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Development of a method for biohydrogen production from wheat straw by dark fermentation
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 6 ( 2 0 1 1 ) 4 1 1 e4 2 0
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Development of a method for biohydrogen production from wheat straw by dark fermentation Nima Nasirian a,*, Morteza Almassi b, Saeid Minaei c, Renatus Widmann d a
Department of Agricultural Mechanization, Shoushtar branch, Islamic Azad University, Shoushtar, Iran1 Department of Agricultural Mechanization, Science and Research Branch, Islamic Azad University, Simon Bolivar Blvd, Ashrafi Esfehani highway, Tehran, Iran c Department of Mechanics of Agricultural Machinery, Tarbiat Modares University, Tehran, Iran d Department of Urban Water and Waste Management, University of Duisburg-Essen, Universita¨tsstr. 15, 45141 Essen, Germany b
article info
abstract
Article history:
Lignocellulosic biomass contains approximately 70e80% carbohydrates. If properly
Received 8 July 2010
hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen
Received in revised form
production. In this research, batch tests of biohydrogen production from acid-pretreated
22 September 2010
wheat straw were conducted to analyze the effects of various associated bioprocesses. The
Accepted 23 September 2010
objective of the pretreatment phase was to investigate the effects of various sulfuric acid
Available online 16 October 2010
pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 C,
Keywords:
they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated
Biohydrogen
under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test
Wheat straw
apparatus. Five different conditions were evaluated in the tests, which were conducted in
Acid pretreatment
accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated
Dark fermentation
straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermenta-
Agricultural waste
tion (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method
Bioenergy
proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H2/mol glucose, which resulted from 5% carbon degradation (hC, gas) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H2/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Ongoing fossil fuel use to meet global energy needs is causing critical environmental problems all over the world. Concerns
related to these problems include energy, economic and political crises and effects on human, animal and plant health. Since the oil crisis in 1973, considerable progress has been made in the search for alternative energy sources [1].
* Corresponding author. Tel.: þ989166004985; fax: þ982188692870. E-mail address: [email protected] (N. Nasirian). 1 Present address. 0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.09.073
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Nomenclature W DOC SHF VS SSF CSTR C TS hC, gas COD DMC Mw CRD H2
watt dissolved organic carbon Separate Hydrolysis and Fermentation volatile solids Simultaneous Saccharification and Fermentation continuously stirred tank reactor Carbon total solids carbon degradation chemical oxygen demand direct microbial conversion microwave completely randomized design hydrogen
Modern bioenergy has received increased attention in the past decade because it not only provides an effective energy source from a technical point of view but also utilizes resources that can be sustainably obtained around the globe [2]. It is well known that biomass residues such as crop stalks are persistent environmental pollutants. The annual global yield of biomass residue exceeds 220 billion tons, which equals the energy of 60e80 billion tons of crude oil [3,8]. Cellulosic biomass is a promising resource due to its abundance and low cost; it has been used in a variety of fermentation processes to produce biofuels like ethanol and hydrogen [4,5]. Hawkes et al. [6] suggested that hydrogen production from fermentable biomass has an advantage over ethanol production because the microorganisms that produce hydrogen can use a wider range of cellulosic hydrolysates than the yeast on which ethanol production is chiefly based. Hydrogen, an entirely carbon-free fuel with a high combustion enthalpy (141.9 kJ/g), is considered a feasible alternative to fossil fuels, and the technology for using hydrogen as a transport fuel is already well established. Hydrogen is a promising, ideal fuel of the future that offers many social, economic and environmental benefits. In the long term, it has the potential to reduce the global dependence on imported oil and also to decrease carbon and other emissions from the transportation sector. The idea of a post-fossil fuel, hydrogen-based economy began to gain mainstream interest only in the last decade [7]. Hydrogen is currently produced in large amounts in the chemical industry, including from the steam reforming of fossil fuels. Hydrogen must be produced sustainably to be economically feasible. This could be achieved from water through electrolysis powered by photosynthetic or other renewable energy, or by gasification or pyrolysis of biomass. It also may be possible to develop a cost-effective and reliable technology to produce hydrogen directly from renewable biomass or organic waste products through anaerobic fermentation [6]. Strategies for H2 production from plant sources essentially follow two major routes: photochemical conversion of sunlight and dark fermentative processes. Although many scientific issues still require research, the fermentative path currently appears to be closer to practical utilization. The benefits of this approach include the low cost
hPa N2 L CO2 NmL CH4 WS Bio-H2 Resid. R Concent. ANOVA Std. Mg
hekto pascal nitrogen liter carbon dioxide normal milliliter methane wheat straw biohydrogen Residence repeat Concentration analysis of variance Standard mega gram
of biomass and the fact that the byproducts of agricultural food production can be used as feedstock [8e10]. Many substrates have been used for fermentative hydrogen production; glucose, sucrose and starch have been the most widely used. In recent years, however, a few studies have begun to use organic wastes as a substrate for hydrogen production [11]. Unused, lignocellulosic waste biomass from forestry, agriculture, and municipal sources is a potential feedstock for the synthesis of biofuels like hydrogen (H2), which could replace fossil fuels and reduce greenhouse gas emissions [12e14]. Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, and lignin. Cellulose consists of linear, highly ordered chains of glucose and is one of the most abundant biopolymers on the planet [15]. Li et al. (2007) [16], Zhang et al. (2007) [17] and Ivanova et al. (2009) [18] investigated mesophilic microbial communities that convert lignocellulosic biomass like wheat straw and corn stalks into biohydrogen. Still, research into the conversion of cellulosic biomass to biohydrogen is limited. In general, it is difficult to directly convert raw crop stalk wastes into biohydrogen gas by microbe anaerobic fermentation due to the complex chemical composition of the waste, which incorporates cellulose, hemicellulose, lignin, protein, and fat. Although the complex structures of lignocellulosic biomasses are not ideal for fermentative hydrogen production, some pretreatment methods allow this biomass to be easily used by hydrogen-producing bacteria [19]. Fan et al. [9] reported the conversion of wheat straw wastes that were pre-treated with dilute HCl by microwave heating into biohydrogen; Zhang et al. [17] used corn stalks for hydrogen production after pretreatment with 0.5% NaOH at 36 C. Interesting questions remain about the ideal environmental conditions (e.g., pretreatment conditions, substrate concentration and acid concentration) for the subsequent fermentation of the resulting fractions. Pretreatment is an important tool for practical cellulose conversion processes. It alters the structure of cellulosic biomass to make cellulose more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars. Pretreatment also removes lignin and hemicellulose, reduces cellulose crystallinity, and increases the porosity of
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 6 ( 2 0 1 1 ) 4 1 1 e4 2 0
the materials [20]. Successful biological conversion of biomass to hydrogen is strongly dependent on processing the raw materials to produce feedstock that can be fermented by the microorganisms. Three main biofuel fermentation processes are widely used in research on bioethanol production from lignocellulosic biomass materials: separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and direct microbial conversion (DMC) [15]. To the best of our knowledge, the current study is the first report of biohydrogen production from wheat straw residues under various bioprocess systems. As a step toward the ultimate goal of replacing rapidly depleting conventional fossil fuels with environmentally friendly and economically feasible biohydrogen resources, this work intends to establish the feasibility of biohydrogen production from agricultural lignocellulosic wastes (specifically wheat straw), which are widely available for renewable energy production and relatively cheap for potential practical applications. More specifically, this research pursued the following principal goals: 1. Pretreatment of prepared waste materials and yield optimization. 2. Biohydrogen production from waste materials in different bioprocess systems in accordance with the 2nd and 3rd generation of biofuel production processes.
2.
Materials and methods
2.1.
Raw material
The wheat straw used in this study was harvested in September 2009 and collected at a farm in the outskirts of Mo¨nchengladbach, Germany. A straw bale was transported to the laboratory site at Duisburg-Essen University in Essen. The sampled biomass, which primarily contained stalk, straw and wheat residues, was sealed in a plastic bag at room temperature before pretreatment. The main ingredients of the sample were 44.33% carbon and 0.434% nitrogen on a dryweight basis. The volatile solid mass of the fresh straw was about 93% of the total solid. Mechanical treatment of the sample was performed for size reduction using an FRITSCH mill equipped with a 1-cm sieve.
2.2.
Hydrogen-producing microflora
The seed sludge of mixed culture was obtained from a continuously stirring tank reactor that was producing hydrogen by fermenting waste sugar at 36 1 C and pH 5.2e5.4. The reactor had been operating continuously for four months and produced a biogas composed of more than 60% hydrogen.
2.3.
Acid pretreatment
Acid pretreatment was performed in two steps. Initially, sulfuric acid (H2SO4) at concentrations of 0.5, 1, and 2% (w/v) was used to pretreat 3.5 g samples of wheat straw at a solid loading of 10% (w/w). Treatments were performed at 90 C and
413
120 C in a convection oven for residence times of 30, 60, and 90 min. Collected solids were washed with warm water, and portions of the solid residues were divided and weighed to determine their fermentation potential in a microwave test. A full factorial design (3 3 2) was used to conduct the experiments and analyze the results of the different pretreatment methods. Each experiment was repeated three times, which resulted in a total of 54 treatments. Following analysis of the preliminary results, the best method was determined through a one-factor experimental design (CRD). Sulfuric acid (H2SO4) at concentrations of 3, 4 and 5% (w/v) was used to pretreat 3.5 g wheat straw samples at a solid loading of 10% (w/w). Treatments were performed in a convection oven at 120 C for a residence time of 90 min and were repeated three times. A total of 11 treatments were tested in the second step.
2.4.
Acid hydrolysis
Wheat straw samples were hydrolyzed with 2% (w/v) sulfuric acid (H2SO4) at a solid loading of 10% (w/w). Treatments were performed in a convection oven at 120 C for 90 min. The liquid result (hydrolysis product) was used for biohydrogen production by a batch-dark fermentation process.
2.5.
Enzyme hydrolysis
The enzyme mixture Methaplus (Cowatech, Burglengenfel, Germany) was tested in different bioconversion processes. Methaplus is an enzyme mixture of cellulases, xylanases, and b-glucanases that is produced especially for use in biogas plants. Recommended dosages (retrieved from http://www. cowatec.de/e/methaplus.htm) for the tests were 100 g/Mg TS and 0.1 mg/g VS [21].
2.6.
Organic carbon extraction microwave test
The microwave test is a quick method for analyzing the anaerobic degradation behavior of organic substances to identify the substrate that is capable of producing the maximum amount of gas during the fermentation process [22]. Ochs et al. (2005) [22] applied this test method for the first time to discover the proper substrate for an anaerobic digestion process. The microwave test simulates fermentation gas production phenomena in batch mode over a much shorter time; the original process lasts for several days, but the test takes only 30 min. Ochs et al. (2005) [22] discovered a rational relationship between the amount of dissolved organic carbon obtained after a 30-min test in the microwave oven and the maximum congested fermentation gas obtained after a 21-day test. They demonstrated that a smaller amount of dissolved organic carbon (DOC) residue from heating the sample substrate in a special reactor yields more gas production in the original batch test. Based on the results of Ochs et al. (2005) [22], this study applied the microwave test to select the best pretreatment method for wheat straw under different environmental conditions. Testing helped to identify the preprocessing treatment method with an optimum bacterial carbon degradation
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capability that produces more cellulosic residues and glucose for subsequent phases of hydrolysis and fermentation. MDS-2000 microwave equipment (CEM GmbH, Kamp-Linfort, Germany) was used to extract organic carbon from a sample substrate. The device was equipped with six 100-ml reaction vessels (digester vessels) made of Hostaflon (a chemically modified polytetraflouroethylene with a low-pore structure and a smooth surface), secured in protecting capsules made of polypropylene, and installed on a rotating plate. The vessels were sealed with covering sheaths made of a fiberglass-ulten composite material to ensure their resistance to pressure. To avoid carbon leakage in the gas phase during all test series, a sealing screw cap was also installed on the capsules. To gauge the temperature, a fiber-optic temperature sensor was inserted into the reaction vessels through a coated shaft. To monitor and control pressure in the reactors, an electronic pressure sensor (a Teflon hose with Kevlar fabric coating) was attached with a screw cap on one of the six vessels, which was designated the reference reactor. The reactors withstood pressures of up to 40 bar, allowed a maximum temperature of 200 C, and were controllable with respect to these two parameters. Originally, the microwave experiments were conducted using distilled water as the digestion medium. To gain a mixing ratio of about 1:20, 2.5 g dry material (preprocessed wheat) was dissolved in 50 g distilled water. The pre-treated materials were then heated to 190 C over a 12-min run-up time (ramp). Preliminary studies showed that this could be achieved by the microwave oven at maximum power (630 W) for the stated duration. The designated duration of the experiment was 30 min plus the ramp, during which time the temperature was held at 190 C. To avoid gas loss at high temperatures, the reactors needed at least 30 min to cool down to a temperature of less than 100 C before they could be opened for further analysis. Once the vessels were opened, the liquid supernatant was decanted into tubes to determine the resultant DOC.
2.7.
NH4Cl 2600 mg, K2HPO4 250 mg, KH2PO4 250 mg, MgCl2$6H2O 320 mg, FeSO4$7H2O 86 mg, CoCl2$6H2O 15 mg, MnCl2$4H2O 15 mg, (NH4)6Mo7O24$4H2O 14 mg, Na2B4O7$10H2O 12 mg, NiCl2$6H2O 49 mg, ZnCl2 23 mg, CuCl2$2H2O 10 mg and CaCl2$2H2O 66 mg. The solution was stored as a 13-fold concentrate and acidified to pH < 2. During all tests, a nutrient dilution according to Hussy et al. (2003) [24] was provided in addition to the feeding substrate to ensure a proper supply of trace elements, nitrogen and phosphorous.
2.8.
Calculated parameters
The fermentation yields of the batch processes were evaluated according to DIN 38414 S8 [23]. The daily volume of produced gas of each trial was estimated and converted to standard volume (0 C, 1013 hPa). The degree of degradation (hC, gas) was calculated by dividing the carbon weight from fermented gas (produced in CO2 form) by the carbon weight of the input substrate to demonstrate the degradability of input material to fermentation gas. For batch tests, the estimated amounts of input and output carbon are affected by the carbon ingredient of the applied inoculum sludge, which should be subtracted from the calculated amounts. Indeed, the solid/fluid equilibria were very difficult to sustain because the batch tests had very small amounts of input and output. If inaccuracies arose during the test set-up (such as improper mixing of the inoculum or substrate) or during the sampling at the end of the test (such as small sample amounts remaining in the vessels), it was not possible to establish a reliable balance. To compensate for these effects, the degree of degradation was determined based on the CO2 content of the gas phase (Cgas). Equation (1) shows the calculation of the degree of degradation [25]. hC;gas ¼ Cgas =Csolid;input 100
2.9.
(1)
Wheat straw bioconversion processes
Batch test set-up
Batch test results depend mainly on the inoculum microbial activity and the correct determination and interpretation of the accumulated gas amount [VDI 2004]. Therefore, the batch tests were conducted with the equipment described in DIN 38414 S8 [23]. The batch tests took place under mesophilic conditions in 0.5-L fermentation vessels in a tempered climate chamber. The test samples were manually mixed once a day, and the amount of produced gas was measured by the liquid replacement method in eudiometers. The temperature in the climate chamber was set to 35 C, as is common in anaerobic batch tests (DIN 38414 S8). The typical test duration was seven days, including the lag phase. As an additional modification, the inserted inoculum/substrate amount was calculated according to Ochs (2005) [22]; the proportion of inoculum to substrate was fixed to 1:1 in relation to VS. Usually, 1 g VS substrate and 1 g VS inoculum in the form of CSTR biohydrogen-producing sludge were inserted into the test vessels, which were filled to 300 g with tap water. To improve the fermentation conditions, 1 ml nutrient was added to each system. Mineral nutrients were added at the following concentrations (modified based on Hussy et al., 2003) [24]:
Five approaches, including SHF and SSF, were applied to convert wheat straw to biohydrogen (Fig. 1).
2.10.
Analytical methods
The produced gas was collected in special bags, and its volume was measured by the liquid replacement method in
a) Wheat Straw (WS)
b) WS
c) WS
Fermentation
Bio-H2
Pretreatment
Fermentation
Pretreatment
Enzyme Hydrolysis
Bio-H2
Fermentation
Bio-H2 SHF
d) WS
e) WS
Pretreatment
Acid Hydrolysis
Enzyme Hydrolysis Fermentation
Fermentation
Bio-H2 SSF
Bio-H2
Fig. 1 e Overview of process scheme used to convert wheat straw into biohydrogen.
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Table 1 e Composition of sulfuric acid pretreatments and microwave tests. Mean Std. Deviation (n)c Run No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Acid Concent. %w/v
Resid.a time min
Oven temp. C
Mw resid. time min
Mwb Temp. C
DOC mg/L
0.5 0.5 0.5 1 1 1 2 2 2 0.5 0.5 0.5 1 1 1 2 2 2
30 60 90 30 60 90 30 60 90 30 60 90 30 60 90 30 60 90
90 90 90 90 90 90 90 90 90 120 120 120 120 120 120 120 120 120
42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42
190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190
6390.0 563.2 (3) 6158.9 739.1 (3) 6495.2 972.8 (3) 6934.2 480.3 (3) 6811.3 569.6 (3) 6701.9 579.0 (3) 7628.5 978.8 (3) 7272.1 554.9 (3) 6461.5 390.4 (3) 6714.3 744.6 (3) 6604.8 985.2 (3) 6314.1 1140.1 (3) 6616.1 617.3 (3) 6638.3 670.7 (3) 6104.4 204.3 (3) 7067.5 993.3 (3) 6244.9 767.7 (3) 5737.0 648.7 (3)
a Residence. b Mw (Microwave) cooling time < 100 C ¼ 30 min. c Mean standard deviation of (n) replicates.
eudiometers. The volumes of gas produced daily in each trial were converted to standard volumes. The hydrogen content of the produced gas was determined off-line daily with a Conthos 2 process gas analyzer based on thermal conductivity (LFE, Maintal, Germany). Amounts of CO2, CH4 and O2 were measured off-line daily with a GA94 infrared gas analyzer (Geotechnical Instruments/Ansyco, Karlsruhe, Germany). The amounts of organic acid were also measured daily as equivalents of acetic acid by the Hach-Lange cuvette test LCK 365 (50e2500 mg/L). Chemical Oxygen Demand (COD) was determined photometrically using Hach-Lange cuvette tests according to DIN 38409H41 [23]. COD was determined photometrically by measuring the color change by potassium dichromate dilution with an MDA photometer ISIS 9000, type LPG 282 (Hach-Lange Dusseldorf, Germany). Total Solids (TS) and Volatile Solids (VS) of the sludge inocula and input and output test samples were determined by standard methods DIN38414 S2 and S3 [23]. Carbon and nitrogen were determined with a vireo MAX CN analyzer (Elementar Analysis Systems, Hanau, Germany). Dissolved Organic Carbon (DOC) levels were determined with a DIMATOC 2000 DOC analyzer (DIMATEC Analysentechnik GmbH, Essen, Germany). To obtain Total Organic Carbon (TOC), the samples were filtered using a <45-mm celluloseacetate filter (Whatman, Brentford, UK) before analysis.
biohydrogen-producing bacteria. A total of 65 pretreatment experiments were performed with different parameters and compared by the microwave test to determine the best method. To make the methods comparable, the results were first standardized by determining the hydrogen production efficiency and calculating the degree of degradation based on carbon input and dioxide output.
3.1.
Effects of pretreatment
Pretreating lignocellulosic biomass with diluted acid is one of the most effective pretreatment methods and has been extensively researched for decades. After wheat straw was treated with dilute sulfuric acid, the resulting solids were rapidly analyzed in the microwave test oven for comparison. Previous studies of similar chemical approaches demonstrated
8000 7000 6000 5000 4000 DOC [mg/L] 3000 2000 1000
3.
Results and discussion 0.5
The most important information provided by the batch tests is the amount of produced biogas and its composition, which allows identification of the best method for preparing the agricultural lignocellulosic material for degradation by the
0 1
Concentration [w/v]
2 3 4
90 & 90 & 90 60 & 60 & 90 120 30 & 120 5 30 & 90 time & Temperature [min, °C] fresh 120
Minimum DOC value
Fig. 2 e Pretreatment and microwave test results.
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Table 2 e ANOVA of DOC production related to pretreatment methods. Sources Concentration (C) Time (t) Temperature (T) Tt TC tC T t C Error Total *
Table 4 e Analysis of variances of bio-H2 production of different methods.
Degree of freedom
Sum of square
Mean of square
F-value
2 2 1 2 2 4 4 34 54
3133979.1 77,4723 1318290.6 2122056.1 250034.9 2140325.4 389235.8 1.834 107 2.386 109
1566989.5 387361.5 1318290.6 1061028 125017.4 535081.3 97308.9 539429.7
2.905* 0.718 2.444 1.967 0.232 0.992 0.180
Significant at the 0.05 level of probability.
that the process parameters affecting productivity are significantly affected by acid concentration, pH, temperature, residence time and solid loading. Acid pretreatment of wheat straw was performed in a convection oven at 90 C and 120 C with a solids ratio of 10% (w/v). Sulfuric acid concentrations were 0.5, 1 and 2% (w/v) and residence times were 30, 60 and 90 min. Each experiment was repeated three times; accordingly, a total of 54 treatments were performed, as described in Table 1. After pretreatment, the microwave test was used [2e5,9] to determine the best acid pretreatment method (for further studies, refer to Ochs et al., 2005). Liquid supernatants were decanted into tubes, filtered, and evaluated for DOC content. The samples containing smaller amounts of DOC had a greater potential to produce biogas in an anaerobic digestion process. A comparison of DOC results for the tests conducted at 90 C and 120 C indicated that the higher temperature significantly enhanced DOC reduction. It was also noted that an increase in residence time from 30 to 90 min reduced DOC extraction in distilled water. The above scenario is shown in Table 1 and Fig. 2. When the duration of each phenomenon is considered, it can be seen that estimated figures for distilled carbon decrease as time increases. Therefore, the controlling factor that decreases the gradient of the line connecting the points representing 90 min is the increased acid concentration, especially at 120 C. More specifically, Table 2 and Fig. 2 indicate that acid concentration was the most influential factor that decreased
Sources Treatment Error Total
Degree of freedom
Sum of square
Mean of square
F-value
4 10 14
123876.342 891.199 124767.541
30969.086 89.120
347.499**
**
Significant at the 0.01 level of probability.
DOC from 6314 mg/L to 5737 mg/L in the 90 min, 120 C treatments. Therefore, acid concentrations of 3, 4 and 5% (w/v) at 90 min and 120 C were selected for further analysis of the best pretreatment method. The results of the 65 tests of eleven treatments are summarized in Table 3 and Fig. 2. A point was identified at which further increasing acid concentrations started to have a negative effect on the amount of DOC extraction from pretreated materials. Therefore, as highlighted in Fig. 2, the best performing treatment method to prepare wheat straw for hydrolysis and fermentation utilizes an acid concentration of 2% (w/v) and a residence time of 90 min at 120 C.
3.2. Effects of different bioprocess methods on biohydrogen yield After three iterations of each of the tests were executed in a randomized order, analysis of variance (Table 4) was used to test the hypothesis that significant variation existed between the different treatments ( p < 0.01). This result indicates that the selected biofuel production system has a significant effect on biohydrogen production. To standardize the tests, glucose, which was expected to be completely digested and therefore to demonstrate the ultimate capability of the digested sludge, was also used under the same environmental conditions. A Duncan average comparison table was used to identify significant differences among the selected methods (Table 5). This table revealed that all of the methods differed considerably in terms of both hydrogen production and carbon degradation. Duncan’s test can identify significant differences between group means in an analysis of variance setting. Table 5 shows hydrogen production and degrees of degradation based on the amount of CO2 in the gas phase and the
Table 3 e Second step of acid pretreatment. Mean Std. Deviation (n)c Run No.
Acid Concent. % w/v
1 2 3 4
3 4 5 Fresh straw
Resid.a time min
Oven temp. C
Mw resid. time min
Mwb Temp. C
90 90 90 e
120 120 120 e
42 42 42 42
190 190 190 190
a Residence. b Mw (Microwave) cooling time < 100 C ¼ 30 min. c Mean standard deviation of (n) replicates.
DOC mg/L 6951.1 1738.9 7160.5 1348.6 7564.1 352.2 7234.7 651.1
(3) (3) (3) (2)
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Table 5 e Different bioprocess results for biohydrogen production in batch tests. Bioprocess systems Unit Fresh straw (Aa) Pretreatment-only (B) SHF (C) SSF (D) Acid Hydrolysis (E)
hc,
Cumulative H2 Yield
Cumulative H2 yield From Glucose
Yield
NmL/gVS
NmL/g VS
mol H2/mol glucose
% C input
%
5.69 37.11 47.89 125.11 148.83
194.64 194.64 194.64 194.64 194.64
0.04 0.29 0.39 1 1.19
0.98 1.44 1.9 4.88 51.86
2.9 19 25 64 76
Glucose Equivalence
gas
a Comparison of mean (Duncan test at the 0.05 level of probability).
very good parameters for biohydrogen production on a large scale. Based on the acetate/butyrate ratio, 2.5 mol H2/mol glucose can be produced using mixed cultures. Notably, the liquid substrate can be used in continuous system reactors with minimal modifications.
3.3. Effects of different methods on carbon disintegration and hydrogen production For a better demonstration of the differences among the studied methods, the degrees of carbon degradation, hc, gas , and glucose equivalents are shown in Fig. 3. Substrates with very high hydrogen production capabilities like the acidhydrolyzed substrate may achieve a glucose equivalence of around one. In light of the production of saccharides and the homogenous dry substrate in the SSF method, a carbon degradation of more than 5% and a glucose equivalence of 0.5 can be achieved. Similar results were reported by Krupp (2007), who experimented on sugar and starch substrates. Degradation levels of less than 3% were achieved for both the pretreatment-only substrate and the separated hydrolysis and fermentation method (SHF). There was also very little degradation observed for fresh straw. Therefore, the substrates with glucose equivalences above 0.5 and degrees of degradation above 5% have high hydrogen production abilities. Methods that produce substrates with glucose equivalences of around one are promising systems for continuous tests.
60
0.9 0.8
Glucose Equivalence.
Degradation 50
0.5 30 0.4 20
0.3 0.2
10 0.1 0
0 Fresh straw
Pretreatment SHF only Bioprocess method s
SSF
Acid hydrolysi s
Fig. 3 e Glucose equivalence and carbon degradation of different bioprocess methods.
Degradation,
40
0.6
c, gas [%]
0.7 Glucose equivalence [-]
glucose equivalence of the test substrates. For the tested series, the mean value for hydrogen production from glucose of 194.64 NmL/g VS was used. The hydrogen concentration in the produced biogas was calculated to be between 57.2 and 63.9%. For simplification, an average hydrogen concentration in the biogas of 60% was assumed for all tests. As shown in Table 5, fresh wheat straw without pretreatment (designated the first test method) did not undergo any degradation by fermentative bacteria, which means that the bacteria were not able to produce gas from fresh straw. In addition, the comparison of means table (Table 5) reveals that there were significant differences between the treatment methods; the pretreatment-only and SHF methods had significant ( p 0.05) effects on fresh straw degradation. This difference highlights the importance of pretreatment methods for biofuel production from lignocellulosic materials. The next method (pretreatment-only) involved pretreating the wheat straw samples and supplying the prepared substrate with hydrogen-producing bacteria. Results showed that pretreatment alone did not yield an interesting result. However, the degradation of cellulosic substances resulted in the production of 37.11 NmL H2/g VS gas plus 1.44% carbon degradation from the solid to the gas phase. The remarkable contrast between this method and the previous one indicates the positive effect of pretreatment. Next, to isolate the effects of pretreatment, hydrolysis and fermentation on hydrogen production, the SHF test was conducted to identify the optimum conditions for each phenomenon. Despite the significant differences observed in gas yield (47.89 NmL H2/g VS) and carbon degradation (1.9%) for this test, the efficiency of the designed system (0.39 mol H2/mol glucose) was affected by factors like an increase in the amount of glucose in the test environment. The SSF method had a significant ( p 0.05) effect on biohydrogen production and carbon degradation. It proved to be the most effective and economical way to convert wheat straw to biohydrogen. By mixing cellulose and glucose in the same reactor, glucose was rapidly removed during hydrolysis before it inhibited cellulase enzymes. The optimum temperature selected for the reaction (37e38 C) represented a compromise between the optimum temperatures for the enzymes in hydrolysis and the bacteria in dark fermentation. The supernatant derived from acid hydrolysis was a promising substance for continuous tests because it was characterized by roughly 1.19 mol hydrogen per mol glucose, 51% carbon degradation and 76% glucose equivalence, which are
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350 SSF Acid hydrolysis Pretreatment only Fresh straw Glucose SHF sludge
300
H2 (NmL/g V S )
250
200
150
100
50
0 0
1
2
3
4
5
6
Time (day) Fig. 4 e Effect of different bioprocess methods on bio-H2 yield.
Fig. 4 illustrates the effects of different bioprocess methods on the cumulative amount of hydrogen produced in batch tests. Hydrogen production from wheat straw stopped after two days, while sludge produced the same yield in five days. Adding straw to sludge as a carbon resource therefore reduces the lag phase of bacterial growth. Moreover, the microorganisms in the hydrogen CSTR sludge were not able to decompose wheat straw structures. Fig. 4 depicts the significant effect of the selected pretreatment method on the hydrogen yield of fresh straw. The produced hydrogen amount was insufficient for a commercial scale. Although the maximum rates of the SSF, SHF and pretreatment-only methods were the same, the SSF method demonstrated the best hydrogen yield. In the SHF method, gas production did not start immediately after the sludge was added to the system, and the bacteria were not provided with enough glucose to allow further production. This shows that the glucose produced from pre-treated wheat straw inhibited the cellulase enzymes. In addition, the hydrogen yield by the SHF method was significantly lower than that achieved by the SSF method. The highest conversion of carbon from the solid to the gas phase was also achieved by the SSF bioprocess method; its high hydrogen production capacity is demonstrated by its 64% glucose equivalence and 5% carbon degradation. The gas production trend of the acid hydrolyzation method was close to the trend from glucose, which shows that the acid hydrolyzation method is highly capable of being transferred from batch to continuous mode.
3.4.
Comparison with other work
Extensive research has been conducted on the production of hydrogen and other biofuels from saccharides and starches. In
recent years, this work has tended to focus on lignocellulosic sources, especially agricultural waste materials. Nevertheless, we could find very few studies on the production of biohydrogen using pretreatment methods and other biofuel production systems. In this section, the efficiency of our selected method is compared with the results from other available studies. Notably, the majority of other work has used pure cultures rather than mixed cultures and did not mention the hydrogen yield of the applied sludge or its corresponding glucose equivalent hydrogen yield. According to Table 6, H2 yields observed in this study are higher than the H2 yields reported in other studies. As indicated, the use of mixed culture inocula resulted in a slightly higher yield. The highest hydrogen yield was obtained in this study from wheat straw (141 ml/g VS). This yield corresponded to a glucose equivalence of nearly 65%, which is comparable with study results reporting hydrogen production from lignocellulosic materials by dark fermentation (mixed culture). Zhang et al. (2007) [27] studied hydrogen production from corn stalks by dark fermentation and obtained a maximum hydrogen yield of 149 ml/g VS using pure cultures. Results obtained in this study were considerably higher than those reported by Fan et al. (2008) [9], Yuan et al. (2009) [26] and Pen et al. (2009) [29], who used acid pretreatments and mixed cultures at the same temperatures. These previous studies produced biohydrogen from corn stalks, wheat straw and corncobs, respectively. Table 6 shows the efficiencies and pretreatment methods used in other research on lignocellulosic materials. The efficiencies of these methods are substantially different from those of the SSF and acid hydrolysis methods. An analysis of different pretreatment methods shows that the effect of temperature on the
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Table 6 e Comparison of yields with other works. Substrate
Pretreatment method
Inoculum Mix culture Pure culture Pure culture Pure culture Mix culture Mix culture Pure culture Pure culture Pure culture Pure culture Pure culture Pure culture Pure culture Pure culture
Cow dung compost Clostridium paraputrificum _ Clostridium sp. Dairy manure Cow dung compost Clostridium butyricum C. saccharolyticus _ Clostridium butyricum C. saccharolyticus C. saccharolyticus C. saccharolyticus C. saccharolyticus
Pure culture Pure culture Mix culture Mix culture Mix culture Mix culture Mix culture Mix culture Mix culture
C. saccharolyticus _ Anaerobic sludge Anaerobic sludge CSTR H2 sludge CSTR H2 sludge CSTR H2 sludge CSTR H2 sludge CSTR H2 sludge
Wheat straw Corn stalk Corn stalk Corn stalk Corncob Wheat straw Corn straw Maize leaves Corn stalk Corn straw Maize leaves Sweet sorghum plant Sugarcane bagasse Silphium trifoliatum leaves Wheat straw Corn stalk Corn Stover Corn stover Wheat straw Wheat straw Wheat straw Wheat straw Supernatant after wheat straw pretreatment
Max. Yield T ( C) (mL H2/g VS)
Ref.
HCL Enzyme 0.2% HCL boiled 30 min Bio-pretreatment 15 day 1% HCL þ 100 C 30 min No pretreatment No pretreatment No pretreatment No pretreatment 1.5 Mpa 10 min 130 C 30 min 130 C 30 min 130 C 30 min 130 C 30 min
Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch
36 55 36 36 36 36 35 70 36 35 70 70 70 70
62.8 132 149.69 176 107.9 1 9 18 3 68 42 32.4 19.6 10.3
[8] [26] [27] [28] [29] [8] [16] [18] [27] [16] [18] [18] [18] [18]
130 C 30 min 0.5% NaOH 220 C 3 min 1.2% HCL þ 200 C 1 min No pretreatment 2% H2SO4 þ 120 C 90 min 2% H2SO4 þ 120 C 90 min 2% H2SO4 þ 120 C 90 min 2% H2SO4 þ 120 C 90 min
Batch Batch Batch Batch Batch Batch Batch (SHF) Batch (SSF) Batch
70 36 35 35 36 36 36 36 36
49 57 49 66 6.4 41.9 54.1 141 168.4
[18] [27] [30] [30] Current Current Current Current Current
deconstruction of lignocellulosic materials is not significant without the application of chemicals. The methods described in this study should be further developed for the methodological accumulation, preservation, size reduction, pretreatment and chemical deconstruction of lignocellulosic waste materials. It is essential to upgrade biohydrogen production systems, including reactors that are specially designed to produce biohydrogen from lignocellulosic materials, by utilizing different substrates and comparing them with other methods, especially SSF.
4.
Reactor type
Conclusions
Experiments revealed that the pretreatment, hydrolysis and fermentation of wheat straw facilitate its use by different methods of biohydrogen production in batch tests. Fresh straw, pre-treated straw and the supernatant residues of acid hydrolization were applied along with the SSF and SHF bioprocess methods to produce biohydrogen in standard batch tests (DIN 38414 S8). Pretreatment with 2% (w/v) sulfuric acid for a 90-min residence time at 120 C proved to be the most effective treatment. Among the 65 treatments studied, tt caused the most significant decrease of DOC in microwave tests. Acid concentration also proved to be a strongly influential factor in decreasing DOC from 6314 mg/L to 5737 mg/L. Concentrations above 2% had a negative effect on DOC extraction from pre-treated materials. Significant differences were also observed between bioprocess methods; for example, the degradation of fresh straw to fermentation gas was less than 1%. The SSF method was found to be an effective and economical way to convert wheat straw to biohydrogen; it
study study study study study
achieved 1 mol H2/mol glucose hydrogen yield, 64% glucose equivalence and 5% carbon degradation (hC, gas) and proved to have the shortest lag phase during gas production of all of the test runs. Supernatants from acid hydrolysis were promising substances for continuous tests of mass biohydrogen production due to their yield of around 1.19 mol H2/mol glucose (76% glucose equivalent) and 52% carbon degradation.
Acknowledgement The authors very much appreciate the valuable cooperation of the Department of Urban Water and Waste Management, University of Duisburg-Essen, who provided the laboratory facilities that were necessary for this research.
references
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Lentinus tigrinus on wheat straw. Process Biochemistry 2006; 41(3):594e8. Hawkes FR, Dinsdale R, Hawkes DL. Sustainable fermentative hydrogen production: challenges for process optimization. International Journal of Hydrogen Energy 2002; 27:1339e47. Kotay SH, Das D. Biohydrogen as a renewable energy resource e prospects and potential. International Journal of Hydrogen Energy 2007;30:750e61. Fan Y-T, Zhang Y-H, Zhang S-F, Hou H-W, Ren B- Z. Efficient conversion of wheat straw wastes into biohydrogen gas by cow dung compost. Bioresource Technology 2006;97:500e5. Pattra S, Sangyoka S, Boonmee M, Reungsang A. Biohydrogen production from the fermentation of sugarcane bagasse hydrolysate by Clostridium butyricum. International Journal of Hydrogen Energy 2008;33:5256e65. Kyazze G, Dinsdale R, Hawkes FR, Guwy AJ, Premier GC, Donnison IS. Direct fermentation of fodder maize, chicory fructans and perennial ryegrass to hydrogen using mixed microflora. Bioresource Technology 2008;99(18):8833e9. Kapdan IK, Kargi F. Biohydrogen production from waste materials. International Journal of Hydrogen Energy 2006;38: 569e82. Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. International Journal of Hydrogen Energy 2001;26:13e28. Hallenbeck PC. Fundamentals of the fermentative production of hydrogen. Journal of Water Science Technology 2005;52:21e9. Levin DB, Zhu H, Beland M, Cicek N, Holbien BE. Potential for renewable hydrogen and methane biogas energy production from residual biomass in Canada. Bioresource Technology 2007;98:654e60. Levin DB, Carere CR, Cicek N, Sparling R. Challenges for biohydrogen production via direct lignocellulose fermentation. International Journal of Hydrogen Energy 2009;34:7390e403. Li D, Chen H. Biological hydrogen production from steam exploded straw by simultaneous saccharification and fermentation. International Journal of Hydrogen Energy 2007;32(12):1742e8. Zhang ML, Fan Y, Xing Y, Pan C, Zhang G, Lay J. Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures. International Journal of Hydrogen Energy 2007;31(4):250e4. Ivanova G, Ra’khely G, Kova’cs KL. Thermophilic biohydrogen production from energy plants by Caldicellulosiruptor saccharolyticus and comparison with related studies. International Journal of Hydrogen Energy 2009;34(9):3659e70.
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Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Development of a method for biohydrogen production from wheat straw by dark fermentation Nima Nasirian a,*, Morteza Almassi b, Saeid Minaei c, Renatus Widmann d a
Department of Agricultural Mechanization, Shoushtar branch, Islamic Azad University, Shoushtar, Iran1 Department of Agricultural Mechanization, Science and Research Branch, Islamic Azad University, Simon Bolivar Blvd, Ashrafi Esfehani highway, Tehran, Iran c Department of Mechanics of Agricultural Machinery, Tarbiat Modares University, Tehran, Iran d Department of Urban Water and Waste Management, University of Duisburg-Essen, Universita¨tsstr. 15, 45141 Essen, Germany b
article info
abstract
Article history:
Lignocellulosic biomass contains approximately 70e80% carbohydrates. If properly
Received 8 July 2010
hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen
Received in revised form
production. In this research, batch tests of biohydrogen production from acid-pretreated
22 September 2010
wheat straw were conducted to analyze the effects of various associated bioprocesses. The
Accepted 23 September 2010
objective of the pretreatment phase was to investigate the effects of various sulfuric acid
Available online 16 October 2010
pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 C,
Keywords:
they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated
Biohydrogen
under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test
Wheat straw
apparatus. Five different conditions were evaluated in the tests, which were conducted in
Acid pretreatment
accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated
Dark fermentation
straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermenta-
Agricultural waste
tion (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method
Bioenergy
proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H2/mol glucose, which resulted from 5% carbon degradation (hC, gas) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H2/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Ongoing fossil fuel use to meet global energy needs is causing critical environmental problems all over the world. Concerns
related to these problems include energy, economic and political crises and effects on human, animal and plant health. Since the oil crisis in 1973, considerable progress has been made in the search for alternative energy sources [1].
* Corresponding author. Tel.: þ989166004985; fax: þ982188692870. E-mail address: [email protected] (N. Nasirian). 1 Present address. 0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.09.073
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Nomenclature W DOC SHF VS SSF CSTR C TS hC, gas COD DMC Mw CRD H2
watt dissolved organic carbon Separate Hydrolysis and Fermentation volatile solids Simultaneous Saccharification and Fermentation continuously stirred tank reactor Carbon total solids carbon degradation chemical oxygen demand direct microbial conversion microwave completely randomized design hydrogen
Modern bioenergy has received increased attention in the past decade because it not only provides an effective energy source from a technical point of view but also utilizes resources that can be sustainably obtained around the globe [2]. It is well known that biomass residues such as crop stalks are persistent environmental pollutants. The annual global yield of biomass residue exceeds 220 billion tons, which equals the energy of 60e80 billion tons of crude oil [3,8]. Cellulosic biomass is a promising resource due to its abundance and low cost; it has been used in a variety of fermentation processes to produce biofuels like ethanol and hydrogen [4,5]. Hawkes et al. [6] suggested that hydrogen production from fermentable biomass has an advantage over ethanol production because the microorganisms that produce hydrogen can use a wider range of cellulosic hydrolysates than the yeast on which ethanol production is chiefly based. Hydrogen, an entirely carbon-free fuel with a high combustion enthalpy (141.9 kJ/g), is considered a feasible alternative to fossil fuels, and the technology for using hydrogen as a transport fuel is already well established. Hydrogen is a promising, ideal fuel of the future that offers many social, economic and environmental benefits. In the long term, it has the potential to reduce the global dependence on imported oil and also to decrease carbon and other emissions from the transportation sector. The idea of a post-fossil fuel, hydrogen-based economy began to gain mainstream interest only in the last decade [7]. Hydrogen is currently produced in large amounts in the chemical industry, including from the steam reforming of fossil fuels. Hydrogen must be produced sustainably to be economically feasible. This could be achieved from water through electrolysis powered by photosynthetic or other renewable energy, or by gasification or pyrolysis of biomass. It also may be possible to develop a cost-effective and reliable technology to produce hydrogen directly from renewable biomass or organic waste products through anaerobic fermentation [6]. Strategies for H2 production from plant sources essentially follow two major routes: photochemical conversion of sunlight and dark fermentative processes. Although many scientific issues still require research, the fermentative path currently appears to be closer to practical utilization. The benefits of this approach include the low cost
hPa N2 L CO2 NmL CH4 WS Bio-H2 Resid. R Concent. ANOVA Std. Mg
hekto pascal nitrogen liter carbon dioxide normal milliliter methane wheat straw biohydrogen Residence repeat Concentration analysis of variance Standard mega gram
of biomass and the fact that the byproducts of agricultural food production can be used as feedstock [8e10]. Many substrates have been used for fermentative hydrogen production; glucose, sucrose and starch have been the most widely used. In recent years, however, a few studies have begun to use organic wastes as a substrate for hydrogen production [11]. Unused, lignocellulosic waste biomass from forestry, agriculture, and municipal sources is a potential feedstock for the synthesis of biofuels like hydrogen (H2), which could replace fossil fuels and reduce greenhouse gas emissions [12e14]. Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, and lignin. Cellulose consists of linear, highly ordered chains of glucose and is one of the most abundant biopolymers on the planet [15]. Li et al. (2007) [16], Zhang et al. (2007) [17] and Ivanova et al. (2009) [18] investigated mesophilic microbial communities that convert lignocellulosic biomass like wheat straw and corn stalks into biohydrogen. Still, research into the conversion of cellulosic biomass to biohydrogen is limited. In general, it is difficult to directly convert raw crop stalk wastes into biohydrogen gas by microbe anaerobic fermentation due to the complex chemical composition of the waste, which incorporates cellulose, hemicellulose, lignin, protein, and fat. Although the complex structures of lignocellulosic biomasses are not ideal for fermentative hydrogen production, some pretreatment methods allow this biomass to be easily used by hydrogen-producing bacteria [19]. Fan et al. [9] reported the conversion of wheat straw wastes that were pre-treated with dilute HCl by microwave heating into biohydrogen; Zhang et al. [17] used corn stalks for hydrogen production after pretreatment with 0.5% NaOH at 36 C. Interesting questions remain about the ideal environmental conditions (e.g., pretreatment conditions, substrate concentration and acid concentration) for the subsequent fermentation of the resulting fractions. Pretreatment is an important tool for practical cellulose conversion processes. It alters the structure of cellulosic biomass to make cellulose more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars. Pretreatment also removes lignin and hemicellulose, reduces cellulose crystallinity, and increases the porosity of
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 6 ( 2 0 1 1 ) 4 1 1 e4 2 0
the materials [20]. Successful biological conversion of biomass to hydrogen is strongly dependent on processing the raw materials to produce feedstock that can be fermented by the microorganisms. Three main biofuel fermentation processes are widely used in research on bioethanol production from lignocellulosic biomass materials: separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and direct microbial conversion (DMC) [15]. To the best of our knowledge, the current study is the first report of biohydrogen production from wheat straw residues under various bioprocess systems. As a step toward the ultimate goal of replacing rapidly depleting conventional fossil fuels with environmentally friendly and economically feasible biohydrogen resources, this work intends to establish the feasibility of biohydrogen production from agricultural lignocellulosic wastes (specifically wheat straw), which are widely available for renewable energy production and relatively cheap for potential practical applications. More specifically, this research pursued the following principal goals: 1. Pretreatment of prepared waste materials and yield optimization. 2. Biohydrogen production from waste materials in different bioprocess systems in accordance with the 2nd and 3rd generation of biofuel production processes.
2.
Materials and methods
2.1.
Raw material
The wheat straw used in this study was harvested in September 2009 and collected at a farm in the outskirts of Mo¨nchengladbach, Germany. A straw bale was transported to the laboratory site at Duisburg-Essen University in Essen. The sampled biomass, which primarily contained stalk, straw and wheat residues, was sealed in a plastic bag at room temperature before pretreatment. The main ingredients of the sample were 44.33% carbon and 0.434% nitrogen on a dryweight basis. The volatile solid mass of the fresh straw was about 93% of the total solid. Mechanical treatment of the sample was performed for size reduction using an FRITSCH mill equipped with a 1-cm sieve.
2.2.
Hydrogen-producing microflora
The seed sludge of mixed culture was obtained from a continuously stirring tank reactor that was producing hydrogen by fermenting waste sugar at 36 1 C and pH 5.2e5.4. The reactor had been operating continuously for four months and produced a biogas composed of more than 60% hydrogen.
2.3.
Acid pretreatment
Acid pretreatment was performed in two steps. Initially, sulfuric acid (H2SO4) at concentrations of 0.5, 1, and 2% (w/v) was used to pretreat 3.5 g samples of wheat straw at a solid loading of 10% (w/w). Treatments were performed at 90 C and
413
120 C in a convection oven for residence times of 30, 60, and 90 min. Collected solids were washed with warm water, and portions of the solid residues were divided and weighed to determine their fermentation potential in a microwave test. A full factorial design (3 3 2) was used to conduct the experiments and analyze the results of the different pretreatment methods. Each experiment was repeated three times, which resulted in a total of 54 treatments. Following analysis of the preliminary results, the best method was determined through a one-factor experimental design (CRD). Sulfuric acid (H2SO4) at concentrations of 3, 4 and 5% (w/v) was used to pretreat 3.5 g wheat straw samples at a solid loading of 10% (w/w). Treatments were performed in a convection oven at 120 C for a residence time of 90 min and were repeated three times. A total of 11 treatments were tested in the second step.
2.4.
Acid hydrolysis
Wheat straw samples were hydrolyzed with 2% (w/v) sulfuric acid (H2SO4) at a solid loading of 10% (w/w). Treatments were performed in a convection oven at 120 C for 90 min. The liquid result (hydrolysis product) was used for biohydrogen production by a batch-dark fermentation process.
2.5.
Enzyme hydrolysis
The enzyme mixture Methaplus (Cowatech, Burglengenfel, Germany) was tested in different bioconversion processes. Methaplus is an enzyme mixture of cellulases, xylanases, and b-glucanases that is produced especially for use in biogas plants. Recommended dosages (retrieved from http://www. cowatec.de/e/methaplus.htm) for the tests were 100 g/Mg TS and 0.1 mg/g VS [21].
2.6.
Organic carbon extraction microwave test
The microwave test is a quick method for analyzing the anaerobic degradation behavior of organic substances to identify the substrate that is capable of producing the maximum amount of gas during the fermentation process [22]. Ochs et al. (2005) [22] applied this test method for the first time to discover the proper substrate for an anaerobic digestion process. The microwave test simulates fermentation gas production phenomena in batch mode over a much shorter time; the original process lasts for several days, but the test takes only 30 min. Ochs et al. (2005) [22] discovered a rational relationship between the amount of dissolved organic carbon obtained after a 30-min test in the microwave oven and the maximum congested fermentation gas obtained after a 21-day test. They demonstrated that a smaller amount of dissolved organic carbon (DOC) residue from heating the sample substrate in a special reactor yields more gas production in the original batch test. Based on the results of Ochs et al. (2005) [22], this study applied the microwave test to select the best pretreatment method for wheat straw under different environmental conditions. Testing helped to identify the preprocessing treatment method with an optimum bacterial carbon degradation
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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 6 ( 2 0 1 1 ) 4 1 1 e4 2 0
capability that produces more cellulosic residues and glucose for subsequent phases of hydrolysis and fermentation. MDS-2000 microwave equipment (CEM GmbH, Kamp-Linfort, Germany) was used to extract organic carbon from a sample substrate. The device was equipped with six 100-ml reaction vessels (digester vessels) made of Hostaflon (a chemically modified polytetraflouroethylene with a low-pore structure and a smooth surface), secured in protecting capsules made of polypropylene, and installed on a rotating plate. The vessels were sealed with covering sheaths made of a fiberglass-ulten composite material to ensure their resistance to pressure. To avoid carbon leakage in the gas phase during all test series, a sealing screw cap was also installed on the capsules. To gauge the temperature, a fiber-optic temperature sensor was inserted into the reaction vessels through a coated shaft. To monitor and control pressure in the reactors, an electronic pressure sensor (a Teflon hose with Kevlar fabric coating) was attached with a screw cap on one of the six vessels, which was designated the reference reactor. The reactors withstood pressures of up to 40 bar, allowed a maximum temperature of 200 C, and were controllable with respect to these two parameters. Originally, the microwave experiments were conducted using distilled water as the digestion medium. To gain a mixing ratio of about 1:20, 2.5 g dry material (preprocessed wheat) was dissolved in 50 g distilled water. The pre-treated materials were then heated to 190 C over a 12-min run-up time (ramp). Preliminary studies showed that this could be achieved by the microwave oven at maximum power (630 W) for the stated duration. The designated duration of the experiment was 30 min plus the ramp, during which time the temperature was held at 190 C. To avoid gas loss at high temperatures, the reactors needed at least 30 min to cool down to a temperature of less than 100 C before they could be opened for further analysis. Once the vessels were opened, the liquid supernatant was decanted into tubes to determine the resultant DOC.
2.7.
NH4Cl 2600 mg, K2HPO4 250 mg, KH2PO4 250 mg, MgCl2$6H2O 320 mg, FeSO4$7H2O 86 mg, CoCl2$6H2O 15 mg, MnCl2$4H2O 15 mg, (NH4)6Mo7O24$4H2O 14 mg, Na2B4O7$10H2O 12 mg, NiCl2$6H2O 49 mg, ZnCl2 23 mg, CuCl2$2H2O 10 mg and CaCl2$2H2O 66 mg. The solution was stored as a 13-fold concentrate and acidified to pH < 2. During all tests, a nutrient dilution according to Hussy et al. (2003) [24] was provided in addition to the feeding substrate to ensure a proper supply of trace elements, nitrogen and phosphorous.
2.8.
Calculated parameters
The fermentation yields of the batch processes were evaluated according to DIN 38414 S8 [23]. The daily volume of produced gas of each trial was estimated and converted to standard volume (0 C, 1013 hPa). The degree of degradation (hC, gas) was calculated by dividing the carbon weight from fermented gas (produced in CO2 form) by the carbon weight of the input substrate to demonstrate the degradability of input material to fermentation gas. For batch tests, the estimated amounts of input and output carbon are affected by the carbon ingredient of the applied inoculum sludge, which should be subtracted from the calculated amounts. Indeed, the solid/fluid equilibria were very difficult to sustain because the batch tests had very small amounts of input and output. If inaccuracies arose during the test set-up (such as improper mixing of the inoculum or substrate) or during the sampling at the end of the test (such as small sample amounts remaining in the vessels), it was not possible to establish a reliable balance. To compensate for these effects, the degree of degradation was determined based on the CO2 content of the gas phase (Cgas). Equation (1) shows the calculation of the degree of degradation [25]. hC;gas ¼ Cgas =Csolid;input 100
2.9.
(1)
Wheat straw bioconversion processes
Batch test set-up
Batch test results depend mainly on the inoculum microbial activity and the correct determination and interpretation of the accumulated gas amount [VDI 2004]. Therefore, the batch tests were conducted with the equipment described in DIN 38414 S8 [23]. The batch tests took place under mesophilic conditions in 0.5-L fermentation vessels in a tempered climate chamber. The test samples were manually mixed once a day, and the amount of produced gas was measured by the liquid replacement method in eudiometers. The temperature in the climate chamber was set to 35 C, as is common in anaerobic batch tests (DIN 38414 S8). The typical test duration was seven days, including the lag phase. As an additional modification, the inserted inoculum/substrate amount was calculated according to Ochs (2005) [22]; the proportion of inoculum to substrate was fixed to 1:1 in relation to VS. Usually, 1 g VS substrate and 1 g VS inoculum in the form of CSTR biohydrogen-producing sludge were inserted into the test vessels, which were filled to 300 g with tap water. To improve the fermentation conditions, 1 ml nutrient was added to each system. Mineral nutrients were added at the following concentrations (modified based on Hussy et al., 2003) [24]:
Five approaches, including SHF and SSF, were applied to convert wheat straw to biohydrogen (Fig. 1).
2.10.
Analytical methods
The produced gas was collected in special bags, and its volume was measured by the liquid replacement method in
a) Wheat Straw (WS)
b) WS
c) WS
Fermentation
Bio-H2
Pretreatment
Fermentation
Pretreatment
Enzyme Hydrolysis
Bio-H2
Fermentation
Bio-H2 SHF
d) WS
e) WS
Pretreatment
Acid Hydrolysis
Enzyme Hydrolysis Fermentation
Fermentation
Bio-H2 SSF
Bio-H2
Fig. 1 e Overview of process scheme used to convert wheat straw into biohydrogen.
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Table 1 e Composition of sulfuric acid pretreatments and microwave tests. Mean Std. Deviation (n)c Run No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Acid Concent. %w/v
Resid.a time min
Oven temp. C
Mw resid. time min
Mwb Temp. C
DOC mg/L
0.5 0.5 0.5 1 1 1 2 2 2 0.5 0.5 0.5 1 1 1 2 2 2
30 60 90 30 60 90 30 60 90 30 60 90 30 60 90 30 60 90
90 90 90 90 90 90 90 90 90 120 120 120 120 120 120 120 120 120
42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42
190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190
6390.0 563.2 (3) 6158.9 739.1 (3) 6495.2 972.8 (3) 6934.2 480.3 (3) 6811.3 569.6 (3) 6701.9 579.0 (3) 7628.5 978.8 (3) 7272.1 554.9 (3) 6461.5 390.4 (3) 6714.3 744.6 (3) 6604.8 985.2 (3) 6314.1 1140.1 (3) 6616.1 617.3 (3) 6638.3 670.7 (3) 6104.4 204.3 (3) 7067.5 993.3 (3) 6244.9 767.7 (3) 5737.0 648.7 (3)
a Residence. b Mw (Microwave) cooling time < 100 C ¼ 30 min. c Mean standard deviation of (n) replicates.
eudiometers. The volumes of gas produced daily in each trial were converted to standard volumes. The hydrogen content of the produced gas was determined off-line daily with a Conthos 2 process gas analyzer based on thermal conductivity (LFE, Maintal, Germany). Amounts of CO2, CH4 and O2 were measured off-line daily with a GA94 infrared gas analyzer (Geotechnical Instruments/Ansyco, Karlsruhe, Germany). The amounts of organic acid were also measured daily as equivalents of acetic acid by the Hach-Lange cuvette test LCK 365 (50e2500 mg/L). Chemical Oxygen Demand (COD) was determined photometrically using Hach-Lange cuvette tests according to DIN 38409H41 [23]. COD was determined photometrically by measuring the color change by potassium dichromate dilution with an MDA photometer ISIS 9000, type LPG 282 (Hach-Lange Dusseldorf, Germany). Total Solids (TS) and Volatile Solids (VS) of the sludge inocula and input and output test samples were determined by standard methods DIN38414 S2 and S3 [23]. Carbon and nitrogen were determined with a vireo MAX CN analyzer (Elementar Analysis Systems, Hanau, Germany). Dissolved Organic Carbon (DOC) levels were determined with a DIMATOC 2000 DOC analyzer (DIMATEC Analysentechnik GmbH, Essen, Germany). To obtain Total Organic Carbon (TOC), the samples were filtered using a <45-mm celluloseacetate filter (Whatman, Brentford, UK) before analysis.
biohydrogen-producing bacteria. A total of 65 pretreatment experiments were performed with different parameters and compared by the microwave test to determine the best method. To make the methods comparable, the results were first standardized by determining the hydrogen production efficiency and calculating the degree of degradation based on carbon input and dioxide output.
3.1.
Effects of pretreatment
Pretreating lignocellulosic biomass with diluted acid is one of the most effective pretreatment methods and has been extensively researched for decades. After wheat straw was treated with dilute sulfuric acid, the resulting solids were rapidly analyzed in the microwave test oven for comparison. Previous studies of similar chemical approaches demonstrated
8000 7000 6000 5000 4000 DOC [mg/L] 3000 2000 1000
3.
Results and discussion 0.5
The most important information provided by the batch tests is the amount of produced biogas and its composition, which allows identification of the best method for preparing the agricultural lignocellulosic material for degradation by the
0 1
Concentration [w/v]
2 3 4
90 & 90 & 90 60 & 60 & 90 120 30 & 120 5 30 & 90 time & Temperature [min, °C] fresh 120
Minimum DOC value
Fig. 2 e Pretreatment and microwave test results.
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Table 2 e ANOVA of DOC production related to pretreatment methods. Sources Concentration (C) Time (t) Temperature (T) Tt TC tC T t C Error Total *
Table 4 e Analysis of variances of bio-H2 production of different methods.
Degree of freedom
Sum of square
Mean of square
F-value
2 2 1 2 2 4 4 34 54
3133979.1 77,4723 1318290.6 2122056.1 250034.9 2140325.4 389235.8 1.834 107 2.386 109
1566989.5 387361.5 1318290.6 1061028 125017.4 535081.3 97308.9 539429.7
2.905* 0.718 2.444 1.967 0.232 0.992 0.180
Significant at the 0.05 level of probability.
that the process parameters affecting productivity are significantly affected by acid concentration, pH, temperature, residence time and solid loading. Acid pretreatment of wheat straw was performed in a convection oven at 90 C and 120 C with a solids ratio of 10% (w/v). Sulfuric acid concentrations were 0.5, 1 and 2% (w/v) and residence times were 30, 60 and 90 min. Each experiment was repeated three times; accordingly, a total of 54 treatments were performed, as described in Table 1. After pretreatment, the microwave test was used [2e5,9] to determine the best acid pretreatment method (for further studies, refer to Ochs et al., 2005). Liquid supernatants were decanted into tubes, filtered, and evaluated for DOC content. The samples containing smaller amounts of DOC had a greater potential to produce biogas in an anaerobic digestion process. A comparison of DOC results for the tests conducted at 90 C and 120 C indicated that the higher temperature significantly enhanced DOC reduction. It was also noted that an increase in residence time from 30 to 90 min reduced DOC extraction in distilled water. The above scenario is shown in Table 1 and Fig. 2. When the duration of each phenomenon is considered, it can be seen that estimated figures for distilled carbon decrease as time increases. Therefore, the controlling factor that decreases the gradient of the line connecting the points representing 90 min is the increased acid concentration, especially at 120 C. More specifically, Table 2 and Fig. 2 indicate that acid concentration was the most influential factor that decreased
Sources Treatment Error Total
Degree of freedom
Sum of square
Mean of square
F-value
4 10 14
123876.342 891.199 124767.541
30969.086 89.120
347.499**
**
Significant at the 0.01 level of probability.
DOC from 6314 mg/L to 5737 mg/L in the 90 min, 120 C treatments. Therefore, acid concentrations of 3, 4 and 5% (w/v) at 90 min and 120 C were selected for further analysis of the best pretreatment method. The results of the 65 tests of eleven treatments are summarized in Table 3 and Fig. 2. A point was identified at which further increasing acid concentrations started to have a negative effect on the amount of DOC extraction from pretreated materials. Therefore, as highlighted in Fig. 2, the best performing treatment method to prepare wheat straw for hydrolysis and fermentation utilizes an acid concentration of 2% (w/v) and a residence time of 90 min at 120 C.
3.2. Effects of different bioprocess methods on biohydrogen yield After three iterations of each of the tests were executed in a randomized order, analysis of variance (Table 4) was used to test the hypothesis that significant variation existed between the different treatments ( p < 0.01). This result indicates that the selected biofuel production system has a significant effect on biohydrogen production. To standardize the tests, glucose, which was expected to be completely digested and therefore to demonstrate the ultimate capability of the digested sludge, was also used under the same environmental conditions. A Duncan average comparison table was used to identify significant differences among the selected methods (Table 5). This table revealed that all of the methods differed considerably in terms of both hydrogen production and carbon degradation. Duncan’s test can identify significant differences between group means in an analysis of variance setting. Table 5 shows hydrogen production and degrees of degradation based on the amount of CO2 in the gas phase and the
Table 3 e Second step of acid pretreatment. Mean Std. Deviation (n)c Run No.
Acid Concent. % w/v
1 2 3 4
3 4 5 Fresh straw
Resid.a time min
Oven temp. C
Mw resid. time min
Mwb Temp. C
90 90 90 e
120 120 120 e
42 42 42 42
190 190 190 190
a Residence. b Mw (Microwave) cooling time < 100 C ¼ 30 min. c Mean standard deviation of (n) replicates.
DOC mg/L 6951.1 1738.9 7160.5 1348.6 7564.1 352.2 7234.7 651.1
(3) (3) (3) (2)
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Table 5 e Different bioprocess results for biohydrogen production in batch tests. Bioprocess systems Unit Fresh straw (Aa) Pretreatment-only (B) SHF (C) SSF (D) Acid Hydrolysis (E)
hc,
Cumulative H2 Yield
Cumulative H2 yield From Glucose
Yield
NmL/gVS
NmL/g VS
mol H2/mol glucose
% C input
%
5.69 37.11 47.89 125.11 148.83
194.64 194.64 194.64 194.64 194.64
0.04 0.29 0.39 1 1.19
0.98 1.44 1.9 4.88 51.86
2.9 19 25 64 76
Glucose Equivalence
gas
a Comparison of mean (Duncan test at the 0.05 level of probability).
very good parameters for biohydrogen production on a large scale. Based on the acetate/butyrate ratio, 2.5 mol H2/mol glucose can be produced using mixed cultures. Notably, the liquid substrate can be used in continuous system reactors with minimal modifications.
3.3. Effects of different methods on carbon disintegration and hydrogen production For a better demonstration of the differences among the studied methods, the degrees of carbon degradation, hc, gas , and glucose equivalents are shown in Fig. 3. Substrates with very high hydrogen production capabilities like the acidhydrolyzed substrate may achieve a glucose equivalence of around one. In light of the production of saccharides and the homogenous dry substrate in the SSF method, a carbon degradation of more than 5% and a glucose equivalence of 0.5 can be achieved. Similar results were reported by Krupp (2007), who experimented on sugar and starch substrates. Degradation levels of less than 3% were achieved for both the pretreatment-only substrate and the separated hydrolysis and fermentation method (SHF). There was also very little degradation observed for fresh straw. Therefore, the substrates with glucose equivalences above 0.5 and degrees of degradation above 5% have high hydrogen production abilities. Methods that produce substrates with glucose equivalences of around one are promising systems for continuous tests.
60
0.9 0.8
Glucose Equivalence.
Degradation 50
0.5 30 0.4 20
0.3 0.2
10 0.1 0
0 Fresh straw
Pretreatment SHF only Bioprocess method s
SSF
Acid hydrolysi s
Fig. 3 e Glucose equivalence and carbon degradation of different bioprocess methods.
Degradation,
40
0.6
c, gas [%]
0.7 Glucose equivalence [-]
glucose equivalence of the test substrates. For the tested series, the mean value for hydrogen production from glucose of 194.64 NmL/g VS was used. The hydrogen concentration in the produced biogas was calculated to be between 57.2 and 63.9%. For simplification, an average hydrogen concentration in the biogas of 60% was assumed for all tests. As shown in Table 5, fresh wheat straw without pretreatment (designated the first test method) did not undergo any degradation by fermentative bacteria, which means that the bacteria were not able to produce gas from fresh straw. In addition, the comparison of means table (Table 5) reveals that there were significant differences between the treatment methods; the pretreatment-only and SHF methods had significant ( p 0.05) effects on fresh straw degradation. This difference highlights the importance of pretreatment methods for biofuel production from lignocellulosic materials. The next method (pretreatment-only) involved pretreating the wheat straw samples and supplying the prepared substrate with hydrogen-producing bacteria. Results showed that pretreatment alone did not yield an interesting result. However, the degradation of cellulosic substances resulted in the production of 37.11 NmL H2/g VS gas plus 1.44% carbon degradation from the solid to the gas phase. The remarkable contrast between this method and the previous one indicates the positive effect of pretreatment. Next, to isolate the effects of pretreatment, hydrolysis and fermentation on hydrogen production, the SHF test was conducted to identify the optimum conditions for each phenomenon. Despite the significant differences observed in gas yield (47.89 NmL H2/g VS) and carbon degradation (1.9%) for this test, the efficiency of the designed system (0.39 mol H2/mol glucose) was affected by factors like an increase in the amount of glucose in the test environment. The SSF method had a significant ( p 0.05) effect on biohydrogen production and carbon degradation. It proved to be the most effective and economical way to convert wheat straw to biohydrogen. By mixing cellulose and glucose in the same reactor, glucose was rapidly removed during hydrolysis before it inhibited cellulase enzymes. The optimum temperature selected for the reaction (37e38 C) represented a compromise between the optimum temperatures for the enzymes in hydrolysis and the bacteria in dark fermentation. The supernatant derived from acid hydrolysis was a promising substance for continuous tests because it was characterized by roughly 1.19 mol hydrogen per mol glucose, 51% carbon degradation and 76% glucose equivalence, which are
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350 SSF Acid hydrolysis Pretreatment only Fresh straw Glucose SHF sludge
300
H2 (NmL/g V S )
250
200
150
100
50
0 0
1
2
3
4
5
6
Time (day) Fig. 4 e Effect of different bioprocess methods on bio-H2 yield.
Fig. 4 illustrates the effects of different bioprocess methods on the cumulative amount of hydrogen produced in batch tests. Hydrogen production from wheat straw stopped after two days, while sludge produced the same yield in five days. Adding straw to sludge as a carbon resource therefore reduces the lag phase of bacterial growth. Moreover, the microorganisms in the hydrogen CSTR sludge were not able to decompose wheat straw structures. Fig. 4 depicts the significant effect of the selected pretreatment method on the hydrogen yield of fresh straw. The produced hydrogen amount was insufficient for a commercial scale. Although the maximum rates of the SSF, SHF and pretreatment-only methods were the same, the SSF method demonstrated the best hydrogen yield. In the SHF method, gas production did not start immediately after the sludge was added to the system, and the bacteria were not provided with enough glucose to allow further production. This shows that the glucose produced from pre-treated wheat straw inhibited the cellulase enzymes. In addition, the hydrogen yield by the SHF method was significantly lower than that achieved by the SSF method. The highest conversion of carbon from the solid to the gas phase was also achieved by the SSF bioprocess method; its high hydrogen production capacity is demonstrated by its 64% glucose equivalence and 5% carbon degradation. The gas production trend of the acid hydrolyzation method was close to the trend from glucose, which shows that the acid hydrolyzation method is highly capable of being transferred from batch to continuous mode.
3.4.
Comparison with other work
Extensive research has been conducted on the production of hydrogen and other biofuels from saccharides and starches. In
recent years, this work has tended to focus on lignocellulosic sources, especially agricultural waste materials. Nevertheless, we could find very few studies on the production of biohydrogen using pretreatment methods and other biofuel production systems. In this section, the efficiency of our selected method is compared with the results from other available studies. Notably, the majority of other work has used pure cultures rather than mixed cultures and did not mention the hydrogen yield of the applied sludge or its corresponding glucose equivalent hydrogen yield. According to Table 6, H2 yields observed in this study are higher than the H2 yields reported in other studies. As indicated, the use of mixed culture inocula resulted in a slightly higher yield. The highest hydrogen yield was obtained in this study from wheat straw (141 ml/g VS). This yield corresponded to a glucose equivalence of nearly 65%, which is comparable with study results reporting hydrogen production from lignocellulosic materials by dark fermentation (mixed culture). Zhang et al. (2007) [27] studied hydrogen production from corn stalks by dark fermentation and obtained a maximum hydrogen yield of 149 ml/g VS using pure cultures. Results obtained in this study were considerably higher than those reported by Fan et al. (2008) [9], Yuan et al. (2009) [26] and Pen et al. (2009) [29], who used acid pretreatments and mixed cultures at the same temperatures. These previous studies produced biohydrogen from corn stalks, wheat straw and corncobs, respectively. Table 6 shows the efficiencies and pretreatment methods used in other research on lignocellulosic materials. The efficiencies of these methods are substantially different from those of the SSF and acid hydrolysis methods. An analysis of different pretreatment methods shows that the effect of temperature on the
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Table 6 e Comparison of yields with other works. Substrate
Pretreatment method
Inoculum Mix culture Pure culture Pure culture Pure culture Mix culture Mix culture Pure culture Pure culture Pure culture Pure culture Pure culture Pure culture Pure culture Pure culture
Cow dung compost Clostridium paraputrificum _ Clostridium sp. Dairy manure Cow dung compost Clostridium butyricum C. saccharolyticus _ Clostridium butyricum C. saccharolyticus C. saccharolyticus C. saccharolyticus C. saccharolyticus
Pure culture Pure culture Mix culture Mix culture Mix culture Mix culture Mix culture Mix culture Mix culture
C. saccharolyticus _ Anaerobic sludge Anaerobic sludge CSTR H2 sludge CSTR H2 sludge CSTR H2 sludge CSTR H2 sludge CSTR H2 sludge
Wheat straw Corn stalk Corn stalk Corn stalk Corncob Wheat straw Corn straw Maize leaves Corn stalk Corn straw Maize leaves Sweet sorghum plant Sugarcane bagasse Silphium trifoliatum leaves Wheat straw Corn stalk Corn Stover Corn stover Wheat straw Wheat straw Wheat straw Wheat straw Supernatant after wheat straw pretreatment
Max. Yield T ( C) (mL H2/g VS)
Ref.
HCL Enzyme 0.2% HCL boiled 30 min Bio-pretreatment 15 day 1% HCL þ 100 C 30 min No pretreatment No pretreatment No pretreatment No pretreatment 1.5 Mpa 10 min 130 C 30 min 130 C 30 min 130 C 30 min 130 C 30 min
Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch
36 55 36 36 36 36 35 70 36 35 70 70 70 70
62.8 132 149.69 176 107.9 1 9 18 3 68 42 32.4 19.6 10.3
[8] [26] [27] [28] [29] [8] [16] [18] [27] [16] [18] [18] [18] [18]
130 C 30 min 0.5% NaOH 220 C 3 min 1.2% HCL þ 200 C 1 min No pretreatment 2% H2SO4 þ 120 C 90 min 2% H2SO4 þ 120 C 90 min 2% H2SO4 þ 120 C 90 min 2% H2SO4 þ 120 C 90 min
Batch Batch Batch Batch Batch Batch Batch (SHF) Batch (SSF) Batch
70 36 35 35 36 36 36 36 36
49 57 49 66 6.4 41.9 54.1 141 168.4
[18] [27] [30] [30] Current Current Current Current Current
deconstruction of lignocellulosic materials is not significant without the application of chemicals. The methods described in this study should be further developed for the methodological accumulation, preservation, size reduction, pretreatment and chemical deconstruction of lignocellulosic waste materials. It is essential to upgrade biohydrogen production systems, including reactors that are specially designed to produce biohydrogen from lignocellulosic materials, by utilizing different substrates and comparing them with other methods, especially SSF.
4.
Reactor type
Conclusions
Experiments revealed that the pretreatment, hydrolysis and fermentation of wheat straw facilitate its use by different methods of biohydrogen production in batch tests. Fresh straw, pre-treated straw and the supernatant residues of acid hydrolization were applied along with the SSF and SHF bioprocess methods to produce biohydrogen in standard batch tests (DIN 38414 S8). Pretreatment with 2% (w/v) sulfuric acid for a 90-min residence time at 120 C proved to be the most effective treatment. Among the 65 treatments studied, tt caused the most significant decrease of DOC in microwave tests. Acid concentration also proved to be a strongly influential factor in decreasing DOC from 6314 mg/L to 5737 mg/L. Concentrations above 2% had a negative effect on DOC extraction from pre-treated materials. Significant differences were also observed between bioprocess methods; for example, the degradation of fresh straw to fermentation gas was less than 1%. The SSF method was found to be an effective and economical way to convert wheat straw to biohydrogen; it
study study study study study
achieved 1 mol H2/mol glucose hydrogen yield, 64% glucose equivalence and 5% carbon degradation (hC, gas) and proved to have the shortest lag phase during gas production of all of the test runs. Supernatants from acid hydrolysis were promising substances for continuous tests of mass biohydrogen production due to their yield of around 1.19 mol H2/mol glucose (76% glucose equivalent) and 52% carbon degradation.
Acknowledgement The authors very much appreciate the valuable cooperation of the Department of Urban Water and Waste Management, University of Duisburg-Essen, who provided the laboratory facilities that were necessary for this research.
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