result = mean ± standard deviation (SD). a TS: total solid. b VS: volatile solids. c SM: swine manure. d MS: maize stalks. e In: inoculum. f ND: not determined.
T. Zhang et al. / Waste Management 35 (2015) 119–126 Table 2 Experimental design of mix component batch co-digestion sets (WWa/g).
a b
SM ratio/TS%
SM
MS
DWb
In
30% 50% 70%
23.98 39.96 55.94
29.97 22.18 13.31
446.05 437.86 430.75
200 200 200
WW: wet weight. DW: deionized water.
the early fermentation (the first 10 days). From the first day after seeding, aerogenesis started and kept growing. Most treatments reached their peak values in 3–4 days. When the process went into hydrolytic acidification, the daily biogas production reduced gradually as the pH went down below 6.0. Biogas yield increased with
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the raise of pH little by little after acid phase and of different treatments reached the second or third peak value at different times except the group with 30% SM ratio. Only one biogas yield peak value appeared in the treatments with SM ratio of 30%. After 13 days of the fermentation, biogas yields of all treatments were less than 200 mL. Unlike any other treatments in 30% SM ratio group, the daily biogas production peak value of pH 6.0 appeared in the 11th day (Fig. 1a). In the group with SM ratio of 50%, pH 7.0 daily biogas productions were significant higher than other treatments due to the pH rose over 7.0 after 10 days and there were four evident biogas yield peak values on days 2, 12, 17, and 25. But because of the low pH, other treatments’ biogas yield declined under 100 mL/d after 15 days. The rest treatments of this group at the late digestion (from 25 to 35 days) reduced gradually without significant fluctuations (Fig. 1b). The group with SM ratio of 70% was significantly different from the two groups above. The
Fig. 1. Daily biogas production of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
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pH of every treatment had risen up after acid phase, so in the middle (from 11 to 24 days) and late reaction, the daily biogas production in 70% SM ratio group kept active. Each treatment had bigger yield peak values than that in the first 10 days except pH 8.0 and CK treatments, and the durations of the yield peak value were different (Fig. 1c). According to the performance of daily biogas production, it can be concluded that pH can affect the biogas yield effects, such as the number and the durations of the biogas yield peak values, in thermophilic anaerobic co-digestion under different manure ratio. 3.2. Change of ammonia nitrogen under different conditions Compared with MS, SM has higher nitrogen contents. Lots of studies reported ammonia inhibition occurred during the biogas production progress particularly when mono-digestion of manure under thermophilic conditions (Hansen et al., 1998; Bujoczek et al., 2000). Previous studies have shown that ammonia concentrations greater than 1500 mg/L inhibited digestion when pH was higher than 7.4 (Siegrist et al., 2002). It is shown that the higher SM content, the higher ammonia content in response (Fig. 2). The ammonia content was instable in the early process, which led to VFA accumulation lowering the pH (Angelidaki and Ahring, 1993; Yenigün and Demirel, 2013). Ammonia nitrogen contents of all the treatments in SM content 30% groups declined slightly during the acidification phase in the early stages. Subsequently, ammonia contents began to rise and remained stable between 600 and 850 mL/L in the middle and late periods (Fig. 2a). Different from the group of 30% SM, ammonia nitrogen contents of only the pH 6.5 treatments in 50% and 70% SM were declined in the first 10 days, and showed secular upheaval in the rest of treatments (Fig. 2b and c). In the group with SM ratio of 30% and 50%, the trends of ammonia nitrogen content were smooth in the medium and period (Fig. 2a and b). In the treatments with SM ratio of 70%, three cases (pH 7.0, 7.5, and 8.0) had relatively similar ammonia contents to single SM fermentation in the middle of the digestion (Fig. 2c). At the end of digestion, no treatment had higher ammonia content than mono-digestion of manure compared with all the treatments. At the last phase of the fermentation, every ammonia level of the treatments lay in the thresholds of the mono-fermentation with manure and straw (Fig. 2). Overall, as the co-digestion proceeded, ammonia nitrogen content increased gradually. Accumulated ammonia generated in the degradation of the protein in SM would cause a certain degree of ammonia inhibition (Sung and Liu, 2003). In this study, no obvious ammonia inhibition was observed, which is similar to the research of Wang et al. (2012). 3.3. pH trend during fermentation Horiuchi’s et al. study showed that the main products of organic acids were changed by shifting the culture pH in the reactor (Horiuchi et al., 2001). Therefore, the trend of the pH change affected the daily biogas production to a certain degree. With same initial pH values, the pH resilience in the late stage of fermentation improved significantly as SM ratio increased. In the treatments with SM ratio of 30%, pH value of each treatment was lower than 6.0 in the fifth day after inoculation, which seriously affected the activity of methane bacteria. Since methane bacteria cannot survive in pH lower than 6.0 environment, at the late fermentation, the buffering capacity of the substrate was imbalance showing high alkalinity/VFA ratio. In the treatments with SM ratio of 50%, because of the accumulation of organic acids, the pH of each treatment apart from the initial pH 7.0 went to lower than 6.0. Only that of the initial pH 7.0 treatment recovered to larger than 7.0 after 10 days (Fig. 3a), and the alkalinity/VFA ratio was below 0.2 during
Fig. 2. Change of ammonia nitrogen of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
the 35 days’ digestion, so the total biogas yield of pH 7.0 treatment in 50% SM ratio group was much higher than other treatments in the same SM ratio group. In treatments with SM ratio of 70%, pH value of each treatment gradually recovered to above 7.0. Recovery speed of pH 8.0 treatment was the fastest, followed by 6.0, 7.5, 6.5, and 7.0. At the same, the low alkalinity/VFA ratio showed there was no excessive accumulation of organic acids and the environment of the substrate was suitable for bacteria activity. It can be speculated that increasing manure content would help enhance
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Fig. 3. Change of pH of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
the buffering capacity of the substance, reducing the impact of organic acids accumulation of the fermentation process (Fig. 3b). The fermentation of 70% SM group had final pH values ranging from 7.4 to 8.2 (Fig. 3c). Thus, the biogas yield of treatments with 70% SM content was significantly better than other treatments with same pH and in the same SM group the biogas yield of initial pH 7.0 treatments was better than other treatments. 3.4. Effect of SM ratios on VFA/alkalinity Decrease of the buffering capacity caused by the VFAs’ accumulation is earlier than the pH decreases (Chen et al., 2014), so VFA/ alkalinity ratio were used in this research as a more reliable parameter for monitoring fermentation condition imbalance. Studies have shown that the ratio of volatile fatty acid (VFA) to alkalinity in a range of 0.3–0.5 was optimum for methanogens (Liew et al., 2011). The VFA/alkalinity ratios of all treatments in the first 25 days were lower than 0.4 (Fig. 4). The accumulation of organic acids caused the VFA/alkalinity ratios raised and declined the pH, so in the groups with SM ratios of 30% and 50% (except 50% pH 7.0 treatment), VFA/alkalinity ratios raised sharply after the 25th day (Fig. 4a and b). However, the VFA/alkalinity ratios of treatments with SM ratio of 70% remained in the appropriate range (Fig. 4c), which showed the pH of the substrate was suitable for anaerobic digestion. Combined with the daily biogas production, the optimum VFA/alkalinity ratio for co-digestion of SM/MS was 0.1–0.3. When the VFA/alkalinity ratio hovered from 0.3 to 0.8, the buffering capacity of co-digestion system was restricted to a certain extent. While when VFA/alkalinity ratio was larger than 0.8, the buffering capacity of the system was not enough for fermentation. SM could be used as cushioning material to improve the buffering capacity of the co-digestion system. But it is worth noting that ammonia inhibition is prone to accrue when the ratio of SM is too high.
The variance analysis on effects of SM content and initial pH on the total biogas yield was conducted using SAS software. Tests had proved that data followed a normal distribution. The results showed that the SM ratio and pH as well as their interaction had significant impacts on total gas yield. Based on the total biogas yield of each treatment (Table 3), twofactor analysis of variance for pH and manure ratio is showed in Table 4. From the data in Table 4, it is obvious that pH has significant effects on total biogas yield (Table 5) and the ratio and proportion and its interaction effects with pH reach highly significant. Shifting the initial pH in the digesters from 6.0 to 8.0 by step, the total biogas products showed a trend that decrease came after increase. At the same initial pH, the performances of 70% SM ratio were the best. The total biogas yield of 70% SM ratio was significantly higher than that of the other two ratios. It can be speculated that pH has a very significant effect on the thermophilic anaerobic co-digestion and optimal yields could be obtained by optimizing conditions of the manure ratio and pH. The ratio of SM and initial pH value were designed as factors influencing the total methane volume. Design-Expert software for response surface was used to analyze the relationship between these two factors and total biogas yield (Table 6). Correlation regression equation models were achieved, and 3D map was drawn accordingly. 3.6. Two factors correlation regression equation In order to optimize the studied parameters, response surface methodology (RSM) was used in this study. The methane yield per unit volume was dependent output responses. The following second-order polynomial model showing the estimating coefficients based on experimental data was used to describe the functional relationships between responses (Y) and the set of factors (A and B).
Y ¼ b0 þ b1 A þ b2 B þ b3 AB þ b4 A2 þ b5 B2
ð1Þ
3.5. Modeling and optimization of methane yield From Fig. 5, the accumulative biogas production of treatments with various SM content changed differently. Biogas yield increased with the pH rising when the SM content was 30%. When SM content rose to 50%, the biogas yield of pH 7.0 was significantly higher than others, followed by the 6.5, 7.5, CK, 8.0, and 6.0. S-shaped growth curves were appeared in the SM ratio of 70% different from other conditional control group. In addition to CK group, the rest of the treatments had a second high biogas yield after the 15th days. Through the regression analysis on the influences of the two factors on the biogas yield, the following equations were obtained.
where Y represents the predicted response, A is the ratio of SM; B is the pH; b0 is a constant; b1 and b2 are linear coefficients; b3 is interaction coefficients and b4 and b5 are quadratic coefficients. 3.7. Final equation in terms of coded factors
Total methane yield ¼ þ54:63 þ 44:34 A þ 2:50 B 25:72 A B þ 35:07 A2 333:91 B2 ð2Þ The model was implied significant by the F-value of 10.24 and the R-squared was 82.31%. Both interaction factor and quadratic
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Fig. 4. Change of VFA/ alkalinity of treatments with different SM contents (a) 30% SM, (b) 50% SM and (c) 70% SM.
factors emerged a significant effect for the ratio of SM and the pH over the response among all the evaluated factors. Using Design-Expert software to optimize the design of the model, the optimal conditions for maximum methane potential (Fig. 6) were calculated as a SM ratio of 70% and a pH of 6.81. Accordingly, the maximum total methane yield was expected to reach 146.32 mL/g VS. Compared with the results that the highest methane yield of 145.98 mL/g VS was appeared in pH 7.0 treatment of SM 70% in this study, the optimal conditions for maximum
Fig. 5. Accumulative biogas production of treatments with different SM contents (a) 30% SM, (b) 50% SM, and (c) 70% SM.
Table 3 Total biogas yield of each treatment (mL/g VS). SM ratio/TS%
30% 50% 70%
pH 6.0
6.5
7.0
7.5
8.0
CK
62.53 22.19 240.41
88.27 129.11 285.59
84.58 215.01 307.68
92.38 92.49 296.28
87.33 58.75 203.40
89.77 84.90 273.61
T. Zhang et al. / Waste Management 35 (2015) 119–126 Table 4 The two-factor variance analysis of the total biogas yield. Source
df
Sum of squares
Mean square
F value
Model Error Corrected total
17 36 53
412441.2406 18179.8013 430621.0419
Source
df
pH Ratio pH * ratio
5 2 10
Pr > F
24261.2494 504.9945
48.04
Anova SS
Mean square
F value
Pr > F
59006.6033 319187.8381 34246.7992
11801.3207 159593.9190 3424.6799
23.37 316.03 6.78
