Effect of Lipase Hydrolysis on Biomethane Production from Swine

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Effect of Lipase Hydrolysis on Biomethane Production from Swine Slaughterhouse Waste in China Zhifang Ning,† Jinli Ji,† Yanfeng He,† Yan Huang,† Guangqing Liu,*,† and Chang Chen*,†,‡ †

Biomass Energy and Environmental Engineering Research Center, College of Chemical Engineering and ‡College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China ABSTRACT: The purpose of this study was to evaluate biomethane production in anaerobic digestion of swine slaughterhouse waste (SSW) and the effect of lipase pretreatment on biomethane yield. Lipase extracted from porcine pancreas was employed to hydrolyze SSW at 37 °C for 24 h. Three different lipase concentrations of 0.04, 0.2, and 0.4% (w/v) were adopted. Anaerobic digestion was carried out in batch style for 50 days. The results showed that the highest experimental biomethane yield (EMY) of 851.6 mL/g of VSadded was achieved when the lipase concentration of 0.2% (w/v) was used, which was significantly higher (18.4%; α < 0.05) than that of the untreated group. Furthermore, the highest biodegradability (Bd) of 72.7% was also achieved at 0.2% (w/v) lipase-pretreated group. Results proved that the conversion of SSW to biomethane was improved through pretreatment of lipase hydrolysis and offered a promising method for disposal of SSW.

1. INTRODUCTION Pork, as the most commonly consumed meat in China, has played an irreplaceable role in the traditional diet for a long time. China has the highest production of pork around the world,1 and the demand of pork is increasingly growing with the rapid development of the Chinese economy and living standard. Simultaneously, a huge amount of swine slaughterhouse waste (SSW) was generated every year. SSW in China is very different from slaughterhouse waste (SW) in other countries. Besides lipid, SW in other countries might be rich in protein because protein-rich animal entrails, such as lung, liver, heart, intestine, and kidney, are discarded. However, these are favorite cooking materials for food according to traditional eating habits in China, and thus, they are not contained in SSW. SSW in China is lipid-rich waste, which can cause pollution and is very difficult to degrade as a result of its complex chemical structure and high fat content. In addition, some lawbreaking traders recycled SSW and processed it into illegal cooking oil to sell, which is harmful to the human body as a result of existing toxic and cancerogenic substances.2,3 SSW, if not treated properly, not only can pollute the environment but is also a threat to health. Common methods for the disposal of SW in many countries, including rendering, composting, and alkaline hydrolysis, have negative effects, such as health risks from the presence of pathogens and high energy consumption.4 Anaerobic digestion through which organic waste could be decomposed into methane and carbon dioxide has been reported as an advantageous method for treating SW over the other processes.4,5 Anaerobic digestion is a simple operational process for treating biowaste without much pollution and investment cost.6−8 Besides, anaerobic digestion not only produces biogas, which can be converted to energy, but also produces digestate, which can be used as a valuable fertilizer. Anaerobic digestion of SW has been tried in other countries, and most of the substrates were poultry and cattle SW.9−13 However, little research was reported about anaerobic digestion of SSW in China. Considering the huge amount of SSW in © XXXX American Chemical Society

China and the difference between SSW in China and SW in other countries, it is necessary to investigate biomethane production from the lipid-rich SSW through anaerobic digestion. It was reported that lipid-rich waste was first hydrolyzed into long-chain fatty acids (LCFAs) and glycerol and further converted to hydrogen and acetate by acetogenic bacteria and then finally to methane by methanogenic Archaea in anaerobic digestion. However, lipid-rich substrates can inhibit anaerobic digestion efficiency as a result of the complicated structures.14 In recent years, the pretreatment effects of lipase on kitchen waste and oily wastewater were studied by some researchers to enhance bioconversion in anaerobic digestion. For example, Adriano et al. reported that the organic removal rate reached the top (78.2%) in the anaerobic treatment of dairy industrial wastewater after lipase hydrolysis.15 According to Ying et al., biogas production was increased by 4.97−26.50% when kitchen waste was hydrolyzed by lipase.16 Few research, however, was reported thus far about the effects of lipase pretreatment on anaerobic digestion of SSW, which is, therefore, very necessary to be carried out. The purpose of this study was to investigate biomethane production potential from SSW in China and whether lipase hydrolysis is an effective pretreatment method to improve the conversion of SSW into biomethane.

2. MATERIALS AND METHODS 2.1. Materials. Substrate used in this experiment was SSW obtained from a slaughterhouse in Beijing, China, which was then crushed into mud and kept at −20 °C for future use. Inoculum was anaerobic sludge obtained from the Donghuashan Biogas Plant in Beijing, China. A commercial lipase isolated from pig pancreas was purchased from Tokyo Chemical Industry (Shanghai, China) and used Received: May 6, 2016 Revised: July 25, 2016

A

DOI: 10.1021/acs.energyfuels.6b01097 Energy Fuels XXXX, XXX, XXX−XXX

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Energy & Fuels

2.6. Kinetic Modeling. The modified Gompertz model (eq 5) and Cone model (eq 6) have been widely applied in modeling the biomethane yield22 and were adopted in this study. In this way, the potential of cumulative biomethane production could be described and predicted for later analysis

in this study. The optimum temperature and pH of this lipase are around 37 °C and 7, respectively. 2.2. Lipase Hydrolysis of SSW. Hydrolysis reactions were carried out in serum bottles containing 5.46 g of SSW, 125 mL of phosphatebuffered saline (PBS, 0.01 M), and different dosages of lipase. Three different concentrations (0.04, 0.2, and 0.4%, w/v) of lipase were adopted, and then the experiment was conducted in an incubator at 37 °C for 24 h. 2.3. Anaerobic Digestion. To test biodegradability, an organic loading at 20 g of VS/L of SSW was anaerobically digested in 500 mL serum bottles. Seed sludge was loaded into the bottles to adjust a substrate/inoculum (S/I) ratio to 1:1.17 After the required materials were loaded, tap water was added to each bottle to ensure that the working volume reached 250 mL and nitrogen was blowed up to the rest of the space to create an anaerobic condition. After sealed, the bottles were kept in an incubator at a temperature of 37 °C for 50 days and were shaken 5 times a day. Blank experiments including sludge, a corresponding amount of lipase, and PBS for all experimental groups (untreated and 0.04, 0.2, and 0.4% treated) were carried out. Specific biogas production and biomethane content were measured every day. 2.4. Analytical Methods. The total solid (TS) and volatile solid (VS) contents in SSW were determined according to the standard methods.18 The pH values were measured by a pH meter (Mettler Toledo, Columbus, OH). The crude lipid content was determined by the acid hydrolysis method.19 Elemental compositions (C, H, N, and S) were measured using an organic element analyzer (Vario EL cube, Germany). The oxygen content was determined using a 2400 II oxygen analyzer (PerkinElmer Instruments, Waltham, MA). Biogas composition and ammonia nitrogen (NH3 N), total alkalinity (TA), and volatile fatty acid (VFA) concentrations were measured according to reported methods.20 The absolute pressure difference in the head space of each reactor was measured at 37 °C (T = 310.15 K) using a WAL-BMP-Test system pressure gauge (type 3150, Wal, Germany), which has an accuracy of 0.1%. The daily biogas volume under standard temperature and pressure (Vbiogas, L) was calculated on the basis of eq 1 after the measurement of pressure difference21 Vbiogas (L) =

ΔPVheadC RT

⎧ ⎡μ e ⎤⎫ B = B0 exp⎨ − exp⎢ m (λ − t ) + 1⎥⎬ ⎣ B0 ⎦⎭ ⎩

B=

B0 1 + (kt )−n

(6)

characteristic

SSW

inoculum

crude fat (%)a TS (%)a VS (%)a VS/TS (%) C (%)c H (%)c O (%)c N (%)c S (%)c C/N

83.56 91.70 ± 0.05 91.52 ± 0.03 99.80 ± 0.03 74.12 18.03 6.38 1.18 0.05 62.81

NDb 5.49 ± 0.01 2.75 ± 0.02 50.18 ± 0.04 ND ND ND ND ND ND

a

As total weight of the sample. bND = not determined. cAs TS of the sample.

91.52%, respectively. The ratio of VS/TS in SSW reached 99.80%, which indicated a very high organic content and implied a great potential in biomethane production. The crude fat content was 83.56% TS, higher than some widely used feedstock, such as food waste, which might result in a long digestion time. SSW had a carbon/nitrogen (C/N) ratio of 62.81, which was significantly higher than the reported appropriate range of 13.0−28.023 as a result of the high lipid content in SSW in China. TMP was calculated as 1170.92 mL/ g of VSadded, which showed good capacity to produce biomethane in anaerobic digestion. 3.2. Anaerobic Digestion Performance. Daily biogas production of SSW after hydrolysis at different concentrations of lipase is shown in Figure 1a. It was observed that the trends of daily biogas production at different lipase concentrations (untreated and 0.02, 0.2, and 0.4% treated) were similar. Biogas increased every day during the first 20 days. It is obvious that there is a peak in each curve around the 20th day, which represents the highest daily biogas production, and after that, the production of biogas decreased until it stopped. However, four curves showed different trends in detail, which could be

(4n − a − 2b + 3c) H 2O 4 (4n + a − 2b − 3c) (4n − a + 2b + 3c) → CH4 + CO2 8 8 (2) (4n + a − 2b − 3c)

(3)

The experimental biomethane yield (EMY, mL/g of VSadded) means the maximum cumulative biomethane yield, which is the sum of the daily biomethane yield. The daily biomethane yield was calculated on the basis of Vbiogas and methane content. Biodegradability (Bd) was calculated according to TMP and EMY in eq 4.

EMY × 100% TMP

(5)

Table 1. Characteristics of SSW and Inoculum

CnHaOb Nc +

Bd =

⎪

3. RESULTS AND DISCUSSION 3.1. Characteristics of SSW and Inoculum. The characteristics of SSW and inoculum are presented in Table 1. The determined contents of TS and VS were 91.7 and

where ΔP stands for the absolute pressure difference (mbar), Vhead refers to the volume of the head space (L), C is the molar volume [at standard temperature and pressure (STP), 22.41 L mol−1], R means the universal gas constant (83.14 L mbar K−1 mol−1), and T is the absolute temperature (K). 2.5. Methane Production and Biodegradability. TMP (mL/g of VSadded) is the predicted theoretical methane potential. The molecular formula of CnHaObNc obtained by measured elemental composition was used to represent the organic content of the SSW, and then TMP was calculated by the Buswell formula in eqs 2 and 3.20

⎛ mL of CH4 ⎞ 22.4 × 1000 × 8 TMP ⎜ ⎟= 12n + a + 16b + 14c ⎝ g of VS ⎠

⎪

⎪

where B represents the simulated cumulative biomethane yield (mL/g of VSadded), B0 stands for the simulated maximum cumulative biomethane yield (mL/g of VSadded), μm refers to the maximum biomethane production rate (mL g−1 of VSadded day−1), λ is the lag phase time (days), t means the incubation time (days), e is equal to 2.72, k is the rate constant (day−1), and n is the shape factor. 2.7. Statistical Analysis. All of the experiments were conducted in triplicates. One-way analysis of variance (ANOVA) was adopted to statistically analyze the results of this study by OriginPro 8.0 (OriginLab, Northampton, MA).

(1)

+ c NH3

⎪

(4) B

DOI: 10.1021/acs.energyfuels.6b01097 Energy Fuels XXXX, XXX, XXX−XXX

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Figure 1. Anaerobic digestion performance of SSW hydrolyzed by different concentrations of lipase: (a) daily biogas production, (b) biomethane contents, and (c) daily biomethane production.

manifested by the biogas peak values. The daily biogas production peaks of 65.8 and 61.8 mL/g of VSadded were achieved at lipase concentration of 0.4 and 0.2%, respectively, which were significantly higher than 53.9 and 50.7 mL/g of VSadded achieved at concentration of 0.04% and the untreated, respectively. In conclusion, lipase pretreatment caused the increase of biogas production. Biomethane contents and daily biomethane production are shown in panels b and c of Figure 1, respectively. There seemed no obvious difference on biomethane contents among the four groups, as shown in Figure 1b. There was a rapid growth in biomethane contents in the first 20 days, and it remained stable in the range of 60−70% in all groups until the end of the process. No obvious side effect of lipase pretreatment on the biomethane contents was observed. As shown in panels a and c of Figure 1, the trend of daily biomethane production was similar to daily biogas production. The highest daily biomethane productions of 42.7 and 45.8 mL/g of VSadded were achieved around the 20th day at the concentrations of 0.2 and 0.4%, respectively, significantly higher than that of the untreated (33.9 mL/g of VSadded). 3.3. Cumulative Biomethane Yield. The cumulative biomethane yields are shown in Figure 2. In the beginning, the increase of the cumulative biomethane yield in all groups was slow, which was in accordance with other literatures treating lipid-rich waste, and the mechanism was reported as accumulation of LCFAs.24−26 After a lag phase time, the cumulative biomethane yield increased significantly from the 10th day to the 35th day, during which a significant amount of biomethane was produced. However, the growth between the 35th day and the 40th day slowed considerably, and the

Figure 2. Cumulative biomethane yields of SSW pretreated by different concentrations of lipase.

increase of the cumulative biomethane yield during the last 10 days was little. The whole process is consistent with reported literatures treating lipid-rich waste.16,27,28 All groups showed high EMY, which proved a larger potential of biomethane production from SSW than food waste.29 The highest EMY of 851.6 mL/g of VSadded was achieved at the 0.2% lipasepretreated group. Moreover, the highest EMY was significantly higher (18.4%; α < 0.05) than that of the untreated SSW, which showed a positive effect of lipase pretreatment. 3.4. Evaluation of Process Stability. At the end of anaerobic digestion, NH3 N, pH, TA, VFAs, and VFA/TA were analyzed to evaluate the stability of the anaerobic system, and C

DOI: 10.1021/acs.energyfuels.6b01097 Energy Fuels XXXX, XXX, XXX−XXX

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Energy & Fuels Table 2. Effluent Characteristics after Anaerobic Digestion for Lipase-Pretreated SSWa lipase concentration (%, w/v) TS reduction (%) untreated 0.04 0.2 0.4 a

VS reduction (%)

55.9 60.8 67.3 65.7

initial pH final pH NH3 N (mg/L)

73.8 76.9 79.4 78.4

6.96 6.83 7.59 7.62

7.89 7.95 7.95 8.05

1650 1585 1825 1745

± ± ± ±

TA (mg of CaCO3/L)

28 14 17 12

6900 8100 9000 9167

± ± ± ±

283 141 142 289

VFAs (mg/L)

VFA/TA

± ± ± ±

0.0018 0.0021 0.0027 0.0020

35.2 41.1 53.6 39.9

3.2 6.8 5.5 5.8

NH3 N, ammonia nitrogen; TA, total alkalinity; and VFAs, volatile fatty acids.

Figure 3. Modified Gompertz plots and Cone plots of cumulative biomethane yields for lipase-pretreated SSW.

Table 3. Kinetic Parameters of Biomethane Production from SSWa modified Gompertz model

Cone model

lipase concentration (%, w/v)

B0 (mL/g of VSadded)

μm (mL g−1 of VSadded day−1)

λ (days)

R2

B0 (mL/g of VSadded)

k (day−1)

n

R2

EMY (mL/g of VSadded)

Bd (%)

untreated 0.04 0.2 0.4

746.2 773.3 883.3 857.9

30.6 32.8 36.1 36.8

10.1 9.8 9.5 10.2

0.999 0.999 0.998 0.997

778.5 804.1 924.1 889.3

0.042 0.042 0.044 0.044

3.37 3.39 3.24 3.62

0.999 0.999 0.999 0.998

719.1 745.7 851.6 833.2

61.4 63.6 72.7 71.1

a B0, simulated maximum cumulative biomethane yield (mL/g of VSadded); μm, maximum biomethane production rate (mL g−1 of VSadded day−1); λ, lag phase time (days); EMY, experimental biomethane yield (maximum cumulative biomethane yield, mL/g of VSadded); Bd, biodegradability (%); k, rate constant (day−1); and n, shape factor.

yield. The B0 values in the two models were slightly higher than EMY of SSW. In the modified Gompertz model, the simulated maximum cumulative biomethane yield (B0, 883.3 mL/g of VSadded) and the shortest lag phase time (λ, 9.5 days) were appeared at the lipase concentration of 0.2%. In the Cone model, the simulated maximum cumulative biomethane yield (B0) of 924.1 mL/g of VSadded was also achieved when the lipase concentration was 0.2%. The highest Bd reached 72.7% in the 0.2% lipase-pretreated group, which showed a very high conversion efficiency.

the results are shown in Table 2. TS and VS reduction after 0.2% lipase pretreatment reached 67.3 and 79.4%, respectively, which were the highest values in all groups and indicated that lipase pretreatment could remove more TS and VS in SSW. The effluent pH values were in the range of 7.89−8.05, which were all located in the recommended range.30 There was no significant difference between lipase hydrolyzed groups and the untreated group in the NH3 N concentration and TA. VFAs (35.2−53.6 mg/L) were much less than the inhibited concentrations,31 and VFA/TA was also located in a preferred range. From the data point of view, the anaerobic digestion processes in all groups were stable and no obvious side effect was caused by the lipase pretreatment. 3.5. Modified Gompertz Model and Cone Model of Cumulative Biomethane Yield. The modified Gompertz model and Cone model were used to express the cumulative biomethane yield in Figure 3, and the calculated parameters are presented in Table 3. The R2 values of the two models were all between 0.997 and 0.999, which implied that both of the kinetic models were well-fit to describe the cumulative biomethane

4. CONCLUSION SSW in China showed a large potential in biomethane production. The lipase hydrolysis pretreatment significantly improved biomethane production from SSW (18.4%; α < 0.05). After 0.2% (w/v) lipase pretreatment, the highest EMY and Bd reached 851.6 mL/g of VSadded and 72.7%, respectively. The results of this study proved that lipase hydrolysis provided an effective pretreatment method for anaerobic digestion of D

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from oily effluents and sludges: An overview. Renewable Sustainable Energy Rev. 2015, 45, 351−358. (15) Mendes, A. A.; Pereira, E. B.; de Castro, H. F. Effect of the enzymatic hydrolysis pretreatment of lipids-rich wastewater on the anaerobic biodigestion. Biochem. Eng. J. 2006, 32 (3), 185−190. (16) Meng, Y.; Li, S.; Yuan, H.; Zou, D.; Liu, Y.; Zhu, B.; Li, X. Effect of lipase addition on hydrolysis and biomethane production of Chinese food waste. Bioresour. Technol. 2015, 179, 452−459. (17) Slimane, K.; Fathya, S.; Assia, K.; Hamza, M. Influence of Inoculums/Substrate Ratios (ISRs) on the Mesophilic Anaerobic Digestion of Slaughterhouse Waste in Batch Mode: Process Stability and Biogas Production. Energy Procedia 2014, 50 (50), 57−63. (18) American Public Health Association (APHA); American Water Works Association (AWWA); Water Environment Federation (WEF); Rice, E. W.; Bridgewater, L. Standard Methods for the Examination of Water and Wastewater; APHA: Washington, D.C., 2012; pp 113. (19) Wei, Y. Y. Determination of fat content in ham sausage by acid hydrolysis method. Meat Ind. 2010, No. 9, 21−22. (20) Li, Y.; Feng, L.; Zhang, R.; He, Y.; Liu, X.; Xiao, X.; Ma, X.; Chen, C.; Liu, G. Influence of inoculum source and pre-incubation on bio-methane potential of chicken manure and corn stover. Appl. Biochem. Biotechnol. 2013, 171 (1), 117−127. (21) El-Mashad, H. M.; Zhang, R. Biogas production from codigestion of dairy manure and food waste. Bioresour. Technol. 2010, 101 (11), 4021−4028. (22) El-Mashad, H. M. Kinetics of methane production from the codigestion of switchgrass and Spirulina platensis algae. Bioresour. Technol. 2013, 132, 305−312. (23) Romano, R. T.; Zhang, R. Co-digestion of onion juice and wastewater sludge using an anaerobic mixed biofilm reactor. Bioresour. Technol. 2008, 99 (3), 631−637. (24) Cavaleiro, A. J.; Pereira, M. A.; Alves, M. Enhancement of methane production from long chain fatty acid based effluents. Bioresour. Technol. 2008, 99 (10), 4086−4095. (25) Cirne, D. G.; Paloumet, X.; Björnsson, L.; Alves, M. M.; Mattiasson, B. Anaerobic digestion of lipid-rich wasteEffects of lipid concentration. Renewable Energy 2007, 32 (6), 965−975. (26) Noutsopoulos, C.; Mamais, D.; Antoniou, K.; Avramides, C.; San Miguel, G.; Rincon, S.; Vagiona, D. Increase of biogas production through co-digestion of lipids and sewage sludge. Global NEST J. 2012, 14 (2), 133−140. (27) Cherif, S.; Aloui, F.; Carrière, F.; Sayadi, S. Lipase PreHydrolysis Enhance Anaerobic Biodigestion of Soap Stock from an Oil Refining Industry. J. Oleo Sci. 2014, 63 (2), 109−114. (28) Prabhudessai, V.; Salgaonkar, B.; Braganca, J.; Mutnuri, S. Pretreatment of cottage cheese to enhance biogas production. BioMed Res. Int. 2014, 2014, 1−6. (29) Long, W.; Baoning, Z.; Hairong, Y.; Yanping, L.; Dexun, Z.; Xiujin, L. Comparative investigations on pilot-scale anaerobic digestion of food waste at 30 °C and 35 °C. Int. J. Agric. Biol. Eng. 2016, 9 (1), 109−117. (30) Brown, D.; Li, Y. Solid state anaerobic co-digestion of yard waste and food waste for biogas production. Bioresour. Technol. 2013, 127, 275−280. (31) Shen, F.; Yuan, H.; Pang, Y.; Chen, S.; Zhu, B.; Zou, D.; Liu, Y.; Ma, J.; Yu, L.; Li, X. Performances of anaerobic co-digestion of fruit & vegetable waste (FVW) and food waste (FW): Single-phase vs. twophase. Bioresour. Technol. 2013, 144, 80−85.

SSW and might be a practical way to be employed in future industrial applications.

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AUTHOR INFORMATION

Corresponding Authors

*(G. Liu) E-mail: [email protected]. *(C. Chen) Fax: +86-10-64442375. E-mail: chenchang@mail. buct.edu.cn, 505A Zonghe Building, 15 North Third Ring East Road, Beijing 100029, China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Technology Support Program of China (2012BAC25B06 and 2012BAC25B05) and the Fundamental Research Funds for the Central Universities (YS1407).

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REFERENCES

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DOI: 10.1021/acs.energyfuels.6b01097 Energy Fuels XXXX, XXX, XXX−XXX