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Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3 e1 6 4 7 8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures Chyi-How Lay a,b, I-Yuan Sung a, Gopalakrishnan Kumar a, Chen-Yeon Chu b,c,d, Chin-Chao Chen e, Chiu-Yue Lin a,b,c,d,* a
Department of Environmental Engineering and Science, Feng Chia University, Taiwan Green Energy Development Center, Feng Chia University, Taiwan c Department of Chemical Engineering, Feng Chia University, Taiwan d Master’s Program of Green Energy Science and Technology, Feng Chia University, Taiwan e Department of Landscape Architecture, Chungchou University of Science and Technology, Taiwan b
article info
abstract
Article history:
The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients.
Received 7 December 2011
After being used for growing mushroom for one to two weeks this bag becomes mushroom
Received in revised form
cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced
19 February 2012
annually in Taiwan and are usually burned or discarded. The cellulosic materials and
Accepted 23 February 2012
nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen
Available online 1 April 2012
fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra
Keywords:
nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature
Biohydrogen
55 C and an initial cultivation pH of 8. The results show that extra nutrient addition
Mushroom cultivation waste
inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using
Anaerobic mixed cultures
cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H2/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H2/L/d and 2.02 mmol H2/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Today’s energy system is mainly based on the fossil fuels which are depleting and cannot be sustainable. In view of the alternative fuels to overcome the future energy demand, hydrogen is an attractive fuel because of various features such
as clean, efficient, renewable, and does not generate any toxic by product on its combustion [1]. Biological hydrogen production process is one of the main alternative methods. Producing H2 via fermentative route is more environmental friendly and less energy intensive, thereby being competitive to conventional H2 production methods such as thermo-
* Corresponding author. Department of Environmental Engineering and Science, Feng Chia University, 100 Wenhwa Road, Seatwen, Taichung, Taiwan 40724, Taiwan. Tel.: þ886 4 24517250x6200; fax: þ886 4 35072114. E-mail address: [email protected] (C.-Y. Lin). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.02.135
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were heat pretreated at 95 C for 1 h to inhibit hydrogenconsuming bacteria. MCW was collected from a mushroom farm in Changhwa (central Taiwan). The waste was dried at 105 C for zero moisture and then powdered to pass 0.297 mm-mesh sieve. The characteristics of the mushroom waste are shown in Table 2.
chemical means [2]. The substrates used in fermentative hydrogen production are generally rich in carbohydrates like glucose, sucrose and starch [3]. Currently, biohydrogen and other biofuels are produced from various agricultural and bioenergy-generation residues. At present the biomass from crops like sweet sorghum, potatoes, sugarcane, soybean and palm oil have been used as raw materials for bioethanol or biohydrogen production [4e8]. The mushroom waste has attained considerable attention because it is available abundantly and easy to collect and use as feedstock for the biohydrogen process. Mushroom bag is a polypropylene bag stuffed with wood flour and nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW) About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. Therefore, proper treatment or usage of this waste could avoid the environmental problems. Moreover, recent studies show that this waste could be a feedstock for biohydrogen production in effective way [11]. The advantages of using MCW feedstock for hydrogen production include cost effective because of this waste being needed to be treated for reducing environmental pollution problems. However, it has disadvantages such as further disposal of fermentation residue and solid fermentation is different from liquid fermentation in operation. Fermentative hydrogen production process is a tedious process and requires optimization of inoculums type, pretreatment, substrate nature and composition, pH and temperature to scale-up the process. Inoculum selection and pretreatment are important. Several types of inoculum have been reported such as sewage sludge [9] and cow dung [10]. Pretreatment of the mushroom cultivation waste by using acid was also studied [11].
2.
Materials and methods
2.1.
Seed inocula and substrate
2.2.
Experimental design and procedure
Batch hydrogen production experiments were performed using serum bottles (125 mL) with anaerobic head space. The vials were first purged with argon gas followed by adding 10 mL of seed inocula, 40 mL of de-ionized water or nutrient solution, 10 mL of pH adjustment solution (1 N HCl or 1 N NaOH) and tested dried MCW agricultural powder ranging 1.2 g per 60 mL working volume (20 g/L). These vials were placed in a reciprocal air-bath shaker (150 rpm) with a cultivation temperature of 55 1 C. The tested initial pH value was 8.0. The nutrient solution contained inorganic supplements (mg/L): NH4HCO3 5240, K2HPO4 125, MgCl2$6H2O 100, MnSO4$6H2O 15, FeSO4$7H2O 25, CuSO4$5H2O 5, CoCl2$5H2O 0.125 and NaHCO3 6720 [12]. Each experimental condition was carried out with duplicate.
2.3.
Analytical method
The analytical procedures of APHA Standard Methods [13] were used to determine pH, oxidation-reduction potential (ORP), total chemical oxygen demand (TCOD), ammonia nitrogen (NH3eN), suspended solids (SS) and volatile SS (VSS). Biogas volume was determined by a gas tight syringe at room temperature (20 C) and a pressure of 760 mm Hg. The biogas composition in the batch enrichment assays was measured with a CHINA Chromatography 8700T gas chromatograph equipped with a packed (packing, Porapak Q), stainless steel column and a thermal conductivity detector. Oven, injector and detector temperatures were 40, 40 and 40 C, respectively and argon as the carrier gas. Same methods are indicated in our previous studies [14]. Anthrone-sufuric acid method was used to measure total carbohydrate concentration [14]. Cellulose, hemicelluloses and lignin were determined by FIBERTECTM 1020 (M6). Elemental analysis was performed on an Elemental Analyzer (Model: Vario EL). Hydrogen production potential (P, mL), maximum hydrogen production rate (Rm, mL/h) and lag phase time (l, h) obtained from the modified Gompertz equation (Eq. (1)) [14]
The seed inocula were collected from two municipal wastewater treatment plants (SS1and SS2), cow dung compost (CD) and pig slurry (PS) located in central Taiwan. The pH, total COD (chemical oxygen demand), soluble COD, total carbohydrate, volatile suspended solids (VSS, to express the biomass concentrations), total solids and NH3eN concentrations of the seed inoculaums are listed in Table 1. The collected sludges
Table 1 e Characteristics of seed inoculms. Seed
pH
Total COD
Soluble COD (mg COD/L)
S1 S2 C P
6.9 7.1 7.4 7.0
0.1 0.1 0.1 0.1
54,400 70,480 65,680 77,200
226 1471 113 1018
2020 2027 7200 17,280
28 46 339 339
Total carbohydrate (mg/L as glucose) 6424 7054 10,641 2380
92 215 226 31
S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung; COD, chemical oxygen demand.
VSS (g/L)
NH3eN (mg/L)
21 32 22 17
75 124 276 243
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Table 2 e Characteristics of mushroom waste. TS* (g/L)
VSa (g/L)
TCODa (G-COD/L)
Total Carbohydratea (g/L)
Celluloseb (%)
Hemicelluloseb (%)
Ligninb (%)
0.84
0.78
1.01
0.46
31.2
6.9
14
Ultimate analysis wt % b
C/N ratiob
C
H
N
S
40.2
5.0
1.4
0.2
28.7
a Tested by the agricultural waste solution with 1 g dried biomass in 1 L solution. b Tested by dried biomass.
were used as the response variable. STATISTIC software (version 6.0, Statsoft Inc., USA) and Sigmaplot software (trial version 9.0, Systat Software Inc., USA) were used for regression and graphical analyses of the data, respectively. Rm $e ðl tÞ þ 1 HðtÞ ¼ P$exp exp P
(1)
H(t) is the cumulative hydrogen production (mL); P is the hydrogen production potential (mL); Rm is the maximum hydrogen production rate (mL/h); e is 2.71828; l is the lag phase time (h) and t is the cultivation time (h). The maximum hydrogen production rate (HPRmax, mmol H2/L-d) was defined as hydrogen production per working volume per cultivation time and calculated based on the maximum hydrogen production rate (Rm, mL/h) obtained from Gompertz equation. Specific hydrogen production rate (SHPRmax, mmol H2/g VSSd) was defined as HPR divided by initial VSS of seed inoculums (5 g/L). Hydrogen production yield (HY, mmol H2/g TVS) was defined as hydrogen production per gram of dried MCW.
3.
Results and discussion
3.1.
Hydrogen generation
Table 3 shows that the nutrient addition could enhance the total biogas production comparing with without extra nutrient for the same seed inoculum. On the contrary, the hydrogen contents in biogas of no nutrient addition were higher than that of with extra nutrients for the same seed inoculum. The
peak hydrogen production of 24.2 mL was obtained using cow dung seed inoculum without any Endo nutrient (Fig. 1a and Table 3). However, the hydrogen production was less than 10 mL for other seed inoculum. Moreover, the maximum biogas and hydrogen production were 150 mL and 39.5 mL, respectively, with nutrient addition and no seed inoculum (EB, Endogenous bacteria) (Fig. 2b and Table 3). A peak HY of 0.73 mmol H2/g TVS was obtained with no inoculum at nutrient addition (Table 3). This HY value was about 3 times of HY 0.28 mmol H2/g TVS which was obtained in beer lees fermentation at 36 C and pH 7.0 by cow dung compost [15]. However, peak HPR and SHPR of 10.11 mmol H2/ L/d and 2.02 mmol H2/g VSS/d, respectively, were obtained by using cow dung inoculum without extra nutrients. This value is higher than that of converting other agricultural wastes into hydrogen (Table 4). Fig. 3 depicts that nutrient addition inhibited HPR and HY when using cow dung and pig slurry seeds. Biohydrogen production requires certain essential micro-nutrients such as nitrogen (N), phosphate (P), and some trace elements for bacterial metabolism, growth and activity [16]. A proper C/N-ratio value for mixed microflora is necessary to optimize anaerobic hydrogen production from organic wastewater. The reason of low HY for cow dung and pig slurry might be that too high nutrient concentration (such as nitrogen) which inhibited the fermentation (Fig. 3). However, nutrient addition elevated HPR and HY in the case of using sewage sludge inoculum and without inoculum. Using cow dung seed has the most significant increase (240%) in HPR and SHPR. On the other hand, the HY increased from 0.04 mmol H2/g TVS with extra nutrients to 0.12 mmol H2/g TVS without
Table 3 e Biogas production using various seed inocula without and with nutrients. Seed
SS1 SS2 CD PS EB
H2 Nutrient Total H2 formulation biogas conc. (mL) (%) (mL)
Without With Without With Without With Without With Without With
21.0 67.3 19.7 73.0 51.5 72.3 20.0 34.3 13.7 150.0
38.1 25.6 38.1 27.1 47.0 16.7 32.0 7.0 30.7 26.3
8.0 17.2 7.5 19.8 24.2 12.1 6.4 2.4 4.2 39.5
Modified Gompertz equation parameter P R (mL) (mL/h)
l (h)
R2
7.8 17.7 7.5 20.4 24.3 12.1 6.8 2.3 4.4 40.5
3.8 3.8 4.6 4.3 26.2 18.1 3.2 9.9 5.9 21.5
0.9707 0.9795 0.9785 0.9831 0.992 0.9981 0.9834 0.9789 0.9674 0.9966
0.20 0.21 0.15 0.20 0.68 0.21 0.05 0.03 0.07 0.27
HPR SHPR HY (mmol H2/L/d) (mmol H2/g VSS/d) (mmol H2/g TVS)
2.97 3.12 2.23 2.97 10.11 3.12 0.74 0.45 1.04 4.02
S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung, EB, Endogenous bacteria.
0.59 0.62 0.45 0.59 2.02 0.62 0.15 0.09 N.A N.A
0.15 0.32 0.14 0.37 0.45 0.22 0.12 0.04 0.08 0.73
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1.0
a
SS1 SS2 CD PD EB
40
Without With
0.8
HY (mmol H2/g TVS)
Accumulative H2 production (mL)
50
30
20
0.6
0.4
0.2 10
0.0 12
0
b
10
HPR (mmol H2/L-d)
Accumulative H2 production (mL)
50
40
30
20
8 6 4 2 0
10
0
0
50
100
150
200
250
300
350
Time (h)
Fig. 1 e Hydrogen production using various seed inoculaums (SS1, Sewage sludge 1 (C.H.); SS2, Sewage sludge 2 (L.M.); CD, Cow dung; PS, Pig slurry; EB, endogens bacteria) (a) without and (b) with nutrient formulation.
SHPR (mmol H2/g VSS-d)
2.5
2.0
1.5
1.0
0.5
0.0 SS1
extra nutrient (Table 3 and Fig. 3). Table 3 also indicates that long lag phase time (l) values were obtained for the fermentation using pig dung seed with and without extra nutrients and endogenous bacteria with extra nutrients. There were low C/N ratios because of low carbohydrate concentration and high NH3eN concentration in these conditions.
3.2.
SS2
CD
PS
EB
seed
Fig. 2 e HY, HPR, and SHPR using various seed inocula (SS1, Sewage sludge 1 (C.H.); SS2, Sewage sludge 2 (L.M.); C, Cow dung; PS, Pig dung; EB, endogenous bacteria) without and with nutrient.
Productions of ethanol and volatile fatty acids
Fig. 3 indicates that the main soluble metabolic product (SMP) was acetate (1440e4570 mg COD/L, 60.4e86.8% of SMP) from mushroom MCW wastes substrate after 308 h cultivation. Similar results were reported during cellulosic materials fermentation [17,18]. Propionate concentration was high (1142 mg COD/L, 20.0% of SMP) using pig slurry inoculum with low hydrogen production performance (0.04 mmol H2/g TVS) (Table 5). The reason was that hydrogen production was inhibited by the cumulated propionate. Similar results were reported by Vavilin et al. [19]. The peak SMP concentration of 5708 mg COD/L without any extra nutrient was obtained using pig slurry inoculum. However, cow dung and pig slurry seed inoculum and endogenous bacteria seed could convert the MCW into high SMP concentrations ranging from 5437 to 5807 mg COD/L.
Table 4 e Biohydrogen production from various raw agriculture wastes. Feedstock
HPR (mmol H2/L/d)
HY (mmol H2/g TVS)
Reference
Mushroom waste Mushroom waste Polar leaves Beer Lees Beer lees Cornstalk
10.11
0.45
This study
4.02
0.73
This study
24.01 46.53 NA NA
0.61 0.13 0.28 0.13
[17] [20] [15] [21]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3 e1 6 4 7 8
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Fig. 3 e Soluble metabolic products using various seed inoculums (SS1, Sewage sludge1 (C.H.); SS2, Sewage sludge2 (L.M.); C, Cow dung; PS, Pig dung; EB, endogenous bacteria) (a) without and (b) with nutrient.
Table 5 e Soluble metabolic products using various seed inoculums without and with nutrient. Seed SS1 SS2 CD PS EB
Nutrient formulation
HY (mmol H2/g TVS)
EtOH (mmol/g TVS)
HAc (mmol/g TVS)
HPr (mmol/g TVS)
HBu (mmol/g TVS)
HVa (mmol/g TVS)
Without With Without With Without With Without With Without With
0.15 0.32 0.14 0.37 0.45 0.22 0.12 0.04 0.08 0.73
0.02 0.01 0.02 0.01 0.03 0.01 0.02 0.03 0.03 0.06
1.54 3.22 1.47 2.90 2.68 3.57 2.55 3.03 1.13 3.29
0.09 0.16 0.10 0.13 0.11 0.19 0.49 0.51 0.09 0.26
0.10 0.12 0.06 0.09 0.08 0.13 0.26 0.16 0.02 0.29
N.D 0.00 N.D 0.00 N.D 0.00 0.04 0.03 N.D N.D
S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung; EB, Endogenous bacteria EtOH, ethanol; HAc, acetic acid; HPr, propionic acid; HBu, butyric acid; HVa, valeric acid; TVFA ¼ HAc þ HPr þ HBu þ HVa; SMP ¼ TVFA þ EtOH.
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The liquid product analysis shows that the major metabolites determined after fermentation were ethanol, acetic, propionic and butyric acids. Peak hydrogen yield was obtained at nutrient addition using endogenous bacteria seed because the substrate was efficiently utilized for hydrogen production by the endogenous bacteria. The possible metabolic pathway of soluble sugar (mainly glucose) to produce hydrogen at the maximum yield is shown as Eq. (2): C6 H12 O6 /0:285H2 þ 1:310CO2 þ 0:023C2 H5 OH þ 1:287CH3 COOH þ 0:101C2 H5 COOH þ 0:113C3 H7 COOH
(2)
As shown in Eq. (2), the metabolic pathway was mainly acetic acid fermentation, and the conversion efficiency of H element in glucose into H2 was 4.75%. Theoretically the metabolic pathways of producing acetic acid from glucose and xylose are as follows: C5 H10 O5 þ 1:67H2 O/3:33H2 þ 1:67CH3 COOH þ 1:67CO2
(3)
C6 H12 O6 þ2H2 O/4H2 þ2CH3 COOH þ 2CO2
(4)
The theoretical metabolic pathway followed mainly acetic acid fermentation which favors high hydrogen production. Comparing Eqs. (2)e(4), the acetic acid production from mushroom wastes was 77.1% and 64.4% from glucose and xylose. Shifting the metabolic pathway to acetate-butyrate fermentation could lead to higher hydrogen production.
4.
Conclusions
Mushroom cultivation waste could be used as the feedstock for hydrogen production using waste sludges (sewage sludge, cow dung and pig slurry) with or without adding any extra nutrients. Peak HY of 0.73 mmol H2/g TVS was obtained with nutrient addition by the endogenous bacteria of mushroom waste. Cow dung inoculum could directly degrade mushroom waste without any extra nutrients and has peak HPR of 10.11 mmol H2/L/d and SHPR of 2.02 mmol H2/g VSS/d. The main soluble metabolic product was acetate (1440e4570 mg COD/L, 60.4e86.8% of SMP) after 308 h cultivation.
Acknowledgments The authors gratefully acknowledge the financial support by Taiwan’s Bureau of Energy (grant no. 99-D0204-3), Taiwan’s National Science Council (NSC-99-2221-E-035-024-MY3, NSC99-2221-E-035-025-MY3, NSC-99-2632-E-035-001-MY3), Feng Chia University (FCU-10G27101) and APEC Research Center for Advanced Biohydrogen Technology.
references
[1] Hansel A, Lindblad P. Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen, a clean and renewable energy source. Appl Microbiol Biotechnol 1998;5:153e60.
[2] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001; 26:13e28. [3] Lin CY, Lay CH. Research and development of biohydrogen production in Taiwan. In: Fang HHP, editor. Environmental anaerobic technology. London: Imperial College Press; 2010. p. 331e44. [4] Antoni D, Zverlov V, Schwarz W. Biofuels from microbes. Appl Microbiol Biotechnol 2007;77:23e35. [5] Antonopoulou G, Gavala HN, Skiadas IV, Angelopoulos K, Lyberatos G. Biofuels generation from sweet sorghum: fermentative hydrogen production and anaerobic digestion of the remaining biomass. Bioresour Technol 2008;99:110e9. [6] Ivanova G, Ra´khely G, Kova´cs KL. Thermophilic biohydrogen production from energy plants by Caldicellulosiruptor saccharolyticus and comparison with related studies. Int J Hydrogen Energy 2009;34:3659e70. [7] Kim S, Dale BE. Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 2004;26: 361e75. [8] Xie B, Cheng J, Zhou J, Song W, Liu J, Cen K. Production of hydrogen and methane from potatoes by two-phase anaerobic fermentation. Bioresour Technol 2008;99:5942e6. [9] Lay CH, Wu JH, Hsiao CL, Chang JJ, Chen CC, Lin CY. Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation. Int J Hydrogen Energy 2010;35:13445e51. [10] Vijayaraghavan K, Ahmad D, Bin Ibrahim MK. Biohydrogen. Generation from jackfruit peel using anaerobic contact filter. Int J Hydrogen Energy 2006;31:569e79. [11] Li YC, Wu SY, Chu CY, Huang HC. Hydrogen production from mushroom farm waste with a two step acid hydrolysis process. Int J Hydrogen Energy 2011;36:14245e51. [12] Endo G, Noike T, Matsumoto T. Characteristics of cellulose and glucose decomposition in acidogenic phase of anaerobic digestion (In Japanese). Proc Soc Civ Eng 1982;325:61e8. [13] APHA. Standard methods for the examination of water and wastewater. New York, USA: American Public Health Association; 1995. [14] Lay CH, Chang FY, Chu CY, Chen CC, Chi YC, Hsieh TT, et al. Enhancement of anaerobic biohydrogen/methane production from cellulose using heat-treated activated sludge. Water Sci Technol 2011;63:1849e54. [15] Fan YT, Zhang GS, Guo XY, Xing Y, Fan MH. Biohydrogenproduction from beer lees biomass by cow dung compost. Biomass Bioenergy 2006;30:493e6. [16] Lin CY, Lay CH. Effects of carbonate and phosphate concentrations on hydrogen production using anaerobic sewage sludge microflora. Int J Hydrogen Energy 2004;29: 275e81. [17] Cui M, Yuan Z, Zhi X, Wei L, Shen J. Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria. Int J Hydrogen Energy 2010;35: 4041e7. [18] Sugaya K, Tuse´ D, Jones JL. Production of acetic acid by Clostridium thermoaceticum in batch and continuous fermentations. Biotechnol Bioengg 1986;28:678e83. [19] Vavilin VA, Rytow SV, Lokshina LY. Modelling hydrogen partial pressure change as a result of competition between the butyric and propionic groups of acidogenic bacteria. Bioresour Technol 1995;54(2):171e7. [20] Cui M, Yuan Z, Zhi X, Shen J. Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria. Int J Hydrogen Energy 2009;34:7971e8. [21] Zhang ML, Fan YT, Xing Y, Pan CM, Zhang GS, Lay JJ. Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures. Biomass Bioenergy 2007;31:250e4.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures Chyi-How Lay a,b, I-Yuan Sung a, Gopalakrishnan Kumar a, Chen-Yeon Chu b,c,d, Chin-Chao Chen e, Chiu-Yue Lin a,b,c,d,* a
Department of Environmental Engineering and Science, Feng Chia University, Taiwan Green Energy Development Center, Feng Chia University, Taiwan c Department of Chemical Engineering, Feng Chia University, Taiwan d Master’s Program of Green Energy Science and Technology, Feng Chia University, Taiwan e Department of Landscape Architecture, Chungchou University of Science and Technology, Taiwan b
article info
abstract
Article history:
The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients.
Received 7 December 2011
After being used for growing mushroom for one to two weeks this bag becomes mushroom
Received in revised form
cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced
19 February 2012
annually in Taiwan and are usually burned or discarded. The cellulosic materials and
Accepted 23 February 2012
nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen
Available online 1 April 2012
fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra
Keywords:
nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature
Biohydrogen
55 C and an initial cultivation pH of 8. The results show that extra nutrient addition
Mushroom cultivation waste
inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using
Anaerobic mixed cultures
cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H2/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H2/L/d and 2.02 mmol H2/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Today’s energy system is mainly based on the fossil fuels which are depleting and cannot be sustainable. In view of the alternative fuels to overcome the future energy demand, hydrogen is an attractive fuel because of various features such
as clean, efficient, renewable, and does not generate any toxic by product on its combustion [1]. Biological hydrogen production process is one of the main alternative methods. Producing H2 via fermentative route is more environmental friendly and less energy intensive, thereby being competitive to conventional H2 production methods such as thermo-
* Corresponding author. Department of Environmental Engineering and Science, Feng Chia University, 100 Wenhwa Road, Seatwen, Taichung, Taiwan 40724, Taiwan. Tel.: þ886 4 24517250x6200; fax: þ886 4 35072114. E-mail address: [email protected] (C.-Y. Lin). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.02.135
16474
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were heat pretreated at 95 C for 1 h to inhibit hydrogenconsuming bacteria. MCW was collected from a mushroom farm in Changhwa (central Taiwan). The waste was dried at 105 C for zero moisture and then powdered to pass 0.297 mm-mesh sieve. The characteristics of the mushroom waste are shown in Table 2.
chemical means [2]. The substrates used in fermentative hydrogen production are generally rich in carbohydrates like glucose, sucrose and starch [3]. Currently, biohydrogen and other biofuels are produced from various agricultural and bioenergy-generation residues. At present the biomass from crops like sweet sorghum, potatoes, sugarcane, soybean and palm oil have been used as raw materials for bioethanol or biohydrogen production [4e8]. The mushroom waste has attained considerable attention because it is available abundantly and easy to collect and use as feedstock for the biohydrogen process. Mushroom bag is a polypropylene bag stuffed with wood flour and nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW) About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. Therefore, proper treatment or usage of this waste could avoid the environmental problems. Moreover, recent studies show that this waste could be a feedstock for biohydrogen production in effective way [11]. The advantages of using MCW feedstock for hydrogen production include cost effective because of this waste being needed to be treated for reducing environmental pollution problems. However, it has disadvantages such as further disposal of fermentation residue and solid fermentation is different from liquid fermentation in operation. Fermentative hydrogen production process is a tedious process and requires optimization of inoculums type, pretreatment, substrate nature and composition, pH and temperature to scale-up the process. Inoculum selection and pretreatment are important. Several types of inoculum have been reported such as sewage sludge [9] and cow dung [10]. Pretreatment of the mushroom cultivation waste by using acid was also studied [11].
2.
Materials and methods
2.1.
Seed inocula and substrate
2.2.
Experimental design and procedure
Batch hydrogen production experiments were performed using serum bottles (125 mL) with anaerobic head space. The vials were first purged with argon gas followed by adding 10 mL of seed inocula, 40 mL of de-ionized water or nutrient solution, 10 mL of pH adjustment solution (1 N HCl or 1 N NaOH) and tested dried MCW agricultural powder ranging 1.2 g per 60 mL working volume (20 g/L). These vials were placed in a reciprocal air-bath shaker (150 rpm) with a cultivation temperature of 55 1 C. The tested initial pH value was 8.0. The nutrient solution contained inorganic supplements (mg/L): NH4HCO3 5240, K2HPO4 125, MgCl2$6H2O 100, MnSO4$6H2O 15, FeSO4$7H2O 25, CuSO4$5H2O 5, CoCl2$5H2O 0.125 and NaHCO3 6720 [12]. Each experimental condition was carried out with duplicate.
2.3.
Analytical method
The analytical procedures of APHA Standard Methods [13] were used to determine pH, oxidation-reduction potential (ORP), total chemical oxygen demand (TCOD), ammonia nitrogen (NH3eN), suspended solids (SS) and volatile SS (VSS). Biogas volume was determined by a gas tight syringe at room temperature (20 C) and a pressure of 760 mm Hg. The biogas composition in the batch enrichment assays was measured with a CHINA Chromatography 8700T gas chromatograph equipped with a packed (packing, Porapak Q), stainless steel column and a thermal conductivity detector. Oven, injector and detector temperatures were 40, 40 and 40 C, respectively and argon as the carrier gas. Same methods are indicated in our previous studies [14]. Anthrone-sufuric acid method was used to measure total carbohydrate concentration [14]. Cellulose, hemicelluloses and lignin were determined by FIBERTECTM 1020 (M6). Elemental analysis was performed on an Elemental Analyzer (Model: Vario EL). Hydrogen production potential (P, mL), maximum hydrogen production rate (Rm, mL/h) and lag phase time (l, h) obtained from the modified Gompertz equation (Eq. (1)) [14]
The seed inocula were collected from two municipal wastewater treatment plants (SS1and SS2), cow dung compost (CD) and pig slurry (PS) located in central Taiwan. The pH, total COD (chemical oxygen demand), soluble COD, total carbohydrate, volatile suspended solids (VSS, to express the biomass concentrations), total solids and NH3eN concentrations of the seed inoculaums are listed in Table 1. The collected sludges
Table 1 e Characteristics of seed inoculms. Seed
pH
Total COD
Soluble COD (mg COD/L)
S1 S2 C P
6.9 7.1 7.4 7.0
0.1 0.1 0.1 0.1
54,400 70,480 65,680 77,200
226 1471 113 1018
2020 2027 7200 17,280
28 46 339 339
Total carbohydrate (mg/L as glucose) 6424 7054 10,641 2380
92 215 226 31
S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung; COD, chemical oxygen demand.
VSS (g/L)
NH3eN (mg/L)
21 32 22 17
75 124 276 243
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Table 2 e Characteristics of mushroom waste. TS* (g/L)
VSa (g/L)
TCODa (G-COD/L)
Total Carbohydratea (g/L)
Celluloseb (%)
Hemicelluloseb (%)
Ligninb (%)
0.84
0.78
1.01
0.46
31.2
6.9
14
Ultimate analysis wt % b
C/N ratiob
C
H
N
S
40.2
5.0
1.4
0.2
28.7
a Tested by the agricultural waste solution with 1 g dried biomass in 1 L solution. b Tested by dried biomass.
were used as the response variable. STATISTIC software (version 6.0, Statsoft Inc., USA) and Sigmaplot software (trial version 9.0, Systat Software Inc., USA) were used for regression and graphical analyses of the data, respectively. Rm $e ðl tÞ þ 1 HðtÞ ¼ P$exp exp P
(1)
H(t) is the cumulative hydrogen production (mL); P is the hydrogen production potential (mL); Rm is the maximum hydrogen production rate (mL/h); e is 2.71828; l is the lag phase time (h) and t is the cultivation time (h). The maximum hydrogen production rate (HPRmax, mmol H2/L-d) was defined as hydrogen production per working volume per cultivation time and calculated based on the maximum hydrogen production rate (Rm, mL/h) obtained from Gompertz equation. Specific hydrogen production rate (SHPRmax, mmol H2/g VSSd) was defined as HPR divided by initial VSS of seed inoculums (5 g/L). Hydrogen production yield (HY, mmol H2/g TVS) was defined as hydrogen production per gram of dried MCW.
3.
Results and discussion
3.1.
Hydrogen generation
Table 3 shows that the nutrient addition could enhance the total biogas production comparing with without extra nutrient for the same seed inoculum. On the contrary, the hydrogen contents in biogas of no nutrient addition were higher than that of with extra nutrients for the same seed inoculum. The
peak hydrogen production of 24.2 mL was obtained using cow dung seed inoculum without any Endo nutrient (Fig. 1a and Table 3). However, the hydrogen production was less than 10 mL for other seed inoculum. Moreover, the maximum biogas and hydrogen production were 150 mL and 39.5 mL, respectively, with nutrient addition and no seed inoculum (EB, Endogenous bacteria) (Fig. 2b and Table 3). A peak HY of 0.73 mmol H2/g TVS was obtained with no inoculum at nutrient addition (Table 3). This HY value was about 3 times of HY 0.28 mmol H2/g TVS which was obtained in beer lees fermentation at 36 C and pH 7.0 by cow dung compost [15]. However, peak HPR and SHPR of 10.11 mmol H2/ L/d and 2.02 mmol H2/g VSS/d, respectively, were obtained by using cow dung inoculum without extra nutrients. This value is higher than that of converting other agricultural wastes into hydrogen (Table 4). Fig. 3 depicts that nutrient addition inhibited HPR and HY when using cow dung and pig slurry seeds. Biohydrogen production requires certain essential micro-nutrients such as nitrogen (N), phosphate (P), and some trace elements for bacterial metabolism, growth and activity [16]. A proper C/N-ratio value for mixed microflora is necessary to optimize anaerobic hydrogen production from organic wastewater. The reason of low HY for cow dung and pig slurry might be that too high nutrient concentration (such as nitrogen) which inhibited the fermentation (Fig. 3). However, nutrient addition elevated HPR and HY in the case of using sewage sludge inoculum and without inoculum. Using cow dung seed has the most significant increase (240%) in HPR and SHPR. On the other hand, the HY increased from 0.04 mmol H2/g TVS with extra nutrients to 0.12 mmol H2/g TVS without
Table 3 e Biogas production using various seed inocula without and with nutrients. Seed
SS1 SS2 CD PS EB
H2 Nutrient Total H2 formulation biogas conc. (mL) (%) (mL)
Without With Without With Without With Without With Without With
21.0 67.3 19.7 73.0 51.5 72.3 20.0 34.3 13.7 150.0
38.1 25.6 38.1 27.1 47.0 16.7 32.0 7.0 30.7 26.3
8.0 17.2 7.5 19.8 24.2 12.1 6.4 2.4 4.2 39.5
Modified Gompertz equation parameter P R (mL) (mL/h)
l (h)
R2
7.8 17.7 7.5 20.4 24.3 12.1 6.8 2.3 4.4 40.5
3.8 3.8 4.6 4.3 26.2 18.1 3.2 9.9 5.9 21.5
0.9707 0.9795 0.9785 0.9831 0.992 0.9981 0.9834 0.9789 0.9674 0.9966
0.20 0.21 0.15 0.20 0.68 0.21 0.05 0.03 0.07 0.27
HPR SHPR HY (mmol H2/L/d) (mmol H2/g VSS/d) (mmol H2/g TVS)
2.97 3.12 2.23 2.97 10.11 3.12 0.74 0.45 1.04 4.02
S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung, EB, Endogenous bacteria.
0.59 0.62 0.45 0.59 2.02 0.62 0.15 0.09 N.A N.A
0.15 0.32 0.14 0.37 0.45 0.22 0.12 0.04 0.08 0.73
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1.0
a
SS1 SS2 CD PD EB
40
Without With
0.8
HY (mmol H2/g TVS)
Accumulative H2 production (mL)
50
30
20
0.6
0.4
0.2 10
0.0 12
0
b
10
HPR (mmol H2/L-d)
Accumulative H2 production (mL)
50
40
30
20
8 6 4 2 0
10
0
0
50
100
150
200
250
300
350
Time (h)
Fig. 1 e Hydrogen production using various seed inoculaums (SS1, Sewage sludge 1 (C.H.); SS2, Sewage sludge 2 (L.M.); CD, Cow dung; PS, Pig slurry; EB, endogens bacteria) (a) without and (b) with nutrient formulation.
SHPR (mmol H2/g VSS-d)
2.5
2.0
1.5
1.0
0.5
0.0 SS1
extra nutrient (Table 3 and Fig. 3). Table 3 also indicates that long lag phase time (l) values were obtained for the fermentation using pig dung seed with and without extra nutrients and endogenous bacteria with extra nutrients. There were low C/N ratios because of low carbohydrate concentration and high NH3eN concentration in these conditions.
3.2.
SS2
CD
PS
EB
seed
Fig. 2 e HY, HPR, and SHPR using various seed inocula (SS1, Sewage sludge 1 (C.H.); SS2, Sewage sludge 2 (L.M.); C, Cow dung; PS, Pig dung; EB, endogenous bacteria) without and with nutrient.
Productions of ethanol and volatile fatty acids
Fig. 3 indicates that the main soluble metabolic product (SMP) was acetate (1440e4570 mg COD/L, 60.4e86.8% of SMP) from mushroom MCW wastes substrate after 308 h cultivation. Similar results were reported during cellulosic materials fermentation [17,18]. Propionate concentration was high (1142 mg COD/L, 20.0% of SMP) using pig slurry inoculum with low hydrogen production performance (0.04 mmol H2/g TVS) (Table 5). The reason was that hydrogen production was inhibited by the cumulated propionate. Similar results were reported by Vavilin et al. [19]. The peak SMP concentration of 5708 mg COD/L without any extra nutrient was obtained using pig slurry inoculum. However, cow dung and pig slurry seed inoculum and endogenous bacteria seed could convert the MCW into high SMP concentrations ranging from 5437 to 5807 mg COD/L.
Table 4 e Biohydrogen production from various raw agriculture wastes. Feedstock
HPR (mmol H2/L/d)
HY (mmol H2/g TVS)
Reference
Mushroom waste Mushroom waste Polar leaves Beer Lees Beer lees Cornstalk
10.11
0.45
This study
4.02
0.73
This study
24.01 46.53 NA NA
0.61 0.13 0.28 0.13
[17] [20] [15] [21]
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Fig. 3 e Soluble metabolic products using various seed inoculums (SS1, Sewage sludge1 (C.H.); SS2, Sewage sludge2 (L.M.); C, Cow dung; PS, Pig dung; EB, endogenous bacteria) (a) without and (b) with nutrient.
Table 5 e Soluble metabolic products using various seed inoculums without and with nutrient. Seed SS1 SS2 CD PS EB
Nutrient formulation
HY (mmol H2/g TVS)
EtOH (mmol/g TVS)
HAc (mmol/g TVS)
HPr (mmol/g TVS)
HBu (mmol/g TVS)
HVa (mmol/g TVS)
Without With Without With Without With Without With Without With
0.15 0.32 0.14 0.37 0.45 0.22 0.12 0.04 0.08 0.73
0.02 0.01 0.02 0.01 0.03 0.01 0.02 0.03 0.03 0.06
1.54 3.22 1.47 2.90 2.68 3.57 2.55 3.03 1.13 3.29
0.09 0.16 0.10 0.13 0.11 0.19 0.49 0.51 0.09 0.26
0.10 0.12 0.06 0.09 0.08 0.13 0.26 0.16 0.02 0.29
N.D 0.00 N.D 0.00 N.D 0.00 0.04 0.03 N.D N.D
S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung; EB, Endogenous bacteria EtOH, ethanol; HAc, acetic acid; HPr, propionic acid; HBu, butyric acid; HVa, valeric acid; TVFA ¼ HAc þ HPr þ HBu þ HVa; SMP ¼ TVFA þ EtOH.
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The liquid product analysis shows that the major metabolites determined after fermentation were ethanol, acetic, propionic and butyric acids. Peak hydrogen yield was obtained at nutrient addition using endogenous bacteria seed because the substrate was efficiently utilized for hydrogen production by the endogenous bacteria. The possible metabolic pathway of soluble sugar (mainly glucose) to produce hydrogen at the maximum yield is shown as Eq. (2): C6 H12 O6 /0:285H2 þ 1:310CO2 þ 0:023C2 H5 OH þ 1:287CH3 COOH þ 0:101C2 H5 COOH þ 0:113C3 H7 COOH
(2)
As shown in Eq. (2), the metabolic pathway was mainly acetic acid fermentation, and the conversion efficiency of H element in glucose into H2 was 4.75%. Theoretically the metabolic pathways of producing acetic acid from glucose and xylose are as follows: C5 H10 O5 þ 1:67H2 O/3:33H2 þ 1:67CH3 COOH þ 1:67CO2
(3)
C6 H12 O6 þ2H2 O/4H2 þ2CH3 COOH þ 2CO2
(4)
The theoretical metabolic pathway followed mainly acetic acid fermentation which favors high hydrogen production. Comparing Eqs. (2)e(4), the acetic acid production from mushroom wastes was 77.1% and 64.4% from glucose and xylose. Shifting the metabolic pathway to acetate-butyrate fermentation could lead to higher hydrogen production.
4.
Conclusions
Mushroom cultivation waste could be used as the feedstock for hydrogen production using waste sludges (sewage sludge, cow dung and pig slurry) with or without adding any extra nutrients. Peak HY of 0.73 mmol H2/g TVS was obtained with nutrient addition by the endogenous bacteria of mushroom waste. Cow dung inoculum could directly degrade mushroom waste without any extra nutrients and has peak HPR of 10.11 mmol H2/L/d and SHPR of 2.02 mmol H2/g VSS/d. The main soluble metabolic product was acetate (1440e4570 mg COD/L, 60.4e86.8% of SMP) after 308 h cultivation.
Acknowledgments The authors gratefully acknowledge the financial support by Taiwan’s Bureau of Energy (grant no. 99-D0204-3), Taiwan’s National Science Council (NSC-99-2221-E-035-024-MY3, NSC99-2221-E-035-025-MY3, NSC-99-2632-E-035-001-MY3), Feng Chia University (FCU-10G27101) and APEC Research Center for Advanced Biohydrogen Technology.
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