Bioresource Technology 191 (2015) 157–165
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Dry anaerobic co-digestion of organic fraction of municipal waste with paperboard mill sludge and gelatin solid waste for enhancement of hydrogen production M. Elsamadony ⇑, A. Tawfik Environmental Engineering Department, Egypt-Japan University of Science and Technology (E-Just), New Borg El Arab City, 21934 Alexandria, Egypt
h i g h l i g h t s Dry anaerobic co-fermentation of OFMSW with GSW and/or PMS was investigated. Maximum H2 production was obtained at 70% OFMSW + 20% GSW + 10% PMS. C/N ratio and Ca
+2
concentration are the key factors affecting on the H2 production.
Buffering capacity of OFMSW increased via addition of GSW and/or PMS. Mean particle size diameters reduced by a percentage of 87.7% in the digestate.
a r t i c l e
i n f o
Article history: Received 1 April 2015 Received in revised form 7 May 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: Bio-hydrogen Dry anaerobic co-fermentation Organic fraction of municipal solid waste Paperboard mill sludge Gelatin solid waste
a b s t r a c t The aim of this study is to investigate the bio-H2 production via dry anaerobic co-fermentation of organic fraction of municipal solid waste (OFMSW) with protein and calcium-rich substrates such as gelatin solid waste (GSW) and paperboard mill sludge (PMS). Co-fermentation of OFMSW/GSW/PMS significantly enhanced the H2 production (HP) and H2 yield (HY). The maximum HP of 1082.5 ± 91.4 mL and HY of 144.9 ± 9.8 mL/gVSremoved were achieved at a volumetric ratio of 70% OFMSW:20% GSW:10% PMS. COD, carbohydrate, protein and lipids conversion efficiencies were 60.9 ± 4.4%, 71.4 ± 3.5%, 22.6 ± 2.3% and 20.5 ± 1.8% respectively. Co-fermentation process reduced the particle size distribution which is favorably utilized by hydrogen producing bacteria. The mean particle size diameters for feedstock and the digestate were 939.3 and 115.2 lm, respectively with reduction value of 8.15-fold in the mixtures. The volumetric H2 production increased from 4.5 ± 0.3 to 7.2 ± 0.6 LH2 =Lsubstrate at increasing Ca+2 concentrations from 1.8 ± 0.1 to 6.3 ± 0.5 g/L respectively. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Proliferation of biological H2 as a renewable energy carrier that replaces fossil fuel resources would result into cleaner environment. Since, H2 produces only water as a combustion by-product, in addition to generating a higher energy yields (122 kj/g) as compared to hydrocarbon counterpart (Tawfik and El-Qelish, 2014a,b). In spite of H2 can be produced by the conventional physico-chemical technologies, but they required electricity derived from combustion of the fossil fuels (Zhou et al., 2013). So far, anaerobic digestion (AD) is the most sophisticated technology in which H2 can be produced biologically from various ⇑ Corresponding author. Tel.: +20 03 4599520. E-mail addresses: [email protected], yahoo.com (M. Elsamadony). http://dx.doi.org/10.1016/j.biortech.2015.05.017 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
m_elsamadoney@
biodegradable organic wastes. This is mainly owing to its low energy requirement, low sludge production, cost effective, as well as being eco-friendly and energy recovery (Razaviarani et al., 2013; Tyagi et al., 2014). AD is classified into three categories of wet, semi-dry, and dry one according to total solid (TS) content of the feedstock (610%, 10–20%, and P20%, respectively). However, dry AD claimed to be more favorable than wet AD in term of smaller reactor volume, lower energy requirements for heating, and less material handling (Elsamadony et al., 2015a). Usually AD process is operated under mesophilic or thermophilic condition, in which thermophilic condition reported more efficient method. Since, thermophilic fermentation prevailed higher gas production, metabolic rates two to threefold mesophilic condition and conversion efficiencies besides pathogen disinfection. Meanwhile, thermophilic dry AD faces some drawbacks of high sensitivity to shock load or toxic substances (Gallert et al., 1998).
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M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
Organic fraction of municipal solid waste (OFMSW) in Egypt amounted to 21 million tons/year which accounted for 60% of total municipal solid waste (MSW) (Tawfik and El-Qelish, 2014a,b). OFMSW is a desirable feedstock for dry AD due to its high organic fraction and carbohydrates content which is the preferable substrate for fermentative hydrogen-producing microorganisms and easily hydrolysable waste with high hydrogen production potential (Kim, 2004). However, dry AD of sole OFMSW has some difficulties which need to be addressed to have a fully functional bio-hydrogen production system such as little amount of trace elements content, lack in nitrogen which is an essential nutrient for hydrogen producers. Furthermore, OFMSW has a low buffering capacity (pH ranged from 4 to 5) which makes it more susceptible to volatile fatty acids (VFAs) inhibition (Lin et al., 2013; Zhou et al., 2013). In order to overcome the aforementioned drawbacks, anaerobic co-fermentation of OFMSW with gelatin solid waste (GSW) and paperboard mill sludge (PMS) has been examined for enhancing the dry AD performance and developing hydrogen productivity due to synergistic and complementary effects. Co-fermentation of different substrates could be beneficial due to dilution of toxic chemicals, enhanced balance of nutrients, and synergistic effect of microorganisms (Razaviarani and Buchanan, 2014). Whereas, co-fermentation of OFMSW with protein-rich substrates such as GSW and PMS supplied a more balanced carbon to nitrogen (C/N) ratio (Kim, 2004). Optimized C/N ratio not only balances the nutrients, but also enhances the bacterial productivity of hydrogen and preclude ammonia inhibition (Zhou et al., 2013). Furthermore, when highly nitrogenous wastes co-fermented with OFMSW provides a good buffering capacity making it more accessible to utilize for energy production and increasing the stability of the anaerobic process (Zhang et al., 2014). Additionally, GSW and PMS (CaCO3-rich wastes) substrates provide OFMSW with calcium ions which released slowly into the substrate (Zhang and Wang, 2013) led to highly increase in hydrogen generation and improved performance of bioreactor by increasing the cell density (Yuan et al., 2010). Formerly, Zhou et al., 2013 suggested that optimal C/N ratio ranged from 26 to 31 in a co-digestion consisting of food waste (FW) and sewage sludge. Kim, 2004 found that co-fermentation of FW with sewage sludge resulted in a more favorable carbohydrate-COD/protein-COD for hydrogen production. Moreover several researchers (Wang et al., 2013; Zhang and Wang, 2013; Zhang et al., 2014, 2013, 2011) indicated that an improved pH buffering capacity, balanced C/N ratio and sufficient trace element could account for the enhanced hydrogen production achieved in a co-digestion process. Formerly, Zhou et al. (2013) suggested that optimal C/N ratio ranged from 26 to 31 in a co-digestion consisting of FW and sewage sludge. Kim (2004) found that co-fermentation of food waste
with sewage sludge resulted in a more favorable carbohydrate-COD/protein-COD for hydrogen production. Moreover several researchers (Wang et al., 2013; Zhang and Wang, 2013; Zhang et al., 2014, 2013, 2011) indicated that an improved pH buffering capacity, balanced C/N ratio and sufficient trace element could account for the enhanced hydrogen production achieved in a co-digestion process. Therefore, the aims of this investigation are to (1) assess the feasibility of thermophilic dry anaerobic co-fermentation of OFMSW/GSW, OFMSW/PMS and OFMSW/GSW/PMS. (2) Study the effect of co-fermentation process on particle size distribution and (3) investigate the key factors affecting on co-fermentation process such as C/N ratio, Ca+2 concentrations and buffering capacity in order to have a better understanding for the synergistic effect of co-fermentation.
2. Methods 2.1. Characteristics of feedstocks and inoculum sludge Three types of substrates were used in this study: (1) organic fractions of municipal solid waste (OFMSW) collected from local municipal landfill site located in Alexandria city, Egypt. The bio-wastes mainly contained rice, vegetables, bread, beans, and some meats (bones were removed from OFMSW). The collected OFMSW was crushed into small particles using an electrical grinder avoided any dilution. Furthermore, the resulting residues was sieved through a stainless steel sieve with opening of 2.0 mm. (2) gelatin solid waste (GSW) harvested from gelatin manufacturing company situated in Alexandria city. The gelatin manufacturing processes include cutting the animals’ fibers into small particles (1–2 cm), soaking the resultant fibers into lime solution for a period ranging from 9 to 10 weeks, adjusting pH up to 12 and finally the extracting gelatin process is taken place at a temperature (50–90 °C). (3) paperboard mill sludge (PMS) obtained from sedimentation basin placed at Aldar Albydaa factory (Borg Alarab city, Egypt). The fresh water and lime is mainly used for the production of paper board from recycled waste paper following by centrifugal to remove impurities, bleaching, and finally drying process at a temperature of 135 °C in rolling machine. OFMSW particulate has a spherical shape, while GSW presented in jellylike form and PMS consists of fibers with different length and diameter. The seed sludge used in this study was harvested from thickener tank of wastewater treatment plant located in Alexandria city, Egypt. The mixed culture bacteria was further concentrated by settling for 24 h, and stored for one month under anaerobic conditions. The characteristics of OFMSW, GSW, PMSW and the seed sludge are presented in Table 1.
Table 1 Characteristics of feedstocks and inoculums sludge. Parameters pH TS VS COD Carbohydrate TKj-N C/N NH4-N Lipids Proteins Ca+2 HAc HBu HLa HPr a
Unit % % g/L g/L g/L mg/L g/L g/L g/L g/L g/L g/L g/L
Values represent the average ± STD.
OFMSW
PMS
GSW
Seed sludge
3.88 ± 0.2a 28.7 ± 1.8 26.6 ± 0.7 115.2 ± 9.5 52.3 ± 4.3 2.9 ± 0.2 39.5 ± 0.7 268.2 ± 18.3 3.9 ± 0.4 16.5 ± 0.9 2.3 ± 0.1 2.7 ± 0.9 1.1 ± 0.1 0.9 ± 0.0 0.4 ± 0.1
6.91 ± 0.4 7.5 ± 0.3 5.4 ± 0.5 51.6 ± 4.1 9.6 ± 0.5 1.9 ± 0.1 27.7 ± 0.2 123.2 ± 10.9 1.2 ± 0.1 10.9 ± 0.7 17.9 ± 1.1 0.6 ± 0.1 0.4 ± 0.1 0.1 ± 0.0 0.1 ± 0.0
12.71 ± 0.9 8.1 ± 0.7 6.2 ± 0.3 66.8 ± 4.4 1.1 ± 0.1 5.2 ± 0.4 13.0 ± 0.2 437.2 ± 31.7 4.2 ± 0.5 29.5 ± 1.1 28.5 ± 2.0 1.2 ± 0.1 N.D. 0.4 ± 0.0 0.6 ± 0.1
6.49 ± 0.2 6.3 ± 0.6 5.0 ± 0.2 22.4 ± 1.6 7.5 ± 0.3 1.0 ± 0.1 22.7 ± 0.1 201.6 ± 15.1 0.9 ± 0.2 4.9 ± 0.2 0.8 ± 0.1 0.9 ± 0.1 1.3 ± 0.1 0.2 ± 0.0 0.5 ± 0.0
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165 Table 2 Batch dry anaerobic co-digestion experiments for hydrogen production from OFMSW/ GSW; OFMSW/PMS and OFMSW/GSW/PMS. Batches/substrates
Seed sludge (mL)
OFMSW (mL)
GSW (mL)
PMS (mL)
OFMSW + GSW
50 50 50 50 50
100 75 50 25 0
0 25 50 75 100
0 0 0 0 0
OFMSW + PMS
50 50 50 50 50
90 75 50 25 0
0 0 0 0 0
10 25 50 75 100
OFMSW + GSW + PMS
50 50 50 50 50 50 50
90 80 80 80 70 70 70
5 10 5 15 20 15 10
5 10 15 5 10 15 20
Agar incubated for 72 h at 55 ± 2 °C under anaerobic conditions. All analyses were independently conducted in duplicate to guarantee the reliability of results. 2.5. Analytical methods
2.2. Experimental set-up Batch dry anaerobic co-digestion experiments for hydrogen production from OFMSW/GSW; OFMSW/PMS and OFMSW/GSW/PMS were tested under thermophilic conditions (55 ± 2 °C). The experiments were conducted using 200 mL serum bottles in duplicates. All batches were initially inoculated with mixed culture bacteria and preheated at 70 °C for 30 min to inhibit the bioactivity of hydrogen consumers and to harvest spore-forming anaerobic bacteria (Zhou et al., 2013). Afterwards different volumetric ratios of substrates (OFMSW/GSW; OFMSW/PMS and OFMSW/GSW/PMS) were added as shown in Table 2. The pH of the reactors was 6.0 ± 0.1. The head space of the bottles (50 mL) were flushed with nitrogen gas for 2 min to remove the oxygen from the culture medium and capped tightly with rubber stoppers and aluminum caps. The evolved gas was measured by the displacement method. Moreover, the biogas composition was analyzed using gas chromatography. 2.3. Kinetic studies Cumulative hydrogen production curves were recorded over the time course of the batch experiments and analyzed using the modified Gompertz equation (Elsamadony et al., 2015b):
Rm e H ¼ P exp exp ðk tÞ þ 1 P
159
ð1Þ
where H (mL) is the cumulative hydrogen production at the reaction time t (h), P (mL) is the hydrogen potential, Rm (mL/h) is the maximum hydrogen production rate, e = 2.71828, and k (h) is the lag phase period. 2.4. Bacterial population Samples for detecting and identification of microbial consortium bacteria were harvested from inoculum sludge and the fermenters containing OFMSW/GSW, OFMSW/PMS and OFMSW/GSW/PMS. The collected samples were serially diluted with phosphate buffering saline (PBS) solution. Afterwards, the samples were spread onto the proper agar plates. Clostridium sp. enumeration was carried out using Neomycin nagler (NN) medium and Desoxycholate hydrogen sulfide lactose (DHL) medium was used to enumerate Enterobacter sp. Anaerobic plate counts were manually accomplished for counting of colonies on Plate Count
Total solids (TS), volatile solids (VS), chemical oxygen demand (COD), total Kjeldahl nitrogen (TKj-N), lipids and calcium ion were quantified according to APHA, 2005. Protein content was calculated as follows (6.25 TKj-N ammonia nitrogen)) (Zhang et al., 2011). Carbohydrate was measured according to the phenol–sulfuric acid method (Dubois et al., 1956). Volatile fatty acids (VFAs) were analyzed by high performance liquid chromatography (LC-10AD, Shimadzu, Japan). The temperature of column oven was 40 °C. 4 mM H2SO4 was used as a mobile phase at a flow rate of 0.5 mL/min for 22 min followed by 0.4 mL/min for 8 min. The evolved gas was measured by displacement method. Moreover, Hydrogen content in the biogas was analyzed using a gas chromatograph (GC-2014, Shimadzu, Japan) equipped with a thermal conductivity detector and Shin carbon column. The operational temperatures of the injection port, the column oven and the detector were 100, 120 and 150 °C, respectively. Helium was used as the carrier gas at a flow rate of 25 mL/min. Particle size distribution analysis was carried out using laser diffraction spectroscopy (Beckman Coulter LS230). 3. Results and discussion 3.1. Hydrogen production, yield and microbial population Fig. 1a–c shows the time course of cumulative hydrogen production (CHP) from organic fraction of municipal solid waste (OFMSW) + gelatin solid waste (GSW), OFMSW + paper mill sludge (PMS) and OFMSW + GSW + PMS via dry anaerobic co-digestion process. The results obtained indicated that the peak CHP of 836.8 ± 65.1 mL was achieved from co-fermentation of OFMSW (75%) + GS (25%) (v/v) (Fig. 1a). This exceeds the mono-fermentation of OFMSW by a value of 25.1%. This is mainly due to the gelatin waste represents an excellent source for nitrogen which is necessary for hydrogen producing bacteria. Similar trends were observed by Kim (2004). Breure et al., 1986 showed that the hydrolysis of gelatin was increased as well as biomass concentration due to adding of glucose. Similar findings were reported by Zhou et al., 2013 who found that maximum CHP of 255 mL was achieved from co-fermentation of food waste + primary sludge + waste activated sludge at volumetric ratios of 80:15:5 respectively which surpass the mono fermentation of food waste by 77%. However, the CHP was deteriorated at higher volumetric percentage of GSW where the CHP amounted to 676.7 ± 39.6 mL at OFMSW (50%) + GS (50%). The lowest CHP of 466.2 ± 42.4 and 254.1 ± 28.3 mL was registered for OFMSW (25%) + GS (75%) and GSW (100%), respectively. This indicates that the major portion of hydrogen production was mainly due to the degradation of OFMSW where the CHP amounted to 668.8 ± 52.3 mL for mono-fermentation of OFMSW (100%). Fig. 1b shows the CHP from co-digestion of OFMSW + paper mill sludge (PMS). The results revealed that the maximum CHP was 857.8 ± 57.4 mL for the mixture of 50% OFMSW + 50% PMS which significantly exceeded the anaerobic digestion of OFMSW alone by a value of 28.3%. Apparently, the co-fermentation of OFMSW with PMS had a positive impact on the CHP. Furthermore, decreasing the volumetric ratio of OFMSW in the batches led to decline in hydrogen production. Similar results was reported by Gupta et al., 2014 who found lower hydrogen productivity of 53 mL with cellulose as sole substrate compared to glucose as co-substrate with
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
100% OFMSW : 0% GSW 75% OFMSW : 25% GSW 50% OFMSW : 50% GSW 25% OFMSW : 75% GSW 0% OFMSW : 100% GSW
Cumulative hydrogen production (mL)
160
1000
(a) 800 600 400 200 0
90% OFMSW : 10% PMS 75% OFMSW : 25% PMS 50% OFMSW : 50% PMS 25% OFMSW : 75% PMS 0% OFMSW : 100% PMS
Cumulative hydrogen production (mL)
0
20
80% OFMSW: 5% GSW: 15% PMS 80% OFMSW: 15% GSW: 5% PMS 70% OFMSW: 20% GSW: 10% PMS 70% OFMSW: 15% GSW: 15% PMS 70% OFMSW: 10% GSW: 20% PMS
Cumulative hydrogen production (mL)
80% OFMSW: 10% GSW: 10% PMS
80
20 40 60 fermentation time (hrs)
80
20
80
1000
(b) 800 600 400 200 0 0
90% OFMSW: 5% GSW: 5% PMS
40 60 fermentation time (hrs)
1200
(c)
1000 800 600 400 200 0 0
40 60 fermentation time (hrs)
Fig. 1. Cumulative hydrogen production (CHP) from co-digestion of various substrates, (a) OFMSW + GSW, (b) OFMSW + PMS, and (c) OFMSW + GSW + PMS.
cellulose (303 mL). Lin et al., 2013 found that anaerobic co-fermentation of PMS and FW is beneficial for the hydrogen production at a ratio of 2:2 (w/w) where HP increased by 12.8 and 2.2-folds higher than those recorded for individual substrates of PMS and FW, respectively. The CHP was deteriorated and dropped to 677.3 ± 42.4 mL at increasing the content of PMS up to 75% (v/v). PMS alone recorded the minimum CHP of 388.3 ± 39.6 mL. Co-fermentation synergistic effect of OFMSW, GSW and PMS was investigated at different volumetric ratios for hydrogen production (Fig. 1c). The highest CHP of 1082.5 ± 91.4 mL was obtained at (70% OFMSW + 20% GSW + 10% PMS) which was significantly higher than those registered for OFMSW alone by a value of 61.9%. This can be attributed to the addition of GSW and PMS to OFMSW which could enhance H2 production potential due to balanced carbohydrate/protein ratio. Gelatin solid waste (GSW) and paper mill sludge (PMS) are heavily polluted wastes with high protein content, alkalinity and abundant nitrogenous compounds, and they could be promising co-substrates for enhancement of hydrogen production from OFMSW. Moreover, gelatin solid waste (GSW) and paper mill sludge (PMS) contained a huge amount of calcium which is preferable for hydrogen production via anaerobic digestion process. Table 3 shows the superior of hydrogen production from co-fermentation of OFMSW/GSW/PMS as compared to previous studies.
As depicted from Fig. 2a, the pH of the digestate was largely dropped to a level of 3.86 ± 0.28 with fermentation of OFMSW alone resulting in a low hydrogen production. Increasing the volumetric ratios of GSW and PMS in combination with OFMSW maintained pH level in the digestate between 4.4 and 4.95 as presented in Fig. 2a. This is mainly due to the co-substrates containing protein which led to a release of ammonia nitrogen in the media via anaerobic fermentation process resulted in increasing the pH buffering capacity (Banks and Humphreys, 1998). Recent literatures showed the positive effect of co-fermentation process on the pH buffering capacity during AD process (Lin et al., 2013; Wang et al., 2013; Zhang and Wang, 2013; Zhang et al., 2013). Zhang and Wang (2013) discovered that addition of white mud from ammonia soda process (WMA) to food waste led to an increase the final pH from 3.80 to 6.22. In another study, Zhang et al., 2013 found that addition of lime mud from paper-making process to food waste increase the final pH from 4.5 to 5 without using any external alkali chemicals. Ammonia maintains a high level of bicarbonate in the wastewater as a part of buffering capacity in AD (Gallert et al., 1998). Fig. 2b shows the relationship between ammonia produced and pH drop in the co-fermentation processes with correlation factor (R2 = 0.716). Cumulative hydrogen production data shown in Fig. 1a–c were correlated with the Gompertz model equation (Eq. (1)) of which
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M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165 Table 3 Hydrogen production from co-digestion processes. Mixing ratio
Operational conditions
Improvement*
References
OFMSW + SS
1:1
1.20
Tyagi et al. (2014)
FW + LMP
1:0.03
1.51
Zhang et al. (2013)
FW + WMA
1:0.015
1.54
Zhang et al. (2013)
OFMSW + Cellulose OFMSW + Protein OFMSW + GSW + PMS
1:0.2 1:0.2 0.7:0.2:0.1
Dry batch, 55 °C, initial pH 5.5 Dry batch, 55 °C, Mixing at 150 rpm initial pH 6.5–6.9 Dry batch, 55 °C, Mixing at 150 rpm initial pH 6.6–7.4 Dry batch, 37 °C, Mixing at 180 rpm Dry batch, 55 °C, initial pH 6.0
1.35 1.54 1.62
Ponsá et al. (2011) Ponsá et al. (2011) This study
6
1500
5
1200
4
900
3 600
2
OFMSW:GSW
70:15:15%
70:10:20%
70:20:10%
80:15:5%
80:5:15%
90:5:5%
80:10:10%
25:75%
OFMSW:PMS Final pH
0:100%
75:25%
50:50%
90:10%
100: 0%
0:100%
0
50:50%
0
25:75%
300 75:25%
1
TAN concentration (mg/L)
Improvement is a multiplier factor in gas production due to co-digestion of OFMSW or FW with other substrate.
Final pH
*
Co-substrates
OFMSW:GSW:PMS TAN
Fig. 2a. Final pH versus total ammonia nitrogen (TAN).
Ammonia produced (mg/L)
kinetics parameters were determined by regression analysis. The correlation coefficient between the experimental and simulated data was relatively high resulting in R2 of 0.952. Maximum hydrogen production rate (Rm) of 55.7 ± 2.4, 58.1 ± 3.8, and 73.9 ± 6.3 mL/h were achieved at mixtures of 75% OFMSW + 25% GSW, 50% OFMSW + 50% PMS, and 70% OFMSW + 20% GSW + 10% PMS, respectively. The lag phases of the aforementioned mixtures were 6.9 ± 0.3, 7.2 ± 0.4, and 6.8 ± 0.4, respectively. The lag phases of individual substrates of OFMSW, GSW, and PMS were 7.8 ± 0.5, 9.2 ± 0.6, and 11.5 ± 0.8 h, respectively as indicated in Table 4. Thereby, the synergistic effect of using these different characteristics substrates together not only improved CHP but also accelerated the hydrogen production rate and shortened lag phase period. Hydrogen yields (HY) from co-fermentation of various substrates based on COD, and volatile solids (VS) conversion are presented in Table 5. HY obtained from individual feedstock of OFMSW, GSW, and PMS were 101.9 ± 6.3, 65.4 ± 5.2, and 70.4 ± 5.7 mL/gVSremoved or 133.0 ± 16.6, 96.5 ± 7.6, and 115.7 ± 11.6 mL/gCODremoved respectively. A higher HY of 1200 900 600 300 y = 1251.065e-1.291x R² = 0.716
0 0
0.5
1
1.5
2
2.5
pH drop Fig. 2b. Total ammonia produced versus pH drop.
3
124.6 ± 8.7 mL/gVSremoved or 149.5 ± 6.7 mL/gCODremoved was found for co-digestion of 75% OFMSW + 25% GSW. However, HYs was declined at higher volumetric percentage GSW (Table 5). The highest HY of 132.5 ± 7.4 mL/gVSremoved which corresponds to 146.7 ± 10.2 mL/gCODremoved and 8.4 ± 0.6 L/Lsubstrate was achieved for 50% OFMSW in combination with 50% PMS. Gupta et al., 2014 found that cellulose components fermentation exhibited low HY. The addition of glucose to cellulose improved the fermentation process and increased the HYs by a value of 23%. The maximum HY of 144.9 ± 9.8 mL/gVSremoved or 157.5 ± 12.1 mL/gCODremoved was achieved for the batches containing 70% OFMSW + 20% GSW + 10% PMS. These results are higher than those obtained (69 mL/gVSremoved) by Tyagi et al., 2014 for co-digestion of OFMSW with sludge at a ratio of 1:1 with TS concentration not exceeding 20% and 64.48 mL/g VS for co-fermentation of food waste with pulp & paper sludge (1:1 w/w) (Lin et al., 2013). However, similar findings were reported by Zhang et al., 2013 and Zhang and Wang, 2013. The differences in HY between this study and the above-mentioned studies could be due to the different substrates, feedstock characteristics and operational temperature. Table 6 shows the microbial population counts in the inoculum sludge and co-digestion mixtures (OFMSW/GSW; OFMSW/PMS and OFMSW/GSW/PMS). The results revealed that Clostridium sp. and Enterobacter sp., were dominated in the reactors. However, the microbial population count was dependent on the substrate content. Clostridium sp. counts increased from 1.88 ± 0.04 107 CFU/mL (inoculum sludge) to 2.73 ± 0.01 109 CFU/mL (OFMSW/GSW); 2.40 ± 0.10 109 CFU/mL (OFMSW/PMS) and 3.11 ± 0.08 109 CFU/mL (OFMSW/GSW/PMS). Similar trends were observed for Enterobacter sp. as shown in Table 6. The mixture of 70% OFMSW + 20% GSW + 10% PMS provided the highest Clostridium sp. and Enterobacter sp. counts of
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Table 4 Gompertz kinetics analysis of H2 production from different co-fermentation processes. Batches
Gompertz kinetics P mL
Rmax mL/h
k h
R2
H2 %
Co-fermentation of OFMSW + GSW: 100% OFMSW:0% GSW 75% OFMSW:25% GSW 50% OFMSW:50% GSW 25% OFMSW:75% GSW 0% OFMSW:100% GSW
668.8 ± 52.3 836.8 ± 65.1 696.7 ± 39.6 466.2 ± 42.4 254.1 ± 28.3
43.1 ± 3.9 55.7 ± 2.4 45.1 ± 4.8 28.9 ± 1.9 16.0 ± 2.1
7.8 ± 0.5 6.9 ± 0.3 7.7 ± 0.5 8.0 ± 0.6 9.2 ± 0.6
0.996 0.998 0.997 0.997 0.968
52.1 ± 3.4 63.5 ± 2.7 59.6 ± 4.5 47.4 ± 3.1 43.3 ± 1.9
Co-fermentation of OFMSW + PMS: 90% OFMSW:10% PMS 75% OFMSW:25% PMS 50% OFMSW:50% PMS 25% OFMSW:75% PMS 0% OFMSW:100% PMS
721.5 ± 56.0 805.0 ± 84.9 857.8 ± 57.4 677.3 ± 42.4 388.3 ± 39.6
47.5 ± 3.5 52.6 ± 4.4 58.1 ± 3.8 44.7 ± 4.1 22.6 ± 2.9
7.8 ± 0.4 7.6 ± 0.3 7.2 ± 0.4 9.8 ± 0.5 11.5 ± 0.8
0.995 0.999 0.998 0.998 0.952
54.6 ± 3.4 60.4 ± 5.8 65.1 ± 3.2 56.8 ± 5.7 42.2 ± 4.8
751.7 ± 70.7 810.9 ± 45.3 782.2 ± 28.4 890.9 ± 84.9 1082.5 ± 91.4 823.2 ± 49.5 810.3 ± 72.1
50.5 ± 4.7 55.4 ± 5.1 52.0 ± 4.4 61.2 ± 5.7 73.9 ± 6.3 56.1 ± 3.9 53.4 ± 4.5
7.3 ± 0.3 7.2 ± 0.5 7.3 ± 0.4 7.1 ± 0.3 6.8 ± 0.4 7.3 ± 0.6 7.6 ± 0.4
0.998 0.997 0.996 0.998 0.996 0.998 0.996
54.9 ± 2.2 60.3 ± 4.0 63.7 ± 3.7 61.2 ± 5.5 70.8 ± 6.1 66.3 ± 4.8 64.1 ± 3.6
Co-fermentation of OFMSW + GSW + PMS: 90% OFMSW:5% GSW:5% PMS 80% OFMSW:10% GSW:10% PMS 80% OFMSW:5% GSW:15% PMS 80% OFMSW:15% GSW:5% PMS 70% OFMSW:20% GSW:10% PMS 70% OFMSW:15% GSW:15% PMS 70% OFMSW:10% GSW:20% PMS
Table 5 Hydrogen yield from different co-fermentation combinations. Batches
Hydrogen yield (HY) mL/ gCODremoved
mL/ gVSremoved
100%OFMSW:0%GSW 75%OFMSW:25%GSW 50% OFMSW:50% GSW 25% OFMSW:75% GSW 0% OFMSW:100% GSW
133.0 ± 16.6 149.5 ± 6.7 143.5 ± 14.0 130.5 ± 12.5 96.5 ± 7.6
101.9 ± 6.3 124.6 ± 8.7 119.0 ± 6.0 78.8 ± 3.6 65.4 ± 5.2
6.7 ± 0.5 8.9 ± 0.7 7.0 ± 0.4 5.6 ± 0.4 3.0 ± 0.3
90% OFMSW:10%PMS 75% OFMSW:25% PMS 50% OFMSW:50% PMS 25% OFMSW:75% PMS 0% OFMSW:100% PMS
135.4 ± 8.2 139.6 ± 13.9 146.7 ± 10.2 145.3 ± 19.3 115.7 ± 11.6
102.7 ± 8.8 131.3 ± 11.6 132.5 ± 7.4 110.8 ± 9.4 70.4 ± 5.7
7.8 ± 0.6 8.0 ± 0.8 8.4 ± 0.6 6.8 ± 0.4 3.1 ± 0.4
90% OFMSW:5% GSW:5% PMS 80% OFMSW:10% GSW:10% PMS 80% OFMSW:5% GSW:15% PMS 80% OFMSW:15% GSW:5% PMS 70% OFMSW:20% GSW:10% PMS 70% OFMSW:15% GSW:15% PMS 70% OFMSW:10% GSW:20% PMS
129.1 ± 5.5 136.6 ± 16.8
141.8 ± 10.5 142.8 ± 13.2
7.6 ± 0.7 7.8 ± 0.5
135.6 ± 9.2 137.3 ± 3.9 157.5 ± 12.1
137.1 ± 9.7 138.6 ± 7.1 144.9 ± 9.8
8.1 ± 0.3 8.2 ± 0.8 10.6 ± 0.9
147.2 ± 15.8
129.3 ± 11.5
8.2 ± 0.5
141.2 ± 8.0
128.8 ± 12.4
8.1 ± 0.7
L/ Lsubstrate
3.11 ± 0.08 109 CFU/mL and 1.17 ± 0.04 107 CFU/mL, respectively. This indicates that the mixture of OFMSW/GSW/PMS is capable to harvest more hydrogen producing anaerobes compared with OFMSW/PMS and OFMSW/GSW. Castelló et al. (2011) found that the predominance of Clostridium and Enterobacter sp. in the fermenters fed with cheese whey (protein-rich waste). Enterobacter sp. showed a high increase in the population count
of 1.32 ± 0.06 107 CFU/mL for OFMSW/PMS as compared to OFMSW/GSW (4.85 ± 0.03 106 CFU/mL). Gadow et al., 2013 stated that Enterobacter sp. has a great ability to produce H2 from cellulosic compounds. 3.2. COD, carbohydrate, protein and lipid conversion Fig. 3 presents the COD removal efficiency in addition to the biodegradation efficiency of carbohydrate, protein and lipids during anaerobic co-digestion of different substrates. As depicted in Fig. 3, COD removal efficiency of the mono-fermentation of OFMSW, GSW and PMS were relatively law and amounted to 42.2 ± 4.7, 31.5 ± 3.5 and 47.1 ± 4.8%, respectively. The carbohydrate, protein and lipids degradation efficiency for individual substrates were 55.9 ± 5.4%, 17.1 ± 1.4% and 13.3 ± 1.1% for OFMSW, 78.8 ± 6.4%, 20.3 ± 2.2% and 17.7 ± 1.9% for GSW, 60.4 ± 3.6%, 16.5 ± 2.5% and 12.6 ± 1.6% for PMS. However, the COD removal efficiency was increased up to 50.0 ± 2.1% and 60.4 ± 3.0% with co-fermentation of 75% OFMSW + 25% GSW and 50% OFMSW + 50% PMS respectively. The higher COD removal achieved with adding PMS over GSW may rely to the high lipid content in gelatinous waste which impedes the AD process (Cirne et al., 2007). Similar trends were observed for the removal of carbohydrate, protein and lipids (Fig. 3). The degradation efficiencies of carbohydrate, protein and lipids were significantly increased with co-fermentation of 75% OFMSW + 25% GSW and 50% OFMSW + 50% PMS resulting 65.3 ± 4.7% and 68.7 ± 5.1% for carbohydrate, 23.2 ± 1.8% and 20.8 ± 1.0% for protein, and 19.6 ± 1.6% and 18.8 ± 2.1% for lipids. The COD, carbohydrate, protein and lipids removal was deteriorated at higher and lower dilutions of OFMSW with PMS and GSW. Similar findings were reported by Elbeshbishy and Nakhla (2012) who found that maximum total
Table 6 Microbial population counts in the fermenters. Microbe type
Clostridium sp. (CFU/mL) Enterobacter sp. (CFU/mL)
Inoculum sludge
1.88 ± 0.04 107 2.16 ± 0.05 105
Co-digestion mixture 70% OFMSW/25% GSW
50% OFMSW/50% PMS
70% OFMSW/20% GSW/10% PMS
2.73 ± 0.01 109 4.85 ± 0.03 106
2.40 ± 0.10 109 1.32 ± 0.06 107
3.11 ± 0.08 109 1.17 ± 0.04 107
163
Carbohydrate (%)
80:15:5%
90:5:5%
80:10:10%
0:100%
0:100%
COD removal (%)
OFMSW:GSW:PMS
OFMSW:PMS
OFMSW:GSW
70:10:20%
0
70:15:15%
0
70:20:10%
15 80:5:15%
20 25:75%
30
50:50%
40
75:25%
45
90:10%
60
60
25:75%
80
75:25%
75
50:50%
100
100: 0%
Carbohydrate, protein and lipid removal (%)
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
Protein (%)
Lipid (%)
COD (%)
Fig. 3. COD, carbohydrate, protein and lipid removal efficiency (%).
COD destruction of 59% was achieved with mixture of 80% starch and 20% bovine serum albumin (BSA) compared with 53% and 43% for the fermentation of individual starch and BSA, respectively. This hypothesis was explained by hydrolysis coefficients of carbohydrates and proteins, which registered the highest values at the same mixture (80% starch + 20% BSA). Lin et al., 2013 found that the highest COD removal efficiency of 87% observed at dual fermentation of food waste and paper sludge with ratio of 2:2 (w/w based on VS) which was higher than those obtained for fermentation of individual substrates. In another study Gupta et al., 2014 reported that the COD removal efficiency of a combination of 50% starch to 50% cellulose via AD was higher than the individual substrate of starch and cellulose by percentage of 33% and 72%, respectively. Anaerobic co-fermentation of the three substrates resulted in a positive synergism effect by increasing the COD removal and biodegradation efficiencies of carbohydrates, protein and lipids as shown in Fig. 3. Co-fermentation of 70% OFMSW + 20% GSW + 10% PMS achieved COD removal efficiency of 60.9 ± 4.4% which is 1.53-fold COD removal from OFMSW alone. Carbohydrate, protein and lipids degradation efficiencies were 71.4 ± 3.5, 22.6 ± 2.3 and 20.5 ± 1.8%, respectively for the mixture of 70% OFMSW + 20% GSW + 10% PMS.
followed by HBu was the dominant VFA fractions in the mono-fermentation of OFMSW and PMS. This was not the case for the sole fermentation of GSW whereas HAc and HPr were the highest fractions of VFA. Mono-fermentation of OFMSW, GSW, and PMS produced 5.16 ± 0.38, 3.98 ± 0.27, 4.41 ± 0.16 g/L for HAc, 3.93 ± 0.17, 2.28 ± 0.18, 4.08 ± 0.22 g/L for HBu, 0.58 ± 0.06, 3.02 ± 0.27, 2.81 ± 0.18 g/L for HPr and 1.09 ± 0.13, 0.20 ± 0.03 and 2.47 ± 0.18 g/L for HLa respectively. HAc generation from GSW via dry anaerobic digestion is mainly due to lipids (18.3% of COD) hydrolysis resulting glycerol and long chain fatty acids (LCFAs) which further converted into acetate via the b-oxidation pathway (Cirne et al., 2007). Similar trends were obtained by Fang and Yu, 2002 for gelatin-rich wastewater. They found that the mainstream VFAs were HAc (20.1%), HPr (12.3%) and HBu (9.0%). Gupta et al., 2014 found that mono-fermentation of cellulosic component has relatively low HAc/HBu ratio in addition to a high HPr production. The results in Fig. 4 show that propionate (HPr) fraction significantly increased at increasing the volumetric ratio of GSW in co-fermentation with OFMSW. HLa concentration was dropped at increasing the ratio of GSW. Acetate (HAc) and butyrate (HBu) peaked up to 6.57 ± 0.52 and 4.88 ± 0.35 g/L respectively at 75% OFMSW + 25% GSW and then decreased thereafter. Elbeshbishy and Nakhla, 2012 found that maximum concentration of HAc and HBu achieved at co-fermentation of 80% starch with 20% BSA. Co-fermentation of OFMSW and PMS promoted higher production of HPr and HLa at increasing the volumetric percentage of PMS in the mixture (Fig. 4). In addition, HAc and HBu showed a peak value of 5.56 ± 0.23 and 5.31 ± 0.27 g/L respectively at 50% OFMSW + 50% PMS. However, further increase in volumetric percentage of PMS in the mixture exceeding 50% led to decrease in HAc and HBu generation. Gupta et al., 2014 reported that
3.3. Soluble metabolites components
10.0 8.0 6.0 4.0
OFMSW:GSW Acetate
OFMSW:PMS Buytrate
Propionate
OFMSW:GSW:PMS Lactic
Fig. 4. Net production of soluble metabolites concentrations.
70:10:20%
70:15:15%
80:15:5%
70:20:10%
80:5:15%
80:10:10%
90:5:5%
0:100%
25:75%
50:50%
75:25%
90:10%
0:100%
25:75%
50:50%
0.0
75:25%
2.0 100: 0%
Net production of soluble metabolites (g/L)
Acetate (HAc) and butyrate (HBu), are the desirable soluble products since hydrogen generation occurs via those metabolite products (Elsamadony et al., 2015b). However, HAc, HBu, propionate (HPr), and lactate (HLa) generation was positively affected by the type of feedstock, mono-fermentation, di-fermentation and tri-fermentation processes. As depicted from Fig. 4, HAc
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210
40
HY (mL/g CODremoved)
Before anaerobic digestion After Anaerobic digestion
Intensity (%)
30
20
10
180 150 120 90 60 y = -0.179x2 + 10.175x R² = 0.739
30 0 0
10
0 0
500
1000 1500 Particle diameter (µm)
2000
Fig. 5. Particles size distribution for the mixture of 70% OFMSW + 20% GSW + 10% PMS before and after AD.
co-fermentation of glucose with cellulose (50:50% v/v) led to increase HAc, HBu, and HPr by 5.8, 2.7, and 1.9-fold as compared to fermentation of cellulose alone. Moreover, Lin et al., 2013 found that maximum production of total VFAs (12.3 g/L) achieved at a mixture of FW and PMS with ratio 2:2 (w/w, based on VS). The overall maximum HY of 157.5 ± 12.1 mL/gCODremoved was registered at co-fermentation of 70% OFMSW + 20% GSW + 10% PMS. This corresponds to the soluble metabolite components, in terms of HAc, HBu, HPr and HLa of 8.31 ± 0.58, 6.76 ± 0.30, 1.04 ± 0.14 and 1.16 ± 0.03 g/L, respectively as presented in Fig. 4. 3.4. Effect of co-fermentation process on particle size distribution Limited number of literatures investigated the effect of anaerobic co-fermentation on particles size distribution of the mixture via dry anaerobic digestion (AD). However, particle size of substrates may significantly effect on the hydrolysis rate, the speed and performance of AD process (Zhang and Banks, 2013). Fig. 5 shows the effect of anaerobic co-fermentation of the three substrates (70% OFMSW + 20% GSW + 10% PMS) on particles size distribution. As depicted from Fig. 5, the domain particle size in the mixture before co-fermentation process was 850 lm with intensity of 24.1% followed by 1850 lm (14.8%) then 450 lm (13.4%). However, the effectiveness of co-fermentation on particles size emerged clearly in the digestate, where 34% of particles were within diameter range of 75 lm. This indicates that the co-fermentation process reduced the particle size distribution which is favorably utilized by hydrogen producing bacteria. Izumi et al., 2010 emphasized that the highest gas production rate accomplished at initial diameter of food waste particles with value of 718 lm and finer particles declined the gas production rate. The mean particle size diameters for feedstock and the digestate were 939.3 and 115.2 lm, respectively with reduction of 8.15-fold in the particle size of the mixtures.
20
30
40
COD/TN ratio Fig. 6a. Effect of carbon to nitrogen (C/N) ratio on H2 yields.
considerably varied based on the volumetric ratios of the feedstocks subjected to co-fermentation processes. Fig. 6a shows the influence of initial C/N ratio on HY. The maximum HYs of 149.5 ± 6.7, 146.7 ± 10.2, and 157.5 ± 12.1 mL/gCODremoved were registered at C/N ratios of 28.8 ± 0.2, 32.8 ± 0.4 and 29.4 ± 0.3 for 75% OFMSW + 25% GSW, 50% OFMSW + 50% PMS, and 70% OFMSW + 20% GSW + 10% PMS, respectively. At lower and higher C/N ratios, HYs were decreased followed a 2nd degree polynomial function with correlation coefficient factor (R2 = 0.739). Thus, the positive synergistic effect emphasized through co-fermentation of different co-substrates. According to earlier studies the desired C/N ratio was close to those obtained in this investigation i.e. C/N ratio ranged from 26 to 31 for co-fermentation of food waste with primary and secondary sludge (Zhou et al., 2013) and C/N ratio of 33 for mono-fermentation of food waste and sludge (Sreela-or et al., 2011). In addition, (Tawfik and El-Qelish, 2014a,b) found that increasing COD/N ratio from 12 to 24.1 improved HY from 93.2 to 150.9 mLH2 =gVSremoved d and thereafter a significant drop in HY was noticed beyond this limit. Tyagi et al. (2014) achieved a maximum HY of 51 mL/gVSremoved at 5:1 volumetric ratio between OFMSW and sludge related to optimum COD/N ratio of 31. 3.6. Effect of Ca+2 concentrations on the volumetric H2 production In order to investigate the effect of calcium concentration on volumetric H2 production, volumetric percentages of calcium ions rich wastes such as GSW and PMS were varied and co-fermented with OFMSW. Fig. 6b shows the effect of different initial calcium (Ca+2) concentrations on volumetric H2 production (VHP). The results depict a good 2nd degree polynomial relationship between calcium (Ca+2) concentration and VHP with correlation coefficient factor R2 of 0.7 (Fig. 6b). The results revealed that volumetric H2 production increased from 4.5 ± 0.3 to 7.2 ± 0.6 LH2 =Lsubstrate at increasing Ca+2 concentrations from 1.8 ± 0.1 to 6.3 ± 0.5 g/L respectively. This can be attributed to, (1) calcium ion plays as coagulant aids to enhance the biomass accumulation as it stimulates microbial growth and, consequently, affect specific growth
3.5. Effect of C/N ratio on the H2 yield 8 Volumetric H2 production (LH2/Lreactor)
Carbon to nitrogen (C/N) ratio of feedstock is critically important to facilitate the conversion of organics into hydrogen (Wang et al., 2013). C/N ratio promotes microbial growth due to providing a sufficient nutrient balance (Tyagi et al., 2014). In addition, a proper C/N ratio improves the hydrogen producers’ activity and preclude ammonia inhibition which in turn enhancing the bio-hydrogen production via anaerobic digestion process (Zhou et al., 2013). OFMSW is carbohydrate-rich substrate with a low nitrogen content resulting in a considerable high C/N ratio of 39.5 ± 0.7 as depicted in Table 1. However, the co-fermentation of OFMSW with N-lean substrates such as GSW and PMS provided C/N of 13.0 ± 0.2 and 27.7 ± 0.2, respectively. C/N ratio was
6 4 2 y = -0.021x2 + 0.204x + 4.867 R² = 0.700
0 0
5
10 15 Ca+2 concentration (g/L)
20
25
Fig. 6b. Effect of Ca+2 concentrations on volumetric H2 production.
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
rate (Chen et al., 2008) (2) calcium is essential for catalytic activity, either as a participant in the reaction, or as a structural requirement in order to maintain the appropriate confirmation of the active site (Yuan et al., 2010) and 3. Ca+2 represents a counteract of the inhibitory effect of ammonia (Chen et al., 2008). However, higher concentrations of Ca+2 exceeding 6.3 g/L led to a significant drop in volumetric H2 production (1.7 ± 0.2 LH2 =Lsubstrate ). A higher Ca+2 levels can cause severe inhibition or toxicity in addition to dehydrate bacterial cells due to osmotic pressure (Chen et al., 2008; Zhang et al., 2013). Similar trends was also observed by Zhang et al. (2013) who reported that the addition of lime mud produced from paper-making process (LMP) (which contains 35.44% (w/w) calcium) to food waste in batch system not only improved the hydrogen production 1.5-fold compared with control batch but also accelerated the hydrogen production rate, and shortened lag phase period. Thereafter, the hydrogen production dropped at higher amount of lime mud. Likewise, Yuan et al. (2010) concluded that supplying continuous stirred-tank reactor (CSTR) reactor with 100 mg/L Ca+2 improved the stability of the system as cell retention was multiplied twice in cell density than the case of no calcium addition. As well, addition of 3 g/L of Ca+2 achieved the maximum biogas production and organic removal efficiency via AD of swine wastewater (Ahn et al., 2006). Higher Ca+2 concentration exceeded 7 g/L had an inhibitory effect on anaerobic digestion process. Whereas, anaerobic granules exposure to a significant loss in biological activity at high calcium concentration (El-Mamouni, 1995). 4. Conclusions Dry anaerobic co-fermentation of 70% OFMSW + 20% GSW + 10% PM resulted in a positive synergism effect on the HP and HY by increasing the conversion efficiency of COD (60.9 ± 4.4%), carbohydrates (71.4 ± 3.5%), protein (22.6 ± 2.3) and lipids (20.5 ± 1.8%). However, the HP and HY is C/N ratio, calcium concentration, type of feedstock and volumetric ratio dependant. Maximum HYs of 124.6 ± 8.7, 132.5 ± 7.4, and 144.9 ± 9.8 mL/gVSremoved achieved by co-fermentation of 75% OFMSW + 25% GSW, 50% OFMSW + 50% PMS, and 70% OFMSW + 20% GSW + 10% PMS, respectively. The highest HP was obtained at C/N ratio of 29.4 ± 0.3 and Ca+2 concentration of 6.3 ± 0.5 g/L. Acknowledgements The first author would like to acknowledge Ministry of Higher Education (MoHE) of Egypt for providing a scholarship to conduct this study as well as the Egypt Japan University of Science and Technology (E-JUST) for offering the facility and tools needed to conduct this work. This research is also financed from Science & Technology Development Fund (STDF) in Egypt, project ID: 3665. References Ahn, J.-H., Do, T.H., Kim, S.D., Hwang, S., 2006. The effect of calcium on the anaerobic digestion treating swine wastewater. Biochem. Eng. J. 30, 33–38. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC, USA. Banks, C.J., Humphreys, P.N., 1998. The anaerobic treatment of a ligno-cellulosic substrate offering little natural pH buffering capacity. Water Sci. Technol. 38, 29–35. Breure, A.M., Beeftink, H.H., Verkuijlen, J., van Andel, J.G., 1986. Acidogenic fermentation of protein/carbohydrate mixtures by bacterial populations adapted to one of the substrates in anaerobic chemostat cultures. Appl. Microbiol. Biotechnol. 23, 245–249. Castelló, E., Perna, V., Wenzel, J., Borzacconi, L., Etchebehere, C., 2011. Microbial community composition and reactor performance during hydrogen production in a UASB reactor fed with raw cheese whey inoculated with compost. Water Sci. Technol. 64, 2265–2273.
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Dry anaerobic co-digestion of organic fraction of municipal waste with paperboard mill sludge and gelatin solid waste for enhancement of hydrogen production M. Elsamadony ⇑, A. Tawfik Environmental Engineering Department, Egypt-Japan University of Science and Technology (E-Just), New Borg El Arab City, 21934 Alexandria, Egypt
h i g h l i g h t s Dry anaerobic co-fermentation of OFMSW with GSW and/or PMS was investigated. Maximum H2 production was obtained at 70% OFMSW + 20% GSW + 10% PMS. C/N ratio and Ca
+2
concentration are the key factors affecting on the H2 production.
Buffering capacity of OFMSW increased via addition of GSW and/or PMS. Mean particle size diameters reduced by a percentage of 87.7% in the digestate.
a r t i c l e
i n f o
Article history: Received 1 April 2015 Received in revised form 7 May 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: Bio-hydrogen Dry anaerobic co-fermentation Organic fraction of municipal solid waste Paperboard mill sludge Gelatin solid waste
a b s t r a c t The aim of this study is to investigate the bio-H2 production via dry anaerobic co-fermentation of organic fraction of municipal solid waste (OFMSW) with protein and calcium-rich substrates such as gelatin solid waste (GSW) and paperboard mill sludge (PMS). Co-fermentation of OFMSW/GSW/PMS significantly enhanced the H2 production (HP) and H2 yield (HY). The maximum HP of 1082.5 ± 91.4 mL and HY of 144.9 ± 9.8 mL/gVSremoved were achieved at a volumetric ratio of 70% OFMSW:20% GSW:10% PMS. COD, carbohydrate, protein and lipids conversion efficiencies were 60.9 ± 4.4%, 71.4 ± 3.5%, 22.6 ± 2.3% and 20.5 ± 1.8% respectively. Co-fermentation process reduced the particle size distribution which is favorably utilized by hydrogen producing bacteria. The mean particle size diameters for feedstock and the digestate were 939.3 and 115.2 lm, respectively with reduction value of 8.15-fold in the mixtures. The volumetric H2 production increased from 4.5 ± 0.3 to 7.2 ± 0.6 LH2 =Lsubstrate at increasing Ca+2 concentrations from 1.8 ± 0.1 to 6.3 ± 0.5 g/L respectively. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Proliferation of biological H2 as a renewable energy carrier that replaces fossil fuel resources would result into cleaner environment. Since, H2 produces only water as a combustion by-product, in addition to generating a higher energy yields (122 kj/g) as compared to hydrocarbon counterpart (Tawfik and El-Qelish, 2014a,b). In spite of H2 can be produced by the conventional physico-chemical technologies, but they required electricity derived from combustion of the fossil fuels (Zhou et al., 2013). So far, anaerobic digestion (AD) is the most sophisticated technology in which H2 can be produced biologically from various ⇑ Corresponding author. Tel.: +20 03 4599520. E-mail addresses: [email protected], yahoo.com (M. Elsamadony). http://dx.doi.org/10.1016/j.biortech.2015.05.017 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
m_elsamadoney@
biodegradable organic wastes. This is mainly owing to its low energy requirement, low sludge production, cost effective, as well as being eco-friendly and energy recovery (Razaviarani et al., 2013; Tyagi et al., 2014). AD is classified into three categories of wet, semi-dry, and dry one according to total solid (TS) content of the feedstock (610%, 10–20%, and P20%, respectively). However, dry AD claimed to be more favorable than wet AD in term of smaller reactor volume, lower energy requirements for heating, and less material handling (Elsamadony et al., 2015a). Usually AD process is operated under mesophilic or thermophilic condition, in which thermophilic condition reported more efficient method. Since, thermophilic fermentation prevailed higher gas production, metabolic rates two to threefold mesophilic condition and conversion efficiencies besides pathogen disinfection. Meanwhile, thermophilic dry AD faces some drawbacks of high sensitivity to shock load or toxic substances (Gallert et al., 1998).
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Organic fraction of municipal solid waste (OFMSW) in Egypt amounted to 21 million tons/year which accounted for 60% of total municipal solid waste (MSW) (Tawfik and El-Qelish, 2014a,b). OFMSW is a desirable feedstock for dry AD due to its high organic fraction and carbohydrates content which is the preferable substrate for fermentative hydrogen-producing microorganisms and easily hydrolysable waste with high hydrogen production potential (Kim, 2004). However, dry AD of sole OFMSW has some difficulties which need to be addressed to have a fully functional bio-hydrogen production system such as little amount of trace elements content, lack in nitrogen which is an essential nutrient for hydrogen producers. Furthermore, OFMSW has a low buffering capacity (pH ranged from 4 to 5) which makes it more susceptible to volatile fatty acids (VFAs) inhibition (Lin et al., 2013; Zhou et al., 2013). In order to overcome the aforementioned drawbacks, anaerobic co-fermentation of OFMSW with gelatin solid waste (GSW) and paperboard mill sludge (PMS) has been examined for enhancing the dry AD performance and developing hydrogen productivity due to synergistic and complementary effects. Co-fermentation of different substrates could be beneficial due to dilution of toxic chemicals, enhanced balance of nutrients, and synergistic effect of microorganisms (Razaviarani and Buchanan, 2014). Whereas, co-fermentation of OFMSW with protein-rich substrates such as GSW and PMS supplied a more balanced carbon to nitrogen (C/N) ratio (Kim, 2004). Optimized C/N ratio not only balances the nutrients, but also enhances the bacterial productivity of hydrogen and preclude ammonia inhibition (Zhou et al., 2013). Furthermore, when highly nitrogenous wastes co-fermented with OFMSW provides a good buffering capacity making it more accessible to utilize for energy production and increasing the stability of the anaerobic process (Zhang et al., 2014). Additionally, GSW and PMS (CaCO3-rich wastes) substrates provide OFMSW with calcium ions which released slowly into the substrate (Zhang and Wang, 2013) led to highly increase in hydrogen generation and improved performance of bioreactor by increasing the cell density (Yuan et al., 2010). Formerly, Zhou et al., 2013 suggested that optimal C/N ratio ranged from 26 to 31 in a co-digestion consisting of food waste (FW) and sewage sludge. Kim, 2004 found that co-fermentation of FW with sewage sludge resulted in a more favorable carbohydrate-COD/protein-COD for hydrogen production. Moreover several researchers (Wang et al., 2013; Zhang and Wang, 2013; Zhang et al., 2014, 2013, 2011) indicated that an improved pH buffering capacity, balanced C/N ratio and sufficient trace element could account for the enhanced hydrogen production achieved in a co-digestion process. Formerly, Zhou et al. (2013) suggested that optimal C/N ratio ranged from 26 to 31 in a co-digestion consisting of FW and sewage sludge. Kim (2004) found that co-fermentation of food waste
with sewage sludge resulted in a more favorable carbohydrate-COD/protein-COD for hydrogen production. Moreover several researchers (Wang et al., 2013; Zhang and Wang, 2013; Zhang et al., 2014, 2013, 2011) indicated that an improved pH buffering capacity, balanced C/N ratio and sufficient trace element could account for the enhanced hydrogen production achieved in a co-digestion process. Therefore, the aims of this investigation are to (1) assess the feasibility of thermophilic dry anaerobic co-fermentation of OFMSW/GSW, OFMSW/PMS and OFMSW/GSW/PMS. (2) Study the effect of co-fermentation process on particle size distribution and (3) investigate the key factors affecting on co-fermentation process such as C/N ratio, Ca+2 concentrations and buffering capacity in order to have a better understanding for the synergistic effect of co-fermentation.
2. Methods 2.1. Characteristics of feedstocks and inoculum sludge Three types of substrates were used in this study: (1) organic fractions of municipal solid waste (OFMSW) collected from local municipal landfill site located in Alexandria city, Egypt. The bio-wastes mainly contained rice, vegetables, bread, beans, and some meats (bones were removed from OFMSW). The collected OFMSW was crushed into small particles using an electrical grinder avoided any dilution. Furthermore, the resulting residues was sieved through a stainless steel sieve with opening of 2.0 mm. (2) gelatin solid waste (GSW) harvested from gelatin manufacturing company situated in Alexandria city. The gelatin manufacturing processes include cutting the animals’ fibers into small particles (1–2 cm), soaking the resultant fibers into lime solution for a period ranging from 9 to 10 weeks, adjusting pH up to 12 and finally the extracting gelatin process is taken place at a temperature (50–90 °C). (3) paperboard mill sludge (PMS) obtained from sedimentation basin placed at Aldar Albydaa factory (Borg Alarab city, Egypt). The fresh water and lime is mainly used for the production of paper board from recycled waste paper following by centrifugal to remove impurities, bleaching, and finally drying process at a temperature of 135 °C in rolling machine. OFMSW particulate has a spherical shape, while GSW presented in jellylike form and PMS consists of fibers with different length and diameter. The seed sludge used in this study was harvested from thickener tank of wastewater treatment plant located in Alexandria city, Egypt. The mixed culture bacteria was further concentrated by settling for 24 h, and stored for one month under anaerobic conditions. The characteristics of OFMSW, GSW, PMSW and the seed sludge are presented in Table 1.
Table 1 Characteristics of feedstocks and inoculums sludge. Parameters pH TS VS COD Carbohydrate TKj-N C/N NH4-N Lipids Proteins Ca+2 HAc HBu HLa HPr a
Unit % % g/L g/L g/L mg/L g/L g/L g/L g/L g/L g/L g/L
Values represent the average ± STD.
OFMSW
PMS
GSW
Seed sludge
3.88 ± 0.2a 28.7 ± 1.8 26.6 ± 0.7 115.2 ± 9.5 52.3 ± 4.3 2.9 ± 0.2 39.5 ± 0.7 268.2 ± 18.3 3.9 ± 0.4 16.5 ± 0.9 2.3 ± 0.1 2.7 ± 0.9 1.1 ± 0.1 0.9 ± 0.0 0.4 ± 0.1
6.91 ± 0.4 7.5 ± 0.3 5.4 ± 0.5 51.6 ± 4.1 9.6 ± 0.5 1.9 ± 0.1 27.7 ± 0.2 123.2 ± 10.9 1.2 ± 0.1 10.9 ± 0.7 17.9 ± 1.1 0.6 ± 0.1 0.4 ± 0.1 0.1 ± 0.0 0.1 ± 0.0
12.71 ± 0.9 8.1 ± 0.7 6.2 ± 0.3 66.8 ± 4.4 1.1 ± 0.1 5.2 ± 0.4 13.0 ± 0.2 437.2 ± 31.7 4.2 ± 0.5 29.5 ± 1.1 28.5 ± 2.0 1.2 ± 0.1 N.D. 0.4 ± 0.0 0.6 ± 0.1
6.49 ± 0.2 6.3 ± 0.6 5.0 ± 0.2 22.4 ± 1.6 7.5 ± 0.3 1.0 ± 0.1 22.7 ± 0.1 201.6 ± 15.1 0.9 ± 0.2 4.9 ± 0.2 0.8 ± 0.1 0.9 ± 0.1 1.3 ± 0.1 0.2 ± 0.0 0.5 ± 0.0
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165 Table 2 Batch dry anaerobic co-digestion experiments for hydrogen production from OFMSW/ GSW; OFMSW/PMS and OFMSW/GSW/PMS. Batches/substrates
Seed sludge (mL)
OFMSW (mL)
GSW (mL)
PMS (mL)
OFMSW + GSW
50 50 50 50 50
100 75 50 25 0
0 25 50 75 100
0 0 0 0 0
OFMSW + PMS
50 50 50 50 50
90 75 50 25 0
0 0 0 0 0
10 25 50 75 100
OFMSW + GSW + PMS
50 50 50 50 50 50 50
90 80 80 80 70 70 70
5 10 5 15 20 15 10
5 10 15 5 10 15 20
Agar incubated for 72 h at 55 ± 2 °C under anaerobic conditions. All analyses were independently conducted in duplicate to guarantee the reliability of results. 2.5. Analytical methods
2.2. Experimental set-up Batch dry anaerobic co-digestion experiments for hydrogen production from OFMSW/GSW; OFMSW/PMS and OFMSW/GSW/PMS were tested under thermophilic conditions (55 ± 2 °C). The experiments were conducted using 200 mL serum bottles in duplicates. All batches were initially inoculated with mixed culture bacteria and preheated at 70 °C for 30 min to inhibit the bioactivity of hydrogen consumers and to harvest spore-forming anaerobic bacteria (Zhou et al., 2013). Afterwards different volumetric ratios of substrates (OFMSW/GSW; OFMSW/PMS and OFMSW/GSW/PMS) were added as shown in Table 2. The pH of the reactors was 6.0 ± 0.1. The head space of the bottles (50 mL) were flushed with nitrogen gas for 2 min to remove the oxygen from the culture medium and capped tightly with rubber stoppers and aluminum caps. The evolved gas was measured by the displacement method. Moreover, the biogas composition was analyzed using gas chromatography. 2.3. Kinetic studies Cumulative hydrogen production curves were recorded over the time course of the batch experiments and analyzed using the modified Gompertz equation (Elsamadony et al., 2015b):
Rm e H ¼ P exp exp ðk tÞ þ 1 P
159
ð1Þ
where H (mL) is the cumulative hydrogen production at the reaction time t (h), P (mL) is the hydrogen potential, Rm (mL/h) is the maximum hydrogen production rate, e = 2.71828, and k (h) is the lag phase period. 2.4. Bacterial population Samples for detecting and identification of microbial consortium bacteria were harvested from inoculum sludge and the fermenters containing OFMSW/GSW, OFMSW/PMS and OFMSW/GSW/PMS. The collected samples were serially diluted with phosphate buffering saline (PBS) solution. Afterwards, the samples were spread onto the proper agar plates. Clostridium sp. enumeration was carried out using Neomycin nagler (NN) medium and Desoxycholate hydrogen sulfide lactose (DHL) medium was used to enumerate Enterobacter sp. Anaerobic plate counts were manually accomplished for counting of colonies on Plate Count
Total solids (TS), volatile solids (VS), chemical oxygen demand (COD), total Kjeldahl nitrogen (TKj-N), lipids and calcium ion were quantified according to APHA, 2005. Protein content was calculated as follows (6.25 TKj-N ammonia nitrogen)) (Zhang et al., 2011). Carbohydrate was measured according to the phenol–sulfuric acid method (Dubois et al., 1956). Volatile fatty acids (VFAs) were analyzed by high performance liquid chromatography (LC-10AD, Shimadzu, Japan). The temperature of column oven was 40 °C. 4 mM H2SO4 was used as a mobile phase at a flow rate of 0.5 mL/min for 22 min followed by 0.4 mL/min for 8 min. The evolved gas was measured by displacement method. Moreover, Hydrogen content in the biogas was analyzed using a gas chromatograph (GC-2014, Shimadzu, Japan) equipped with a thermal conductivity detector and Shin carbon column. The operational temperatures of the injection port, the column oven and the detector were 100, 120 and 150 °C, respectively. Helium was used as the carrier gas at a flow rate of 25 mL/min. Particle size distribution analysis was carried out using laser diffraction spectroscopy (Beckman Coulter LS230). 3. Results and discussion 3.1. Hydrogen production, yield and microbial population Fig. 1a–c shows the time course of cumulative hydrogen production (CHP) from organic fraction of municipal solid waste (OFMSW) + gelatin solid waste (GSW), OFMSW + paper mill sludge (PMS) and OFMSW + GSW + PMS via dry anaerobic co-digestion process. The results obtained indicated that the peak CHP of 836.8 ± 65.1 mL was achieved from co-fermentation of OFMSW (75%) + GS (25%) (v/v) (Fig. 1a). This exceeds the mono-fermentation of OFMSW by a value of 25.1%. This is mainly due to the gelatin waste represents an excellent source for nitrogen which is necessary for hydrogen producing bacteria. Similar trends were observed by Kim (2004). Breure et al., 1986 showed that the hydrolysis of gelatin was increased as well as biomass concentration due to adding of glucose. Similar findings were reported by Zhou et al., 2013 who found that maximum CHP of 255 mL was achieved from co-fermentation of food waste + primary sludge + waste activated sludge at volumetric ratios of 80:15:5 respectively which surpass the mono fermentation of food waste by 77%. However, the CHP was deteriorated at higher volumetric percentage of GSW where the CHP amounted to 676.7 ± 39.6 mL at OFMSW (50%) + GS (50%). The lowest CHP of 466.2 ± 42.4 and 254.1 ± 28.3 mL was registered for OFMSW (25%) + GS (75%) and GSW (100%), respectively. This indicates that the major portion of hydrogen production was mainly due to the degradation of OFMSW where the CHP amounted to 668.8 ± 52.3 mL for mono-fermentation of OFMSW (100%). Fig. 1b shows the CHP from co-digestion of OFMSW + paper mill sludge (PMS). The results revealed that the maximum CHP was 857.8 ± 57.4 mL for the mixture of 50% OFMSW + 50% PMS which significantly exceeded the anaerobic digestion of OFMSW alone by a value of 28.3%. Apparently, the co-fermentation of OFMSW with PMS had a positive impact on the CHP. Furthermore, decreasing the volumetric ratio of OFMSW in the batches led to decline in hydrogen production. Similar results was reported by Gupta et al., 2014 who found lower hydrogen productivity of 53 mL with cellulose as sole substrate compared to glucose as co-substrate with
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
100% OFMSW : 0% GSW 75% OFMSW : 25% GSW 50% OFMSW : 50% GSW 25% OFMSW : 75% GSW 0% OFMSW : 100% GSW
Cumulative hydrogen production (mL)
160
1000
(a) 800 600 400 200 0
90% OFMSW : 10% PMS 75% OFMSW : 25% PMS 50% OFMSW : 50% PMS 25% OFMSW : 75% PMS 0% OFMSW : 100% PMS
Cumulative hydrogen production (mL)
0
20
80% OFMSW: 5% GSW: 15% PMS 80% OFMSW: 15% GSW: 5% PMS 70% OFMSW: 20% GSW: 10% PMS 70% OFMSW: 15% GSW: 15% PMS 70% OFMSW: 10% GSW: 20% PMS
Cumulative hydrogen production (mL)
80% OFMSW: 10% GSW: 10% PMS
80
20 40 60 fermentation time (hrs)
80
20
80
1000
(b) 800 600 400 200 0 0
90% OFMSW: 5% GSW: 5% PMS
40 60 fermentation time (hrs)
1200
(c)
1000 800 600 400 200 0 0
40 60 fermentation time (hrs)
Fig. 1. Cumulative hydrogen production (CHP) from co-digestion of various substrates, (a) OFMSW + GSW, (b) OFMSW + PMS, and (c) OFMSW + GSW + PMS.
cellulose (303 mL). Lin et al., 2013 found that anaerobic co-fermentation of PMS and FW is beneficial for the hydrogen production at a ratio of 2:2 (w/w) where HP increased by 12.8 and 2.2-folds higher than those recorded for individual substrates of PMS and FW, respectively. The CHP was deteriorated and dropped to 677.3 ± 42.4 mL at increasing the content of PMS up to 75% (v/v). PMS alone recorded the minimum CHP of 388.3 ± 39.6 mL. Co-fermentation synergistic effect of OFMSW, GSW and PMS was investigated at different volumetric ratios for hydrogen production (Fig. 1c). The highest CHP of 1082.5 ± 91.4 mL was obtained at (70% OFMSW + 20% GSW + 10% PMS) which was significantly higher than those registered for OFMSW alone by a value of 61.9%. This can be attributed to the addition of GSW and PMS to OFMSW which could enhance H2 production potential due to balanced carbohydrate/protein ratio. Gelatin solid waste (GSW) and paper mill sludge (PMS) are heavily polluted wastes with high protein content, alkalinity and abundant nitrogenous compounds, and they could be promising co-substrates for enhancement of hydrogen production from OFMSW. Moreover, gelatin solid waste (GSW) and paper mill sludge (PMS) contained a huge amount of calcium which is preferable for hydrogen production via anaerobic digestion process. Table 3 shows the superior of hydrogen production from co-fermentation of OFMSW/GSW/PMS as compared to previous studies.
As depicted from Fig. 2a, the pH of the digestate was largely dropped to a level of 3.86 ± 0.28 with fermentation of OFMSW alone resulting in a low hydrogen production. Increasing the volumetric ratios of GSW and PMS in combination with OFMSW maintained pH level in the digestate between 4.4 and 4.95 as presented in Fig. 2a. This is mainly due to the co-substrates containing protein which led to a release of ammonia nitrogen in the media via anaerobic fermentation process resulted in increasing the pH buffering capacity (Banks and Humphreys, 1998). Recent literatures showed the positive effect of co-fermentation process on the pH buffering capacity during AD process (Lin et al., 2013; Wang et al., 2013; Zhang and Wang, 2013; Zhang et al., 2013). Zhang and Wang (2013) discovered that addition of white mud from ammonia soda process (WMA) to food waste led to an increase the final pH from 3.80 to 6.22. In another study, Zhang et al., 2013 found that addition of lime mud from paper-making process to food waste increase the final pH from 4.5 to 5 without using any external alkali chemicals. Ammonia maintains a high level of bicarbonate in the wastewater as a part of buffering capacity in AD (Gallert et al., 1998). Fig. 2b shows the relationship between ammonia produced and pH drop in the co-fermentation processes with correlation factor (R2 = 0.716). Cumulative hydrogen production data shown in Fig. 1a–c were correlated with the Gompertz model equation (Eq. (1)) of which
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M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165 Table 3 Hydrogen production from co-digestion processes. Mixing ratio
Operational conditions
Improvement*
References
OFMSW + SS
1:1
1.20
Tyagi et al. (2014)
FW + LMP
1:0.03
1.51
Zhang et al. (2013)
FW + WMA
1:0.015
1.54
Zhang et al. (2013)
OFMSW + Cellulose OFMSW + Protein OFMSW + GSW + PMS
1:0.2 1:0.2 0.7:0.2:0.1
Dry batch, 55 °C, initial pH 5.5 Dry batch, 55 °C, Mixing at 150 rpm initial pH 6.5–6.9 Dry batch, 55 °C, Mixing at 150 rpm initial pH 6.6–7.4 Dry batch, 37 °C, Mixing at 180 rpm Dry batch, 55 °C, initial pH 6.0
1.35 1.54 1.62
Ponsá et al. (2011) Ponsá et al. (2011) This study
6
1500
5
1200
4
900
3 600
2
OFMSW:GSW
70:15:15%
70:10:20%
70:20:10%
80:15:5%
80:5:15%
90:5:5%
80:10:10%
25:75%
OFMSW:PMS Final pH
0:100%
75:25%
50:50%
90:10%
100: 0%
0:100%
0
50:50%
0
25:75%
300 75:25%
1
TAN concentration (mg/L)
Improvement is a multiplier factor in gas production due to co-digestion of OFMSW or FW with other substrate.
Final pH
*
Co-substrates
OFMSW:GSW:PMS TAN
Fig. 2a. Final pH versus total ammonia nitrogen (TAN).
Ammonia produced (mg/L)
kinetics parameters were determined by regression analysis. The correlation coefficient between the experimental and simulated data was relatively high resulting in R2 of 0.952. Maximum hydrogen production rate (Rm) of 55.7 ± 2.4, 58.1 ± 3.8, and 73.9 ± 6.3 mL/h were achieved at mixtures of 75% OFMSW + 25% GSW, 50% OFMSW + 50% PMS, and 70% OFMSW + 20% GSW + 10% PMS, respectively. The lag phases of the aforementioned mixtures were 6.9 ± 0.3, 7.2 ± 0.4, and 6.8 ± 0.4, respectively. The lag phases of individual substrates of OFMSW, GSW, and PMS were 7.8 ± 0.5, 9.2 ± 0.6, and 11.5 ± 0.8 h, respectively as indicated in Table 4. Thereby, the synergistic effect of using these different characteristics substrates together not only improved CHP but also accelerated the hydrogen production rate and shortened lag phase period. Hydrogen yields (HY) from co-fermentation of various substrates based on COD, and volatile solids (VS) conversion are presented in Table 5. HY obtained from individual feedstock of OFMSW, GSW, and PMS were 101.9 ± 6.3, 65.4 ± 5.2, and 70.4 ± 5.7 mL/gVSremoved or 133.0 ± 16.6, 96.5 ± 7.6, and 115.7 ± 11.6 mL/gCODremoved respectively. A higher HY of 1200 900 600 300 y = 1251.065e-1.291x R² = 0.716
0 0
0.5
1
1.5
2
2.5
pH drop Fig. 2b. Total ammonia produced versus pH drop.
3
124.6 ± 8.7 mL/gVSremoved or 149.5 ± 6.7 mL/gCODremoved was found for co-digestion of 75% OFMSW + 25% GSW. However, HYs was declined at higher volumetric percentage GSW (Table 5). The highest HY of 132.5 ± 7.4 mL/gVSremoved which corresponds to 146.7 ± 10.2 mL/gCODremoved and 8.4 ± 0.6 L/Lsubstrate was achieved for 50% OFMSW in combination with 50% PMS. Gupta et al., 2014 found that cellulose components fermentation exhibited low HY. The addition of glucose to cellulose improved the fermentation process and increased the HYs by a value of 23%. The maximum HY of 144.9 ± 9.8 mL/gVSremoved or 157.5 ± 12.1 mL/gCODremoved was achieved for the batches containing 70% OFMSW + 20% GSW + 10% PMS. These results are higher than those obtained (69 mL/gVSremoved) by Tyagi et al., 2014 for co-digestion of OFMSW with sludge at a ratio of 1:1 with TS concentration not exceeding 20% and 64.48 mL/g VS for co-fermentation of food waste with pulp & paper sludge (1:1 w/w) (Lin et al., 2013). However, similar findings were reported by Zhang et al., 2013 and Zhang and Wang, 2013. The differences in HY between this study and the above-mentioned studies could be due to the different substrates, feedstock characteristics and operational temperature. Table 6 shows the microbial population counts in the inoculum sludge and co-digestion mixtures (OFMSW/GSW; OFMSW/PMS and OFMSW/GSW/PMS). The results revealed that Clostridium sp. and Enterobacter sp., were dominated in the reactors. However, the microbial population count was dependent on the substrate content. Clostridium sp. counts increased from 1.88 ± 0.04 107 CFU/mL (inoculum sludge) to 2.73 ± 0.01 109 CFU/mL (OFMSW/GSW); 2.40 ± 0.10 109 CFU/mL (OFMSW/PMS) and 3.11 ± 0.08 109 CFU/mL (OFMSW/GSW/PMS). Similar trends were observed for Enterobacter sp. as shown in Table 6. The mixture of 70% OFMSW + 20% GSW + 10% PMS provided the highest Clostridium sp. and Enterobacter sp. counts of
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Table 4 Gompertz kinetics analysis of H2 production from different co-fermentation processes. Batches
Gompertz kinetics P mL
Rmax mL/h
k h
R2
H2 %
Co-fermentation of OFMSW + GSW: 100% OFMSW:0% GSW 75% OFMSW:25% GSW 50% OFMSW:50% GSW 25% OFMSW:75% GSW 0% OFMSW:100% GSW
668.8 ± 52.3 836.8 ± 65.1 696.7 ± 39.6 466.2 ± 42.4 254.1 ± 28.3
43.1 ± 3.9 55.7 ± 2.4 45.1 ± 4.8 28.9 ± 1.9 16.0 ± 2.1
7.8 ± 0.5 6.9 ± 0.3 7.7 ± 0.5 8.0 ± 0.6 9.2 ± 0.6
0.996 0.998 0.997 0.997 0.968
52.1 ± 3.4 63.5 ± 2.7 59.6 ± 4.5 47.4 ± 3.1 43.3 ± 1.9
Co-fermentation of OFMSW + PMS: 90% OFMSW:10% PMS 75% OFMSW:25% PMS 50% OFMSW:50% PMS 25% OFMSW:75% PMS 0% OFMSW:100% PMS
721.5 ± 56.0 805.0 ± 84.9 857.8 ± 57.4 677.3 ± 42.4 388.3 ± 39.6
47.5 ± 3.5 52.6 ± 4.4 58.1 ± 3.8 44.7 ± 4.1 22.6 ± 2.9
7.8 ± 0.4 7.6 ± 0.3 7.2 ± 0.4 9.8 ± 0.5 11.5 ± 0.8
0.995 0.999 0.998 0.998 0.952
54.6 ± 3.4 60.4 ± 5.8 65.1 ± 3.2 56.8 ± 5.7 42.2 ± 4.8
751.7 ± 70.7 810.9 ± 45.3 782.2 ± 28.4 890.9 ± 84.9 1082.5 ± 91.4 823.2 ± 49.5 810.3 ± 72.1
50.5 ± 4.7 55.4 ± 5.1 52.0 ± 4.4 61.2 ± 5.7 73.9 ± 6.3 56.1 ± 3.9 53.4 ± 4.5
7.3 ± 0.3 7.2 ± 0.5 7.3 ± 0.4 7.1 ± 0.3 6.8 ± 0.4 7.3 ± 0.6 7.6 ± 0.4
0.998 0.997 0.996 0.998 0.996 0.998 0.996
54.9 ± 2.2 60.3 ± 4.0 63.7 ± 3.7 61.2 ± 5.5 70.8 ± 6.1 66.3 ± 4.8 64.1 ± 3.6
Co-fermentation of OFMSW + GSW + PMS: 90% OFMSW:5% GSW:5% PMS 80% OFMSW:10% GSW:10% PMS 80% OFMSW:5% GSW:15% PMS 80% OFMSW:15% GSW:5% PMS 70% OFMSW:20% GSW:10% PMS 70% OFMSW:15% GSW:15% PMS 70% OFMSW:10% GSW:20% PMS
Table 5 Hydrogen yield from different co-fermentation combinations. Batches
Hydrogen yield (HY) mL/ gCODremoved
mL/ gVSremoved
100%OFMSW:0%GSW 75%OFMSW:25%GSW 50% OFMSW:50% GSW 25% OFMSW:75% GSW 0% OFMSW:100% GSW
133.0 ± 16.6 149.5 ± 6.7 143.5 ± 14.0 130.5 ± 12.5 96.5 ± 7.6
101.9 ± 6.3 124.6 ± 8.7 119.0 ± 6.0 78.8 ± 3.6 65.4 ± 5.2
6.7 ± 0.5 8.9 ± 0.7 7.0 ± 0.4 5.6 ± 0.4 3.0 ± 0.3
90% OFMSW:10%PMS 75% OFMSW:25% PMS 50% OFMSW:50% PMS 25% OFMSW:75% PMS 0% OFMSW:100% PMS
135.4 ± 8.2 139.6 ± 13.9 146.7 ± 10.2 145.3 ± 19.3 115.7 ± 11.6
102.7 ± 8.8 131.3 ± 11.6 132.5 ± 7.4 110.8 ± 9.4 70.4 ± 5.7
7.8 ± 0.6 8.0 ± 0.8 8.4 ± 0.6 6.8 ± 0.4 3.1 ± 0.4
90% OFMSW:5% GSW:5% PMS 80% OFMSW:10% GSW:10% PMS 80% OFMSW:5% GSW:15% PMS 80% OFMSW:15% GSW:5% PMS 70% OFMSW:20% GSW:10% PMS 70% OFMSW:15% GSW:15% PMS 70% OFMSW:10% GSW:20% PMS
129.1 ± 5.5 136.6 ± 16.8
141.8 ± 10.5 142.8 ± 13.2
7.6 ± 0.7 7.8 ± 0.5
135.6 ± 9.2 137.3 ± 3.9 157.5 ± 12.1
137.1 ± 9.7 138.6 ± 7.1 144.9 ± 9.8
8.1 ± 0.3 8.2 ± 0.8 10.6 ± 0.9
147.2 ± 15.8
129.3 ± 11.5
8.2 ± 0.5
141.2 ± 8.0
128.8 ± 12.4
8.1 ± 0.7
L/ Lsubstrate
3.11 ± 0.08 109 CFU/mL and 1.17 ± 0.04 107 CFU/mL, respectively. This indicates that the mixture of OFMSW/GSW/PMS is capable to harvest more hydrogen producing anaerobes compared with OFMSW/PMS and OFMSW/GSW. Castelló et al. (2011) found that the predominance of Clostridium and Enterobacter sp. in the fermenters fed with cheese whey (protein-rich waste). Enterobacter sp. showed a high increase in the population count
of 1.32 ± 0.06 107 CFU/mL for OFMSW/PMS as compared to OFMSW/GSW (4.85 ± 0.03 106 CFU/mL). Gadow et al., 2013 stated that Enterobacter sp. has a great ability to produce H2 from cellulosic compounds. 3.2. COD, carbohydrate, protein and lipid conversion Fig. 3 presents the COD removal efficiency in addition to the biodegradation efficiency of carbohydrate, protein and lipids during anaerobic co-digestion of different substrates. As depicted in Fig. 3, COD removal efficiency of the mono-fermentation of OFMSW, GSW and PMS were relatively law and amounted to 42.2 ± 4.7, 31.5 ± 3.5 and 47.1 ± 4.8%, respectively. The carbohydrate, protein and lipids degradation efficiency for individual substrates were 55.9 ± 5.4%, 17.1 ± 1.4% and 13.3 ± 1.1% for OFMSW, 78.8 ± 6.4%, 20.3 ± 2.2% and 17.7 ± 1.9% for GSW, 60.4 ± 3.6%, 16.5 ± 2.5% and 12.6 ± 1.6% for PMS. However, the COD removal efficiency was increased up to 50.0 ± 2.1% and 60.4 ± 3.0% with co-fermentation of 75% OFMSW + 25% GSW and 50% OFMSW + 50% PMS respectively. The higher COD removal achieved with adding PMS over GSW may rely to the high lipid content in gelatinous waste which impedes the AD process (Cirne et al., 2007). Similar trends were observed for the removal of carbohydrate, protein and lipids (Fig. 3). The degradation efficiencies of carbohydrate, protein and lipids were significantly increased with co-fermentation of 75% OFMSW + 25% GSW and 50% OFMSW + 50% PMS resulting 65.3 ± 4.7% and 68.7 ± 5.1% for carbohydrate, 23.2 ± 1.8% and 20.8 ± 1.0% for protein, and 19.6 ± 1.6% and 18.8 ± 2.1% for lipids. The COD, carbohydrate, protein and lipids removal was deteriorated at higher and lower dilutions of OFMSW with PMS and GSW. Similar findings were reported by Elbeshbishy and Nakhla (2012) who found that maximum total
Table 6 Microbial population counts in the fermenters. Microbe type
Clostridium sp. (CFU/mL) Enterobacter sp. (CFU/mL)
Inoculum sludge
1.88 ± 0.04 107 2.16 ± 0.05 105
Co-digestion mixture 70% OFMSW/25% GSW
50% OFMSW/50% PMS
70% OFMSW/20% GSW/10% PMS
2.73 ± 0.01 109 4.85 ± 0.03 106
2.40 ± 0.10 109 1.32 ± 0.06 107
3.11 ± 0.08 109 1.17 ± 0.04 107
163
Carbohydrate (%)
80:15:5%
90:5:5%
80:10:10%
0:100%
0:100%
COD removal (%)
OFMSW:GSW:PMS
OFMSW:PMS
OFMSW:GSW
70:10:20%
0
70:15:15%
0
70:20:10%
15 80:5:15%
20 25:75%
30
50:50%
40
75:25%
45
90:10%
60
60
25:75%
80
75:25%
75
50:50%
100
100: 0%
Carbohydrate, protein and lipid removal (%)
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
Protein (%)
Lipid (%)
COD (%)
Fig. 3. COD, carbohydrate, protein and lipid removal efficiency (%).
COD destruction of 59% was achieved with mixture of 80% starch and 20% bovine serum albumin (BSA) compared with 53% and 43% for the fermentation of individual starch and BSA, respectively. This hypothesis was explained by hydrolysis coefficients of carbohydrates and proteins, which registered the highest values at the same mixture (80% starch + 20% BSA). Lin et al., 2013 found that the highest COD removal efficiency of 87% observed at dual fermentation of food waste and paper sludge with ratio of 2:2 (w/w based on VS) which was higher than those obtained for fermentation of individual substrates. In another study Gupta et al., 2014 reported that the COD removal efficiency of a combination of 50% starch to 50% cellulose via AD was higher than the individual substrate of starch and cellulose by percentage of 33% and 72%, respectively. Anaerobic co-fermentation of the three substrates resulted in a positive synergism effect by increasing the COD removal and biodegradation efficiencies of carbohydrates, protein and lipids as shown in Fig. 3. Co-fermentation of 70% OFMSW + 20% GSW + 10% PMS achieved COD removal efficiency of 60.9 ± 4.4% which is 1.53-fold COD removal from OFMSW alone. Carbohydrate, protein and lipids degradation efficiencies were 71.4 ± 3.5, 22.6 ± 2.3 and 20.5 ± 1.8%, respectively for the mixture of 70% OFMSW + 20% GSW + 10% PMS.
followed by HBu was the dominant VFA fractions in the mono-fermentation of OFMSW and PMS. This was not the case for the sole fermentation of GSW whereas HAc and HPr were the highest fractions of VFA. Mono-fermentation of OFMSW, GSW, and PMS produced 5.16 ± 0.38, 3.98 ± 0.27, 4.41 ± 0.16 g/L for HAc, 3.93 ± 0.17, 2.28 ± 0.18, 4.08 ± 0.22 g/L for HBu, 0.58 ± 0.06, 3.02 ± 0.27, 2.81 ± 0.18 g/L for HPr and 1.09 ± 0.13, 0.20 ± 0.03 and 2.47 ± 0.18 g/L for HLa respectively. HAc generation from GSW via dry anaerobic digestion is mainly due to lipids (18.3% of COD) hydrolysis resulting glycerol and long chain fatty acids (LCFAs) which further converted into acetate via the b-oxidation pathway (Cirne et al., 2007). Similar trends were obtained by Fang and Yu, 2002 for gelatin-rich wastewater. They found that the mainstream VFAs were HAc (20.1%), HPr (12.3%) and HBu (9.0%). Gupta et al., 2014 found that mono-fermentation of cellulosic component has relatively low HAc/HBu ratio in addition to a high HPr production. The results in Fig. 4 show that propionate (HPr) fraction significantly increased at increasing the volumetric ratio of GSW in co-fermentation with OFMSW. HLa concentration was dropped at increasing the ratio of GSW. Acetate (HAc) and butyrate (HBu) peaked up to 6.57 ± 0.52 and 4.88 ± 0.35 g/L respectively at 75% OFMSW + 25% GSW and then decreased thereafter. Elbeshbishy and Nakhla, 2012 found that maximum concentration of HAc and HBu achieved at co-fermentation of 80% starch with 20% BSA. Co-fermentation of OFMSW and PMS promoted higher production of HPr and HLa at increasing the volumetric percentage of PMS in the mixture (Fig. 4). In addition, HAc and HBu showed a peak value of 5.56 ± 0.23 and 5.31 ± 0.27 g/L respectively at 50% OFMSW + 50% PMS. However, further increase in volumetric percentage of PMS in the mixture exceeding 50% led to decrease in HAc and HBu generation. Gupta et al., 2014 reported that
3.3. Soluble metabolites components
10.0 8.0 6.0 4.0
OFMSW:GSW Acetate
OFMSW:PMS Buytrate
Propionate
OFMSW:GSW:PMS Lactic
Fig. 4. Net production of soluble metabolites concentrations.
70:10:20%
70:15:15%
80:15:5%
70:20:10%
80:5:15%
80:10:10%
90:5:5%
0:100%
25:75%
50:50%
75:25%
90:10%
0:100%
25:75%
50:50%
0.0
75:25%
2.0 100: 0%
Net production of soluble metabolites (g/L)
Acetate (HAc) and butyrate (HBu), are the desirable soluble products since hydrogen generation occurs via those metabolite products (Elsamadony et al., 2015b). However, HAc, HBu, propionate (HPr), and lactate (HLa) generation was positively affected by the type of feedstock, mono-fermentation, di-fermentation and tri-fermentation processes. As depicted from Fig. 4, HAc
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210
40
HY (mL/g CODremoved)
Before anaerobic digestion After Anaerobic digestion
Intensity (%)
30
20
10
180 150 120 90 60 y = -0.179x2 + 10.175x R² = 0.739
30 0 0
10
0 0
500
1000 1500 Particle diameter (µm)
2000
Fig. 5. Particles size distribution for the mixture of 70% OFMSW + 20% GSW + 10% PMS before and after AD.
co-fermentation of glucose with cellulose (50:50% v/v) led to increase HAc, HBu, and HPr by 5.8, 2.7, and 1.9-fold as compared to fermentation of cellulose alone. Moreover, Lin et al., 2013 found that maximum production of total VFAs (12.3 g/L) achieved at a mixture of FW and PMS with ratio 2:2 (w/w, based on VS). The overall maximum HY of 157.5 ± 12.1 mL/gCODremoved was registered at co-fermentation of 70% OFMSW + 20% GSW + 10% PMS. This corresponds to the soluble metabolite components, in terms of HAc, HBu, HPr and HLa of 8.31 ± 0.58, 6.76 ± 0.30, 1.04 ± 0.14 and 1.16 ± 0.03 g/L, respectively as presented in Fig. 4. 3.4. Effect of co-fermentation process on particle size distribution Limited number of literatures investigated the effect of anaerobic co-fermentation on particles size distribution of the mixture via dry anaerobic digestion (AD). However, particle size of substrates may significantly effect on the hydrolysis rate, the speed and performance of AD process (Zhang and Banks, 2013). Fig. 5 shows the effect of anaerobic co-fermentation of the three substrates (70% OFMSW + 20% GSW + 10% PMS) on particles size distribution. As depicted from Fig. 5, the domain particle size in the mixture before co-fermentation process was 850 lm with intensity of 24.1% followed by 1850 lm (14.8%) then 450 lm (13.4%). However, the effectiveness of co-fermentation on particles size emerged clearly in the digestate, where 34% of particles were within diameter range of 75 lm. This indicates that the co-fermentation process reduced the particle size distribution which is favorably utilized by hydrogen producing bacteria. Izumi et al., 2010 emphasized that the highest gas production rate accomplished at initial diameter of food waste particles with value of 718 lm and finer particles declined the gas production rate. The mean particle size diameters for feedstock and the digestate were 939.3 and 115.2 lm, respectively with reduction of 8.15-fold in the particle size of the mixtures.
20
30
40
COD/TN ratio Fig. 6a. Effect of carbon to nitrogen (C/N) ratio on H2 yields.
considerably varied based on the volumetric ratios of the feedstocks subjected to co-fermentation processes. Fig. 6a shows the influence of initial C/N ratio on HY. The maximum HYs of 149.5 ± 6.7, 146.7 ± 10.2, and 157.5 ± 12.1 mL/gCODremoved were registered at C/N ratios of 28.8 ± 0.2, 32.8 ± 0.4 and 29.4 ± 0.3 for 75% OFMSW + 25% GSW, 50% OFMSW + 50% PMS, and 70% OFMSW + 20% GSW + 10% PMS, respectively. At lower and higher C/N ratios, HYs were decreased followed a 2nd degree polynomial function with correlation coefficient factor (R2 = 0.739). Thus, the positive synergistic effect emphasized through co-fermentation of different co-substrates. According to earlier studies the desired C/N ratio was close to those obtained in this investigation i.e. C/N ratio ranged from 26 to 31 for co-fermentation of food waste with primary and secondary sludge (Zhou et al., 2013) and C/N ratio of 33 for mono-fermentation of food waste and sludge (Sreela-or et al., 2011). In addition, (Tawfik and El-Qelish, 2014a,b) found that increasing COD/N ratio from 12 to 24.1 improved HY from 93.2 to 150.9 mLH2 =gVSremoved d and thereafter a significant drop in HY was noticed beyond this limit. Tyagi et al. (2014) achieved a maximum HY of 51 mL/gVSremoved at 5:1 volumetric ratio between OFMSW and sludge related to optimum COD/N ratio of 31. 3.6. Effect of Ca+2 concentrations on the volumetric H2 production In order to investigate the effect of calcium concentration on volumetric H2 production, volumetric percentages of calcium ions rich wastes such as GSW and PMS were varied and co-fermented with OFMSW. Fig. 6b shows the effect of different initial calcium (Ca+2) concentrations on volumetric H2 production (VHP). The results depict a good 2nd degree polynomial relationship between calcium (Ca+2) concentration and VHP with correlation coefficient factor R2 of 0.7 (Fig. 6b). The results revealed that volumetric H2 production increased from 4.5 ± 0.3 to 7.2 ± 0.6 LH2 =Lsubstrate at increasing Ca+2 concentrations from 1.8 ± 0.1 to 6.3 ± 0.5 g/L respectively. This can be attributed to, (1) calcium ion plays as coagulant aids to enhance the biomass accumulation as it stimulates microbial growth and, consequently, affect specific growth
3.5. Effect of C/N ratio on the H2 yield 8 Volumetric H2 production (LH2/Lreactor)
Carbon to nitrogen (C/N) ratio of feedstock is critically important to facilitate the conversion of organics into hydrogen (Wang et al., 2013). C/N ratio promotes microbial growth due to providing a sufficient nutrient balance (Tyagi et al., 2014). In addition, a proper C/N ratio improves the hydrogen producers’ activity and preclude ammonia inhibition which in turn enhancing the bio-hydrogen production via anaerobic digestion process (Zhou et al., 2013). OFMSW is carbohydrate-rich substrate with a low nitrogen content resulting in a considerable high C/N ratio of 39.5 ± 0.7 as depicted in Table 1. However, the co-fermentation of OFMSW with N-lean substrates such as GSW and PMS provided C/N of 13.0 ± 0.2 and 27.7 ± 0.2, respectively. C/N ratio was
6 4 2 y = -0.021x2 + 0.204x + 4.867 R² = 0.700
0 0
5
10 15 Ca+2 concentration (g/L)
20
25
Fig. 6b. Effect of Ca+2 concentrations on volumetric H2 production.
M. Elsamadony, A. Tawfik / Bioresource Technology 191 (2015) 157–165
rate (Chen et al., 2008) (2) calcium is essential for catalytic activity, either as a participant in the reaction, or as a structural requirement in order to maintain the appropriate confirmation of the active site (Yuan et al., 2010) and 3. Ca+2 represents a counteract of the inhibitory effect of ammonia (Chen et al., 2008). However, higher concentrations of Ca+2 exceeding 6.3 g/L led to a significant drop in volumetric H2 production (1.7 ± 0.2 LH2 =Lsubstrate ). A higher Ca+2 levels can cause severe inhibition or toxicity in addition to dehydrate bacterial cells due to osmotic pressure (Chen et al., 2008; Zhang et al., 2013). Similar trends was also observed by Zhang et al. (2013) who reported that the addition of lime mud produced from paper-making process (LMP) (which contains 35.44% (w/w) calcium) to food waste in batch system not only improved the hydrogen production 1.5-fold compared with control batch but also accelerated the hydrogen production rate, and shortened lag phase period. Thereafter, the hydrogen production dropped at higher amount of lime mud. Likewise, Yuan et al. (2010) concluded that supplying continuous stirred-tank reactor (CSTR) reactor with 100 mg/L Ca+2 improved the stability of the system as cell retention was multiplied twice in cell density than the case of no calcium addition. As well, addition of 3 g/L of Ca+2 achieved the maximum biogas production and organic removal efficiency via AD of swine wastewater (Ahn et al., 2006). Higher Ca+2 concentration exceeded 7 g/L had an inhibitory effect on anaerobic digestion process. Whereas, anaerobic granules exposure to a significant loss in biological activity at high calcium concentration (El-Mamouni, 1995). 4. Conclusions Dry anaerobic co-fermentation of 70% OFMSW + 20% GSW + 10% PM resulted in a positive synergism effect on the HP and HY by increasing the conversion efficiency of COD (60.9 ± 4.4%), carbohydrates (71.4 ± 3.5%), protein (22.6 ± 2.3) and lipids (20.5 ± 1.8%). However, the HP and HY is C/N ratio, calcium concentration, type of feedstock and volumetric ratio dependant. Maximum HYs of 124.6 ± 8.7, 132.5 ± 7.4, and 144.9 ± 9.8 mL/gVSremoved achieved by co-fermentation of 75% OFMSW + 25% GSW, 50% OFMSW + 50% PMS, and 70% OFMSW + 20% GSW + 10% PMS, respectively. The highest HP was obtained at C/N ratio of 29.4 ± 0.3 and Ca+2 concentration of 6.3 ± 0.5 g/L. Acknowledgements The first author would like to acknowledge Ministry of Higher Education (MoHE) of Egypt for providing a scholarship to conduct this study as well as the Egypt Japan University of Science and Technology (E-JUST) for offering the facility and tools needed to conduct this work. This research is also financed from Science & Technology Development Fund (STDF) in Egypt, project ID: 3665. References Ahn, J.-H., Do, T.H., Kim, S.D., Hwang, S., 2006. The effect of calcium on the anaerobic digestion treating swine wastewater. Biochem. Eng. J. 30, 33–38. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC, USA. Banks, C.J., Humphreys, P.N., 1998. The anaerobic treatment of a ligno-cellulosic substrate offering little natural pH buffering capacity. Water Sci. Technol. 38, 29–35. Breure, A.M., Beeftink, H.H., Verkuijlen, J., van Andel, J.G., 1986. Acidogenic fermentation of protein/carbohydrate mixtures by bacterial populations adapted to one of the substrates in anaerobic chemostat cultures. Appl. Microbiol. Biotechnol. 23, 245–249. Castelló, E., Perna, V., Wenzel, J., Borzacconi, L., Etchebehere, C., 2011. Microbial community composition and reactor performance during hydrogen production in a UASB reactor fed with raw cheese whey inoculated with compost. Water Sci. Technol. 64, 2265–2273.
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