Effect of acid, heat and combined acid-heat pretreatments of anaerobic sludge on hydrogen production by 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 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3

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Effect of acid, heat and combined acid-heat pretreatments of anaerobic sludge on hydrogen production by anaerobic mixed cultures Thitirut Assawamongkholsiri a, Alissara Reungsang a,b,*, Sakchai Pattra c a

Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University, Khon Kaen 40002, Thailand c Program of Public Health, Department of Science and Technology, Faculty of Arts and Science, Chaiyaphum Rajabhat University, Chaiyaphum 36000, Thailand b

article info

abstract

Article history:

Activated sludge (AS) from wastewater treatment plant of brewery industry was used as

Received 10 September 2012

substrate for hydrogen production by anaerobic mixed cultures in batch fermentation

Received in revised form

process. The AS (10% TS) was pretreated by acid, heat and combined acid and heat.

27 December 2012

Combined acid- heat treatment (0.5% (w/v) HCl, 110
C, 60 min) gave the highest soluble

Accepted 31 December 2012

COD (sCOD) of 1785.6  27.1 mg/L with the highest soluble protein and carbohydrate of

Available online 29 January 2013

8.1  0.1 and 38.5  0.8 mg/L, respectively. After the pretreatment, the pretreated sludge was used to produce hydrogen by heat treated upflow anaerobic sludge blanket (UASB)

Keywords:

granules. A maximum hydrogen production potential of 481 mL H2/L was achieved from

Bio-hydrogen

the AS pretreated with acid (0.5% (w/v) HCl) for 6 h.

Activated sludge

Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Heat Acid

1.

Introduction

Nowadays, the environmental problems have brought the interests toward the development of alternative energy [1]. Among the alternative energy sources, hydrogen has been receiving an increasing attention due to its environmentally friendly characteristic and high energy content. Hydrogen can be biologically produced from various kinds of substrate ranges from simple sugars such as glucose [2,4]; xylose [3]; arabinose [4]; starch containing waste such as cassava wastewater [5]; lignocellulosic materials such as sugarcane bagasse [6]; and activated sludge (AS) from

wastewater treatment plant, such as, poultry slaughterhouse [7,8]; and diary industry [9]. Among the aforementioned substrates, AS, the major solid waste from aerobically biological wastewater treatment processes [10], has recently received an attention as substrate for producing renewable fuel i.e., methane [11] and hydrogen [11,12] due to its rich polysaccharide and protein content indicating its potential as substrate for hydrogen production [13]. It is reported that in aerobic degradation, one unit of substrate carbon is converted to 0.5 units of CO2 and 0.5 units of cell carbon [14]. Therefore, a utilization of AS as the substrate for renewable energy production not only contributes to a safe and clean energy (i.e.,

* Corresponding author. Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University, Khon Kaen 40002, Thailand. Tel./fax: þ66 43 362 121. E-mail addresses: [email protected], [email protected] (A. Reungsang). 0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2012.12.138

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hydrogen in this study) but also one of the solutions to get rid of this abundant waste. AS from the wastewater treatment plant has microbial cells as the main component [13]. Most of the organics are contained within microbial cell membranes and the semi rigid structure of the cell envelope prevents the osmotic lysis of the cells [15]. In addition, AS contains a significant amount of extracellular polymeric substances of [16] which only 30e50% can be biodegraded [17]. Thus, the hydrolysis of the microbial cells is needed in order to disrupt the cells and release the organics into liquid so as to enhance the anaerobic digestion of AS [18], subsequently increase in hydrogen yield (HY) and shorten the HRT [19]. The AS can be pretreated/hydrolyzed using one or a combination of a number of methods. The pretreatment methods can be divided into 5 categories including thermal, mechanical, chemical, ultrasound and biological hydrolysis (bacterial and enzyme hydrolysis) [20]. These pretreatments are distinctively different in their modes of action. Thermal pretreatment depends on the high temperature. Cell wall and cell membrane of the microorganisms were disrupted by heat that was applied during thermal pretreatment, resulting in a solubilization of the cell components. Mechanical pretreatment physically disintegrate the cells and the cell contents are partially solubilized. Chemical pretreatment include acid and alkaline hydrolysis. Acid hydrolysis depends on the free Hþ while an alkaline hydrolysis depends on free OH [21]. This method hydrolyzes the cell wall and cell membrane. Consequently, the solubility of the organic matter contained within cells is increased [20]. Ultrasonic pretreatment depends on the shear produced in the sonication [22,23]. Lastly, biological hydrolysis breaks the cell wall compounds by an enzyme catalyze reaction. As a result, the organic matter inside the cells is released. In this study, acid and thermal (heat) methods were chosen to pretreat the AS due to its simplicity, no special equipments required, and less timeconsuming. Xiong et al. [24] reported that an increase in the reaction temperature improved the efficiency of hydrolytic and acidogenic waste AS in anaerobic fermentation. Thus, it is hoped that a combined acid-heat pretreatment would enhance the lysis of the microbial cells and more hydrogen would be produced. Therefore, the main objective of this study was to investigate the effects of pretreatment (acid, heat and combined acid-heat) on solubilization of organic matter from AS as well as on the hydrogen production. It is hoped that the pretreatment methods would improve the rate of digestion and facilitate the hydrogen production process.

2.

Materials and methods

2.1.

Seed and inoculum preparation

The anaerobic seed sludge was upflow anaerobic sludge blanket (UASB) granules from Khon Kaen Brewery CO., Ltd., Khon Kaen, Thailand. The UASB reactor is used to treat wastewater from beer production process. Anaerobic sludge was heated at 105
C for 3 h in order to inhibit methanogenic bacteria. For inoculum preparation, 5% (w/v) heat treated UASB granules were enriched with 20 g/L glucose, 0.5 g/L yeast extract, 1 mL/L

nutrient stock solution [25], and 1.29 mL/L stock solution of 155 g/L NH4HCO3. The initial pH of the culture was adjusted to 5.5 by 4 mol/L HCl. The culture was incubated at room temperature and shaken at 150 rpm. After 24 h of incubation, 50% (v/v) of the culture was transferred into fresh medium. After ten cycles of subculture, the culture was used as an inoculum in batch experiment. The volatile suspended solids (VSS) concentration of the inoculum was 14.26  0.21 g-VSS/L.

2.2.

Feedstock

The feedstock is AS obtained from the wastewater treatment of Khon Kaen Brewery CO., Ltd., Khon Kaen, Thailand. The AS was stored at 4
C prior usage. Characteristics of raw AS were shown in Table 1.

2.3.

Pretreatment of AS

AS (10% total solid (TS)) was pretreated by acid, heat and combined acid-heat. For the acid pretreatment, the AS was treated by 0.5% (w/v) HCl and incubated at room temperature for 0, 6, 12, 18 and 24 h. For heat pretreatment, the AS was heated in a hot air oven at 110
C for 15, 30, 45 and 60 min. For combined acid-heat pretreatment, the AS was added with 0.5% (w/v) HCl and then heated in a hot air oven at 110
C for 15, 30, 45 and 60 min. AS (10% TS) without pretreatment (raw AS) was used as a control. The treatment codes are showed in Table 2.

2.4.

Biohydrogen production

Biohydrogen production was conducted in 120 mL serum bottles with a working volume of 70 mL. The serum bottle contained 65 mL of pretreated AS (10% TS), 4.5 mL of seed inoculums (14.26  0.21 g-VSS/L), 1 mL/L of nutrients stock solution [25] and 1.29 mL/L of NH4HCO3 stock solution. An initial pH was adjusted to 5.5 by 2 mol/L HCl or 2 mol/L NaOH. The serum bottle was closed with a rubber stopper and capped with aluminum cap. After replacement of gas phase with nitrogen to create anaerobic condition, the serum bottles were incubated at room temperature (30  2
C) and horizontally shaken at 150 rpm. The fermentation process was continued until no biogas generated. At each time interval, the total gas

Table 1 e Characteristics of raw activated sludge. pH (5% TS AS) Total solid (TS) (g/kg sludge) Volatile solid (VS) (g/kg sludge) Total chemical oxygen demand (tCOD) (g/kg TS) Soluble chemical oxygen demand (sCOD) (mg/kg TS) Carbohydrate concentration (mg/kg TS) Protein concentration (mg/kg TS) Fat concentration (mg/kg TS) Total Nitrogen (mg/kg TS) NHþ 3 (mg/kg TS) Total Phosphorus (mg/kg TS)

7.05 867.95  0.16 76.23  0.06 113.91  14.78 407  47 10326  654 4000  866 1949  534 1644 572 677

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Table 2 e Estimated values of parameters by the modified Gompertz equation and hydrogen yield. Treatment

Time

Code

Hmax (mL H2/L)

Rm (mL H2/L h)

l (h)

R2

H2 yield (mL H2/g VSadded)

Raw sludge Acid

0 min 0h 6h 12 h 18 h 24 h 0 min 15 min 30 min 45 min 60 min 0 min 15 min 30 min 45 min 60 min

raw sludge A0 A6 A12 A18 A24 raw sludge H15 H30 H45 H60 AH0 AH15 AH30 AH45 AH60

66AB 167BC,a 481G,bc 212CD,a 340DE,ab 600G,c 66AB,b 79AB,b 65AB,b 37AB,a 20A,a 167BC,a 284CDE,b 367EF,d 324DE,c 328DE,c

8.38B 13.63CDE,a 15.17DE,a 18.74F,b 34.56H,c 12.76CD,a 8.38B,bc 16.49EF,d 11.06BC,c 4.86A,ab 3.23A,a 13.63CDE,b 29.79G,d 10.74BC,a 13.49CDE,b 27.26G,c

7.40A 44.80D,a 84.07GH,bc 81.90FG,bc 78.20F,b 87.30HI,c 7.40A,a 7.63A,a 8.27A,a 16.35B,c 26.77C,d 44.83D,a 79.14F,c 138.30J,e 66.53E,b 89.37I,d

0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99

9AB 12ABCD,a 34EF,bc 14BCD,ab 23D,ab 41F,c 9AB,b 11ABC,b 9ABC,b 3A,a 2A,a 12D,b 20BCD,a 20BCD,a 23D,b 21CD,ab

Heat

Acid combined heat

Difference capital letters indicated the differences among treatments in column by Duncan test (P < 0.05); Difference lower case letters indicated the difference results obtained by the same pretreatment method affected by difference pretreatment time. Hmax: maximum cumulative hydrogen production; Rm: maximum hydrogen production rate; l: lag phase time; R2: the determination coefficient.

volume was measured by releasing the pressure in the bottles using wetted glass syringe [26]. All treatments were conducted in four replicates.

2.5.

Analytical methods

Biogas composition was analyzed using a gas chromatograph (GC) (GC-2014, Shimadzu, Japan) equipped with a thermal conductivity detector (TCD) and a 0.2 m  3 mm diameter stainless column packed with Shin carbon (50/80 mesh). The operational protocols followed the method of Saraphirom and Reungsang [27]. Volatile fatty acids (VFAs) and ethanol concentrations in the liquid samples were analyzed by high performance liquid chromatography (HPLC) (LC-10AD, Shimadzu, Japan) with ultraviolet detector using a VertiSep polymer-based HPLC column (7.8 mm  300 mm, VertiSep OA 8 mm HPLC column, Vertical). 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. Preparation of the liquid samples prior the analysis by GC and HPLC followed the method of Saraphirom and Reungsang [27]. Closed reflux method was used to determine total COD (tCOD) and soluble COD (sCOD) concentrations [28]. Phenol sulfuric method [29] with glucose as a standard was used to determine total and soluble carbohydrate concentrations. Lowry method [30] with a bovine serum albumin as standard was used to measure total and soluble protein concentrations. pH was measured by a digital pH meter (Sartorius, Germany). Concentrations of TS, volatile solid (VS), total suspended solid (TSS) and VSS were measured according to standard method [28].

2.6.

hydrogen produced at each time interval, using the mass balance equation [31]. A modified Gompertz equation [32] was used to fit the cumulative hydrogen production curve for biohydrogen production.

Kinetic analysis

Hydrogen gas production was calculated from the headspace measurement of gas composition and the total volume of

3.

Results and discussion

3.1.

Effect of pretreatment on AS solubilization

Every pretreatment method could partially break down and solubilize the solid components of AS to the liquid phase as indicated by an increase in concentrations of sCOD (Fig. 1a), soluble carbohydrate (Fig. 1b) and soluble protein (Fig. 1c). Carbohydrate was more solubilized than protein for all pretreatment methods. Effect of acid (A6eA24) and heat (H15eH60) pretreatments on carbohydrate solubilization was not markedly different. Significant higher amount of increased sCOD was observed in heat pretreatment than acid pretreatment. Combined acid-heat pretreatment (AH15eAH60) is the most effective method for solubilization of organic matters from AS as indicated by highest percentage increased in sCOD, soluble carbohydrate and soluble protein concentrations. Combination of acid and heat pretreatment provided a stronger reaction than heat or acid pretreatment alone hence larger amount of organic matters could be degraded and solubilized to the liquid phase. Treatment time significantly affected the increase in sCOD concentration for all pretreatments. In the acid pretreatment, increased sCOD observed in A24 (111.49%) is significantly higher than A6eA18 (39.27e76.90%). For heat pretreatment, an increase in the treatment time from 15 (H15) to 45 (H45) min significantly enhanced the percentage increase in sCOD concentration from 30.91% to 218.32%; further prolong the heat treatment time to 60 min did not affect COD solubilization. Percentage increase in soluble carbohydrate concentration significantly rose from 100% to 162.16% when acid treatment

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that obtained in treatment AH45 (408.57%) as well as treatments AH30 (374.60%) and AH15 (212.30%), respectively. A distinct result from combined acid-heat pretreatment of sludge obtained from this study was on the %increase in solubilization of sCOD, soluble carbohydrate, and soluble protein that was shown to be greater when compared to other studies (Fig. 2). This high % increase in solubilization indicated that more organic matters in the sludge was solubilized as the result of combined acid-heat hydrolysis, which could be the result of more acidic in our study (approximately pH of 1) than the other studies used for a comparison (pH of 2 [33] and 3 [34]). It should be noted that our study used HCl while the other studies used H2SO4 to pretreat the sludge. Smith and Goransson [35] reported that thermal acidic hydrolysis by using HCl gave slightly higher solubilization rate than H2SO4. A hydrolysis yield obtained was 30e50%. Unfortunately, there is limited information in the literature with regard to the use of combined acid-heat pretreatment of the sludge. Most of the previous studies were on the utilization of combined alkalineheat to pretreat the sludge. It is important to note that our findings showed a similar trend to the results of a combined alkaline-heat pretreatment in which the degree of solubilization of sCOD increased with increasing pH (more basic condition) [36,37] (or a solubilization rate increase with decreasing pH (more acidic condition)). The increase in acid or base concentration in the acidic or basic-hydrolyzing process, respectively, could provide a strong or complete reaction for breaking down the cell wall or cell membrane of the microorganisms present in the sludge yielding a better solubilization of sCOD, soluble carbohydrate and soluble protein. Thus, a strong acidic or basic condition is recommended for pretreatment of the sludge.

3.2.

Effect of pretreatments on hydrogen fermentation

Pretreatment methods significantly affected hydrogen production (Table 2). Significant higher maximum cumulative hydrogen production (Hmax) and hydrogen yield (HY) were obtained from acid and AH pretreated sludge when compared to heat pretreatment (Table 2). Treating the sludge by acid for Fig. 1 e Percentages increase in soluble COD (a), soluble protein (b) and soluble carbohydrate (c) for difference pretreatment methods.

time was extended from 6 (A6) to 12 (A12) h; further extend the treatment time to 18 (A18) and 24 (A24) h did not affect carbohydrate solubilization. The increased soluble carbohydrate was statistically increased as the treatment time was extended from 15 to 30, 45 and 60 min for both heat and combined acid-heat (AH) pretreatments. Protein solubilization in acid pretreatment was not affected by the treatment time. For heat pretreatment, higher percentage increase in soluble protein was observed at treatment time of 30 min (H30) (168.89%) than 15 min (H15) (23.57%); prolong the treatment time to 45 (H45) and 60 (H60) min did not increase protein solubilization. Treatment AH60 gave the highest percentage increase in soluble protein of 586.90% which was significantly higher than

Fig. 2 e Comparison of percentages increase in solubilization.

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6 h (A6) gave the Hmax and HY of 480 mL H2/L and 34 mL H2/g VSadded. Under these conditions, the maximum hydrogen production rate (Rm) of 15.17 mLH2/L h was achieved with the lag time (l) of 84.07 h. The highest Hmax and HY of 600 mLH2/L and 41 mL H2/g VSadded were obtained from the treatment A24 (treating AS by acid for 24 h) with the Rm and l of 12.76 mLH2/ L h and 87.30 h, respectively. However, these results were not statistically different from the treatment A6 (Table 2). Considering that a shorter treatment time is needed for economical efficient and practical application, treatment A6 was the most efficient pretreatment of AS prior the hydrogen fermentation. Our results on hydrogen production from sludge pretreated with acid for 6 h showed superior results over the other literature search (Table 3). This might be due to the treatment temperature used in our study (30
C) was higher than Xiao and Liu (15e24
C) [38] and Wang et al. (4
C) [13] resulted in more release of soluble organic matter which was subsequently used as a substrate to produce hydrogen. It is worth noting that it is difficult to compare our results with other researchers’ results because the sludge and the seed inoculum used in each study are different in characteristics which can lead to the differences in hydrogen production efficiency. Fig.3 depicted the ratios of sCOD/tCOD at initial and at the end of hydrogen fermentation. The initial sCOD/tCOD for acid pretreatment was approximately 0.16 and was not significantly affected by treatment time. For heat treatment, sCOD/tCOD significantly increased with the prolong treatment time within the range of 0.20e0.26. Though initial sCOD/ tCOD for AH pretreatments were not statistically different, the trend of increased initial sCOD/tCOD with the extended treatment time was observed. After fermentation, significant increase in sCOD/tCOD was observed in all treatments, except for treatment A12. The results indicated that microorganisms in the fermentation system could further break the microbial cells in AS. As a result, organic matters were released or solubilzed to liquid phase causing an increase in sCOD concentration. The results also indicated that solubilization of organic matter was more effective than utilization. Nonmarkedly difference between initial and final sCOD/tCOD observed in treatment A12 indicated the balance of organic matter solubilization and utilization. Treatments H45-H60 and AH45-AH60 had a significant higher final sCOD/tCOD than other treatments. This is because a long heat treatment time of AS (45e60 min) could change the structures of solid portion in AS and made the protein to be easier to digest by microorganisms in the fermentation system. However, there is no correlation between hydrogen production efficiency and initial or final sCOD/tCOD concentration. Our results were different from other published report which mostly found that

Fig. 3 e Changes of sCOD/tCOD in the hydrogen fermentation system.

hydrogen production efficiency increased when soluble organic matter was more solubilized [39,40]. Different pretreatment methods affected the concentration of soluble organic compounds and consequently affected hydrogen production. Increased soluble carbohydrate concentrations (Table 4), caused by different pretreatment method, resulted in an increase of Hmax and HY (Table 2). Carbohydrate was found as the main substrate for hydrogen production from AS in the previous reports [39]. Increase in solubilization of carbohydrate made it readily to be consumed by hydrogen producing bacteria hence increasing hydrogen production efficiency. Significant higher soluble protein concentration (10.72e14.64 mg/L) obtained from treatments H45 and H60 apparently caused a reduction in Rm (Table 4). Initial concentration of lactic acid (HLa), propionic acid (HPr) and acetic acid (HAc) were not obviously different. Higher initial butyric acid (HBu) concentrations in heat treatment than in acid and AH pretreatments were observed which might be responsible for lower Hmax and HY values in heat treatment. The inhibitory effect of high initial HBu concentration on hydrogen fermentation was reported elsewhere [31,41,42]. Change of soluble organic matter concentrations in the fermentation system reflected the substrate utilization and metabolite production. After fermentation, concentration of carbohydrate and HLa decreased in all treatments implying that they were consumed for microbial growth and/or hydrogen production by the presence microorganisms in the fermentation system. Concentration of HPr in all treatments and HAc and HBu in some treatments were increased which suggested that they were the metabolic products during

Table 3 e Comparison of other literature search on hydrogen production using acid pretreated sludge as the substrate. Substrate Wastewater sludge Sewage sludge Waste activated sludge from brewery industry

Seed inoculums

Pretreatment methods

Digestion time

Hydrogen yield (mLH2/g COD)

Reference

Clostridium bifermentans without extra-seeds Anaerobic mixed culture

HClO4, pH 3.0 6 mol/L HCl, pH 2.0 0.5% (w/v) HCl, pH z 1.0

6 h (at 4
C) 12 h (at room temperature) 6 h (at room temperature)

8.9 2.53 42.72

[13] [38] This study

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Table 4 e Final pH and changes in soluble compounds in the liquid phase in the fermentation system. Treatment Final pH

Raw sludge A0 A6 A12 A18 A24 H15 H30 H45 H60 AH15 AH30 AH45 AH60

sCarbohydrate (mg/L)

sProtein (mg/L)

HLa (mg/L)

Initial Final Initial

HPr (mg/L)

HAc (mg/L)

HBu (mg/L)

Final Initial Final Initial Final Initial

Final

tVFAs (mg/L)

Initial

Final

Initial

Final

5.93

5.83

3.67

1.66

2.09

1843.48

10.98

38.08

379.24

342.14

285.12

167.71

484.62

2391.41 1159.96

5.77 5.86 5.73 5.87 5.86 6.08 6.02 6.08 6.02 5.92 5.75 5.97 5.90

15.28 19.05 16.59 20.36 20.32 6.63 7.66 8.93 12.30 21.31 27.94 40.56 52.54

5.78 6.13 6.08 9.21 7.57 4.73 4.30 4.35 6.14 10.63 15.30 28.22 29.27

2.34 2.53 2.53 2.81 2.86 2.26 5.38 10.72 14.64 2.60 3.43 4.59 5.89

3.33 3.20 3.09 4.27 3.48 2.56 3.27 4.38 5.48 3.52 4.66 3.78 5.45

1969.10 3.44 1992.90 22.09 1810.98 5.95 1942.10 5.55 1445.77 120.22 1818.01 56.28 1845.00 78.45 1758.70 3.48 1629.11 1.89 1928.40 3.28 1703.20 89.98 1828.95 17.62 1846.22 6.01

14.39 18.28 12.25 13.12 8.81 11.29 5.68 16.88 32.72 7.86 11.23 5.99 6.21

155.12 96.64 178.55 66.86 88.04 437.66 295.84 532.22 992.32 106.94 73.19 127.29 105.83

354.37 330.92 341.58 366.35 258.82 288.24 328.12 332.62 322.45 361.36 305.82 342.49 348.80

390.38 179.92 195.00 307.67 353.38 532.16 672.55 908.08 924.38 325.65 426.96 241.67 360.33

94.34 59.89 44.64 54.92 64.88 466.89 578.24 533.76 522.46 40.45 15.68 49.20 59.27

1141.74 1282.11 1162.95 1087.01 1074.24 440.02 420.92 855.32 421.00 1171.76 935.90 1232.33 1079.56

2432.20 2401.98 2209.45 2376.49 1778.28 2584.42 2757.04 2641.96 2506.74 2338.06 2035.94 2226.63 2260.50

1690.68 1580.75 1542.44 1467.10 1635.87 1466.12 1467.76 2299.10 2339.59 1607.64 1526.03 1618.91 1551.74

sCarbohydrate: soluble Carbohydrate; sProtein: soluble Protein; HLa: lactic acid; HPr: propionic acid; HAc: acetic acid; HBu: normal butyric acid. tVFAs (total volatile fatty acids) ¼ HLa þ HPr þ HAc þ HBu; initial: prior hydrogen fermentation; final: after hydrogen fermentation.

hydrogen fermentation. The decrease in concentrations of HAc and HBu might be the results of anaerobic oxidation of these two metabolites to carbon dioxide [43,44]. In addition, the reduction of HLa concentration can also be caused by anaerobic oxidation process. In order to give more explanation about the phenomena on substrate consumption and metabolites production, data on organic matter concentration (Table 4) and hydrogen production (Table 2) were tested for the correlations between hydrogen production and substrate consumption, substrate consumption and SMPs as well as the correlation between hydrogen production and SMPs (Table 5). tCODconsumed represented overall utilization of organic matter in the fermentation system. Soluble carbohydrate, soluble protein and HLa

Table 5 e Correlations between hydrogen production, substrate utilization and soluble metabolite products (SMPs). HPr HAc HBu increased increased increased tCOD consumed sCarbohydrate consumed sProtein consumed HLa consumed HPr increased HAc increased HBu increased

Hmax

were considered as substrate for growth and activity of microorganisms in the fermentation system while HPr, HAc and HBu were considered as the SMPs. tCODconsumed showed a strong positive correlation with Hmax. This result indicated that organic matter was consumed to produce hydrogen in the fermentation system. Level of tCODconsumed correlated positively with the increased level of HBu. This could be explained by the fact that, the production of HBu from organic matters in the AS, for example carbohydrate, released carbon dioxide and hydrogen to the biogas (this also called butyrate-type fermentation) (Eq. (1)) [45] resulting in a decrease of tCOD as reflected by increased tCODconsumed along with the decrease in HBu production. tCODconsumed showed the significant negative correlation with HPr and HAc. The possible pathway to explain this phenomena are HPr production (Eq. (2)) [46,47] and homoacetogenesis pathways (Eq. (3)) [48] which can be proceeded by propionogenic and acetogenic bacteria, respectively. As shown in Eqs. (2) and (3), hydrogen and carbon dioxide in gas phase can be converted to HPr and HAc and accumulated in the liquid phase, as a result, tCOD concentration in the liquid phase increased. These phenomena were reflected by decreased tCODconsumed along with HPr and HAc production.

0.766* 0.655*

0.06* 0.564*

0.794** 0.894**

0.889**

0.852*

0.622*

0.567*

C6H12O6 þ 2H2O / 2H2 þ CH3CH2CH2COOH þ 2CO2

(1)

0.223 e e e

0.407 0.812** e e

0.243 0.796** 0.788** e

0.222 0.732** 0.591* 0.774**

C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O

(2)

4H2 þ 2CO2 / CH3COOH þ 2H2O

(3)

0.991** 0.857**

*Pearson correlation is significant at the 0.05 level (2-tailed), **Pearson correlation is significant at the 0.01 level (2-tailed). tCarbohydrate: total Carbohydrate; sCarbohydrate: soluble Carbohydrate; sProtein: soluble Protein; HLa: lactic acid; HPr: propionic acid; HAc: acetic acid; HBu: normal butyric acid; Hmax: maximum cumulative hydrogen production.

Soluble carbohydrate consumption has a strong positive correlation with Hmax and HBu production. The result indicated that soluble carbohydrate was mainly consumed for hydrogen production by HBu production pathway. The

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negative correlations between soluble carbohydrate consumed and HPr and HAc production was observed. The described possible reason for negative correlation between tCODconsumed and HPr and HAc could also be used to explain these results. Soluble protein consumption negatively correlated with Hmax and HBu production, whereas for HPr and HAc, the correlations were positive. These results implied that protein might be used for HPr and HAc production and degradation of soluble protein might cause negative impact on butyrate-type hydrogen production. However, it should be noted that in some treatments, the rate of protein solubilization was greater than the rate of protein utilization and the soluble protein concentration was increased after fermentation. This might cause the errors in correlation analysis. Hmax had a strong positive correlation with HBu production which confirmed that in this study, hydrogen was produced from butyrate-type fermentation. Significant negative correlation between Hmax and HAc indicated that the HAc was not produced from acetate type fermentation but from acetogenesis pathway which consume hydrogen for HAc production. Hmax had strong negative correlation with HPr confirming that hydrogen was consumed for the production of HPr. Previous research reported that HLa can be used as substrate for hydrogen production by some bacteria species such as Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313) [49], Clostridium acetobutylicum, Butyribacterium methylotrophicum, Clostridium tyrobutyricum [50]. However, it could be seen from our result that HLa consumption has no significant correlation with both Hmax and SMPs. These implied that microorganisms in the fermentation system might utilize HLa not mainly for hydrogen production but for their growth. However, the biomass production was not considered in this study. In this study, initial pH was adjusted to 5.5. Dramatically change of pH was not observed in this fermentation system. After fermentation the pH rose to be in the range of 5.77e6.08 (Table 4). The variation of pH in this hydrogen fermentation system could be explained by the combination of VFAs consumption, solubilized protein from AS, and accumulation of ammonia from protein degradation. Consumption of VFAs during the fermentation (Table 4) could cause an increase in pH in the hydrogen fermentation system. Protein is an amphoteric substance and has large buffering capacity; therefore, the solubilized protein can prevent the dramatic changes of pH in the fermentation system. Formation of ammonia ion led to the increase of pH and alkalinity in the fermentation system and prevented the drop of pH caused by VFAs production [7,19].

4.

Conclusions

Different pretreatment methods i.e., acid, heat and AH affect the solubilization of organic matter from AS to liquid phase in which AH pretreatment gave the highest percentage increase in sCOD, soluble carbohydrate and soluble protein concentration. Prolonged pretreatment time tended to increase the solubilization of organic matter for all investigated pretreatment methods. However, the solubilization efficiency did not

correlate with hydrogen production efficiency. Application of different AS pretreatment method yielded different concentrations of soluble organic matter concentrations which consequently affected hydrogen fermentation. After pretreatment higher HBu concentration was found in heat pretreatment than acid and AH pretreatment; HBu inhibit hydrogen fermentation resulted in lower Hmax and HY for heat pretreatment, accordingly. Correlation analysis revealed that hydrogen was produced by butyrate-type fermentation. HPr and HAc were negatively correlated with Hmax values. Carbohydrate was the substrate which showed the strong positive correlation between its consumption and hydrogen production.

Acknowledgments The authors acknowledge the Royal Golden Jubilee Ph.D. Program (Grant No.PHD/0194/2552) for a Ph.D. scholarship to TA. Additional acknowledge goes to research funds from Fermentation Research Center for Value Added of Agricultural Products and the National Research University Project through Biofuels Research Cluster-Khon Kaen University, Office of the Higher Education Commission.

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