international journal of hydrogen energy 35 (2010) 40924102

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Hydrogen production from cassava wastewater using an anaerobic sequencing batch reactor: Effects of operational parameters, COD:N ratio, and organic acid composition
Thammanoon Sreethawong a,b, Suchawadee Chatsiriwatana a, Pramoch Rangsunvigit a,b, Sumaeth Chavadej a,b,*
a b

The Petroleum and Petrochemical College, Chulalongkorn University, Soi Chula 12, Phyathai Road, Pathumwan, Bangkok 10330, Thailand Center for Petroleum, Petrochemicals, and Advanced Materials, Chulalongkorn University, Bangkok 10330, Thailand

article info
Article history: Received 5 January 2010 Received in revised form 5 February 2010 Accepted 7 February 2010 Available online 15 March 2010 Keywords: Biohydrogen production Cassava wastewater Anaerobic sequencing batch reactor Cycle time COD:N ratio

abstract
In this work, hydrogen production from cassava wastewater using anaerobic sequencing batch reactors (ASBR) was investigated to determine the optimum number of cycles per day, chemical oxygen demand (COD) loading rate, and COD:N ratio. The system operated at a COD loading rate of 30 kg/m3d and 6 cycles per day provided maximum hydrogen production performance in terms of specic hydrogen production rate (SHPR) (388 ml H2/g VSS d or 3800 ml H2/l d) and hydrogen yield (186 ml H2/g COD removed). The effect of nitrogen supplementation was also studied by adding NH4HCO3 into the system at the COD:N ratios of 100:2.2, 100:3.3, and 100:4.4 under the COD loading rate of 30 kg/m3d and 6 cycles per day. The maximum SHPR and hydrogen yield of 524 ml H2/g VSS d (5680 ml H2/l d) and 438 ml H2/g COD removed, respectively, were obtained at the stoichiometric COD:N ratio of 100:2.2. An excess nitrogen was found to promote the productions of higher organic acids and ethanol, resulting in lowering hydrogen production efciency. 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

At present, the excessive utilization of fossil fuels consequently causes global climate change because of the emission of greenhouse pollutants, including COx, NOx, SOx, CxHy, soot, ash, droplets of tars, and other organic compounds, which are released into the atmosphere as a result of their combustion [1,2]. With regards to energy security and the global environment, the use of hydrogen is a potential way to reduce the greenhouse effect, and hydrogen is considered to be the major energy carrier for the future because of its high conversion capability, recyclability, and non-polluting nature [3]. It has a high energy content of 122 kJ/g, which is 2.75 times greater

than those of hydrocarbon fuels. Moreover, it produces only water as its end-product when it burns [4,5]. Hydrogen can be produced from different raw materials, including fossil fuels, biomass, and water. Particularly, hydrogen production by biological processes is of great interest because they can be operated at ambient temperatures and pressures, leading to lowering energy consumption and becoming more environmentally friendly [6]. Biological hydrogen production can be achieved by using anaerobic (dark fermentation) and photosynthetic (photo-fermentation) processes with carbohydrate-rich biomass as a renewable resource. For the anaerobic fermentation, the rst step is the enzymatic hydrolysis of high molecular weight organics to

* Corresponding author. Tel./fax: 66 2 218 4139. E-mail address: sumaeth.c@chula.ac.th (S. Chavadej). 0360-3199/$ see front matter 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.02.030

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water-soluble organics, and the simple organics are further fermented in the second step to produce volatile fatty acids (VFA), hydrogen, and carbon dioxide [5]. The anaerobic fermentation is more advantageous than the photofermentation since it does not depend on energy provided by sunlight, resulting in a high constant hydrogen generation rate throughout the day and night [7]. Hence, the mass production of hydrogen by anaerobic fermentation using high-strength wastewaters as the substrate has been attracting considerable attention. The use of wastewaters as the substrate for hydrogen production with current anaerobic treatment technology is an effective way of producing clean energy from renewable resources in a sustainable approach. This provides dual benets for the direct wastewater treatment along with the sustainable bio-energy production [6]. Thailand is the worlds largest exporter of cassava starch [8], and about 2.5 million tons of starch were exported in 2009 [9]. In the starch production process, 1 kg of fresh cassava root yields about 0.2 kg of starch, 0.40.9 kg of residue, and about 57 liters of wastewater [10]. Cassava wastewater is a carbohydrate-rich starch waste, and has a very high chemical oxygen demand (COD), biochemical oxygen demand (BOD), and total solids [11]. It has already been proved to have high potential for hydrogen production from starch wastewaters via anaerobic fermentation by using batch reactor, repeatedbatch reactor, anaerobic contact reactor, upow anaerobic sludge blanket (UASB) reactor, and agitated granular sludge bed (AGSB) reactor [1224]. However, to our knowledge, there has been only one work reporting the use of an anaerobic sequencing batch reactor (ASBR) for treating a cassava waste, i.e. cassava stillage, for biogas production [25], and also, hydrogen production from real industry-derived cassava wastewater by using an ASBR has not yet been reported. The ASBR process has several advantages compared to other processes since it is easy to control, has high exibility, and has a relatively low investment cost. This is because the ASBR does not require a separate clarier [26]. In this work, the effects of number of cycles per day, COD loading rate, and COD:N ratio on the biological hydrogen production from cassava wastewater using anaerobic sequencing batch reactors (ASBR) were investigated to obtain the optimum conditions that provide maximum hydrogen production. The correlation between the composition of organic acids produced and the process performance in hydrogen production was also investigated.

used to start up the studied anaerobic sequencing batch reactor (ASBR) units.

2.2.

Feed preparation

The cassava wastewater obtained from a cassava starch production plant (Sahamitr Tapioca Chonburi Ltd., Part., Chonburi, Thailand) was ltered through a 0.2 mm sieve to remove any large solid particles before use. The cassava wastewater, having a chemical oxygen demand (COD) of around 20,000 mg/l or slightly higher, was then freshly prepared to obtain the same constant feed COD of 20,000 mg/l throughout the experiments. The cassava wastewater has an original COD-to-nitrogen-to-phosphorus ratio of 100:0.7 1.9:2.33.9, suggesting that it does not have enough nitrogen for the anaerobic decomposition, but the phosphorus content is sufcient for bacterial growth; therefore, phosphorus supplementation was not required. NH4HCO3, as the nitrogen source, was added to the cassava wastewater to obtain different COD:N ratios: 100:2.2 (stoichiometric ratio) [12], 100:3.3 (50% excess of nitrogen), and 100:4.4 (100% excess of nitrogen), in the second part of the experiments in order to study the effect of nitrogen supplementation.

2.3.

Bioreactor operation

Each of two identical anaerobic sequencing batch reactors (ASBR) was constructed from PVC in order to inhibit the activity of photosynthetic bacteria. The reactor had an inner diameter of 13 cm and a height of 30 cm. The reactor was operated with a working volume of 4 liters. Four sampling ports were installed along the height of the column in order to vary the withdrawal volume. A schematic of the studied ASBR unit used in this work is shown in Fig. 1. An ASBR operation is composed of four sequential steps: feed, react, settle, and decant. The time used for each step is controlled by timers, which allow the pump to feed the wastewater into the top of the reactor during the feeding step. Afterwards, in this present work, the liquid in reactor was homogeneously mixed by using a magnetic stirrer at 400 rpm during the reacting step. The system temperature was controlled by using a thermocouple and a heater that were installed inside the reactor. The pH of the mixed solution was controlled automatically by using a pH controller with a 1 M NaOH solution via a diaphragm pump. The ASBR system was

2.
2.1.

Experimental
Anaerobic seed sludge preparation

pH controller

Temperature controller

The anaerobic sludge sample obtained from the rst anaerobic pond treating a cassava wastewater (Sahamitr Tapioca Chonburi Ltd., Part., Chonburi, Thailand) was rst concentrated by sedimentation, and the concentrated sludge was ground and ltered through a 1 mm sieve to remove debris and large sand particles. After that, it was pretreated by boiling at 95  C for 15 min to eradicate the methane-producing bacteria (or hydrogen consumers) and to enrich the hydrogenproducing bacteria [27]. The heat-treated sludge was then

Feed Reactor Heater Stirrer

Water trap

Gas meter

Effluent

NaOH

H2SO4

Fig. 1 Schematic of the ASBR system used in this work.

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operated at high COD loading rates, corresponding to short hydraulic retention times (HRT) in order to minimize hydrogen consumption by methanogenic bacteria [28]. Two series of experiments were consecutively conducted to investigate the effects of the number of cycles per day and the COD:N ratio on the biohydrogen production. In the rst series, cycle times of 4 and 6 h corresponding to 6 and 4 cycles per day, respectively, were selected based on a practical range of possible operation cycles in a single day. The operation times of the four steps (i.e. feed, react, settle, and decant) for the ASBR operation in the present work are shown in Table 1. The feed and decant ow rates were varied at a constant feed COD of 20,000 mg/l, depending on the number of cycles per day. Therefore, the COD loading rate was varied according to the following equation: Table 2 summarizes the conditions for investigating the

Table 2 Conditions for investigating the effect of number of cycles per day. Number of cycles/day Feed and decant volume (l/cycle)
0.50 0.75 1.00 1.25 0.50 0.75 1.00 1.25

Feed and decant ow rate (l/d)

2 3 4 5 3 4.5 6 7.5

HRT COD (h) loading rate (kg/m3d)

10 15 20 25 15 22.5 30 37.5 48 32 24 19.2 32 21.3 16 12.8

COD loading rate; kg=m d

Feed COD; mg=lFeed flow rate; l=d 1000Working volume; l

(1)

effects of number of cycles per day and COD loading rate. For the 4 cycles per day, the COD loading rate was adjusted at 10, 15, 20, and 25 kg/m3d, with 5 kg/m3d increments, whereas for the 6 cycles per day, the COD loading rate was adjusted at 15, 22.5, 30, and 37.5 kg/m3d, with 7.5 kg/m3d increments. Fang and Liu [29] found that the optimum pH for hydrogen production was observed at a pH value of 5.5. Hence, the system pH in this present work was controlled at this value by using the pH controller, with a 1 M NaOH solution. The operating temperature was controlled at a mesophilic condition of 37  C. In the second series, the optimum conditions obtained in the rst series were used to operate the ASBR units to investigate the effect of nitrogen supplementation by varying the feed COD:N ratio: 100:2.2 (stoichiometric ratio), 100:3.3 (50% excess of nitrogen), and 100:4.4 (100% excess of nitrogen). The system pH was also controlled at 5.5, and the operating temperature was also maintained at 37  C.

distilled samples was analyzed by another gas chromatograph (PR2100, Perichrom) equipped with a ame ionization detector and a DB-WAXetr capillary column (J & W Scientic). The MLVSS (mixed liquor volatile suspended solids) used to represent the microbial concentration in the ASBR units at steady state was measured according to the standard methods [30]. The COD in the feed or efuent sample was determined by the dichromate method using a COD analyzer (DR 2700, HACH). The total nitrogen in the feed solutions was determined by the TNT persulfate digestion method. For each studied condition, the ASBR system was operated about 10 d to reach steady state before taking the data. The steady state condition was justied when the gas production rate and efuent COD were invariant. The experimental data taken at steady state from at least a few runs were averaged and used to assess the process performance of the studied ASBR system.

2.4.

Measurements and analytical methods

3.
3.1.

Results and discussion

Effects of COD loading rate and cycle time

The gas production rate of each ASBR unit was measured using a wet gas meter. The composition of the produced gas was analyzed by a gas chromatograph (AutoSystem GC, PerkinElmer) equipped with a thermal conductivity detector and a stainless-steel HayeSep D 100/120 mesh packed column (Alltech). The total amount of volatile fatty acids (VFA) in the feed and efuent samples was determined by the distillation and titration method [30]. The organic acid composition of the

Table 1 Operating conditions for the ASBR system. Operating parameters

Cycle time (min) Feed React Settle Decant Total

4 cycles/day
15 210 120 15 360

6 cycles/day
15 90 120 15 240

The variation of COD removal efciency with the COD loading rate and the number of cycles per day is depicted in Fig. 2a. For any given cycles per day, the COD removal increased with increasing COD loading rate and reached a maximum. After that, it decreased with further increasing COD loading rate. The COD removal efciency reached the maximum of 67% at a COD loading rate of 20 and 30 kg/m3d for 4 and 6 cycles per day, respectively. The results also suggest that the cassava wastewater is easily degraded anaerobically. The gas production rate at different COD loading rates for both 4 and 6 cycles per day is shown in Fig. 2b. For the system with 4 cycles per day, the gas production rate was nearly constant when the COD loading rate increased from 10 to 20 kg/m3d. After that, it decreased with further increasing COD loading rate from 20 to 25 kg/m3d. When the system was operated at 6 cycles per day, the gas production rate rapidly

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Fig. 2 Effects of COD loading rate and cycle time on (a) COD removal efciency, (b) gas production rate, (c) gas composition, and (d) MLVSS (pH [ 5.5, temperature [ 37 8C, and without nitrogen supplementation).

increased with increasing COD loading rate from 15 to 30 kg/ m3d, but it signicantly decreased at a COD loading rate of 37.5 kg/m3d. The decrease in gas production rate at high COD loading rates was observed in both the 4 and 6 cycles per day systems. The comparative results at different COD loading rates for 4 and 6 cycles per day showed that the gas production rate for the system with 6 cycles per day was much higher than that for the system with 4 cycles per day. This is possibly because the more cycles per day there are, the higher the uniform distribution of organic concentration in the bioreactor, leading to the higher gas production rate. The composition of the produced gas at different COD loading rates for 4 and 6 cycles per day is shown in Fig. 2c. For the system with 4 cycles per day, the percentage of hydrogen in the produced gas increased with increasing COD loading rate from 0.4% at a COD loading rate of 10 kg/m3d to 37.3% at a COD loading rate of 20 kg/m3d. After that, it dropped to 30.8% at a COD loading rate of 25 kg/m3d because of the negative effect of toxicity from volatile fatty acid (VFA) accumulation, which will be discussed later. Methane was detected at low COD loading rates in the range of 1015 kg/m3d for 4 cycles per day since the methanogens in the bioreactor can use both

hydrogen and organic acids to form methane. As expected, the methane content was found to decrease dramatically with increasing COD loading rate. It approached zero level when the COD loading rate increased to be higher than 20 kg/m3d. This is because the system contained a higher VFA concentration at a higher COD loading rate, which will be further discussed later. At 6 cycles per day, the percentages of hydrogen and methane exhibited almost the same trend as those for the system with 4 cycles per day, but slightly shifted to higher COD loading rates. For a maximum hydrogen content, the system operated at 6 cycles per day possessed a higher optimum COD loading rate of 30 kg/m3d, which was higher than that of the system operated at 4 cycles per day (20 kg/m3d). The present results agree well with the work of Chen et al. [31]. They found that the highest hydrogen content in the produced gas from a synthetic sucrose-containing wastewater was obtained when their studied ASBR was operated at 6 cycles per day. The percentage of carbon dioxide slightly varied with COD loading rate, and it was found to have an opposite trend to the hydrogen content for both cycle times. In the case of 6 cycles per day, it reached the minimum value of 58.6% at the same COD

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loading rate of 30 kg/m3d. In a comparison between the two cycle times, the system with 6 cycles per day provided a higher optimum COD loading rate (30 kg/m3d) than that with 4 cycles per day (of which 20 kg/m3d was considered an optimum value). The microbial concentrations in the bioreactor in terms of MLVSS for both 4 and 6 cycles per day operations are shown in Fig. 2d. The MLVSS decreased with increasing COD loading rate at both 4 and 6 cycles per day. The results suggest that an increase in COD loading rate leads to increasing microbial washout from the system because of the increasing toxicity of VFA accumulation, which will be discussed later. Interestingly, for any given COD loading rate, the system with 6 cycles per day possessed a higher microbial concentration than that with 4 cycles per day. This is a reason why the system with higher cycles per day exhibited a better process performance. The dependence of the total VFA concentration in the ASBR on both COD loading rate and cycle time is depicted in Fig. 3a. The total VFA concentration in the ASBR increased substantially with increasing COD loading rate for both 4 and 6 cycles per day. The highest VFA concentrations were obtained at the highest COD loading rates of 25 and 37.5 kg/m3d for the 4 and 6 cycles per day, respectively. The compositions of VFA at

different COD loading rates and cycle times are shown in Fig. 3b and c. It can be noticed that the compositions of VFA varied markedly with an increase in COD loading rate for both 4 and 6 cycles per day. For both cycle times, at any given COD loading rate, the organic acid composition was in the following order: butyric acid > valeric acid > acetic acid > propionic acid. Expectedly, propionic acid (HPr) could be observed throughout the experiments, with lesser amounts than the other organic acids. The HPr concentration increased from 73 mg/l at a COD loading rate of 10 kg/m3d to 360 mg/l at a COD loading rate of 15 kg/m3d for the 4 cycles per day operation. After that, it was nearly constant when the COD loading rate increased from 15 to 25 kg/m3d; whereas, it continuously increased with increasing COD loading rate for the 6 cycles per day operation. It is reported that propionate fermentation is easily carried out either by hydrogenproducing bacteria or by other microbial populations [32,33]. The valeric acid (HVa) concentration rapidly increased from 185 mg/l at a COD loading rate of 10 kg/m3d to 840 mg/l at a COD loading rate of 15 kg/m3d, and afterwards it gradually increased to 1280 mg/l at a COD loading rate of 25 kg/m3d for the 4 cycles per day. For the system with 6 cycles per day, the HVa concentration prole showed the same trend as the HPr

Fig. 3 Effects of COD loading rate and cycle time on (a) total VFA concentration, (b) organic acid and alcohol composition at 4 cycles per day, and (c) organic acid and alcohol composition at 6 cycles per day (pH [ 5.5, temperature [ 37 8C, and without nitrogen supplementation).

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and butyric acid (HBu) concentration proles. The acetic acid (HAc) concentration also increased from 380 to 870 mg/l for the 6 cycles per day. However, for the 4 cycles per day, it initially increased from 155 to 734 mg/l when increasing the COD loading rate from 10 to 15 kg/m3d, but it slightly decreased to 473 mg/l with further increasing the COD loading rate to 25 kg/m3d. For both 4 and 6 cycles per day, the HBu concentration changed most remarkably with increasing COD loading rate, compared to the others. For the 4 cycles per day, a rapid increase in HBu concentration from 533 mg/l at a COD loading rate of 10 kg/m3d to 2110 mg/l at a COD loading rate of 15 kg/m3d was observed, followed by a further increase to 3730 mg/l at a COD loading rate of 25 kg/m3d; however, it even more signicantly increased with increasing COD loading rate for the 6 cycles per day. The results showed that HBu was the major product for both the 4 and 6 cycles per day systems using cassava wastewater as the substrate. It has been reported that a system with high VFA concentrations might lead to the production of alcohols. Under the studied conditions, ethanol (EtOH) was found throughout the experiments in the least amount (less than 10 mg/l), and the EtOH concentration decreased with increasing COD loading

rate or number of cycles per day, suggesting that at a higher COD loading rate, the organics in the cassava wastewater are converted predominantly to hydrogen and carbon dioxide, with organic acids as the main products [34]. The higher the COD loading rate, the higher the contents of higher molecularweight organic acids. The hydrogen production rate calculated from the gas production rate and the gas composition as a function of COD loading rate and cycle time is shown in Fig. 4a. For both 4 and 6 cycles per day, the hydrogen production rate was low at a low COD loading rate due to a low hydrogen percentage in the produced gas and a low gas production rate. It increased with increasing COD loading rate, reached a maximum, and afterwards decreased at a very high COD loading rate. The results can be explained in that at low COD loading rates, an increase in COD loading rate simply increases the amount of organics available for the microbes to convert to hydrogen. However, at high COD loading rates, a decrease in hydrogen production rate is due to the toxicity of VFA accumulation. At a cycle time of 6 cycles per day, the hydrogen production rate reached as high as 0.63 l/h at a COD loading rate of 30 kg/m3d. It can be rapidly increased by 56% when compared with the highest

Fig. 4 Effects of COD loading rate and cycle time on (a) hydrogen production rate, (b) SHPR, (c) hydrogen yield (ml H2/g COD applied), and (d) hydrogen yield (ml H2/g COD removed) (pH [ 5.5, temperature [ 37 8C, and without nitrogen supplementation).

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hydrogen production rate of 0.28 l/h at a COD loading rate of 20 kg/m3d for the system with 4 cycles per day. As discussed above, the number of cycles per day, or cycle time, has a strong impact on the hydrogen production performance of the ASBR. From the results, the greater number of cycles per day can increase the production rate of hydrogen. The reason for this increase in the hydrogen production rate is that an increase in the number of cycles per day can cause less variation in the ASBR conditions. As a result, the microbes can grow more efciently, as shown by the higher microbial concentration (MLVSS) for the 6 cycles per day operation as compared to that for the 4 cycles per day operation (Fig. 2d). The specic hydrogen production rate (SHPR) is also used to express the hydrogen production ability of the bacteria under the studied conditions of the ASBR system. Fig. 4b shows the effects of COD loading rate and cycle time on the SHPR. The maximum SHPR of 388 ml H2/g VSS d (or 3800 ml H2/l d) was obtained at a COD loading rate of 30 kg/m3d for the 6 cycles per day operation since the maximum gas production rate and hydrogen percentage in the produced gas were achieved at these conditions (Fig. 2b and c). The hydrogen yield is a measure of the capability of the microorganisms to convert organics (in terms of COD) to hydrogen gas. The effects of COD loading rate and cycle time on hydrogen yield are shown in Fig. 4c and d. For both 4 and 6 cycles per day, there was a similar trend of the hydrogen yield. Firstly, it increased when the COD loading rate was varied from 1015 kg/m3d to 1522.5 kg/m3d, and then decreased as the COD loading rate was over 20 and 30 kg/m3d for the 4 and 6 cycles per day systems, respectively. By comparing the results of the two cycle times, the optimum hydrogen yield (125 ml H2/g COD applied or 186 ml H2/g COD removed) at a COD loading rate of 30 kg/m3d for the 6 cycles per day operation was higher than that (95 ml H2/g COD applied or 170 ml H2/g COD removed) at a COD loading rate of 15 kg/m3d for the 4 cycles per day operation. From the results, a COD loading rate of 30 kg/m3d and a cycle time of 6 cycles per day, which are considered to be the optimum conditions for the maximum hydrogen production rate, the maximum SHPR, and the maximum hydrogen yield, were selected for further investigation. From the present results as shown in Figs. 24, the correlation between the total VFA and the hydrogen production efciency in terms of hydrogen production rate, specic hydrogen production rates, and hydrogen yields can be used to operate and control this type of bioreactor (ASBR) for hydrogen production. To maximize the hydrogen production from cassava wastewater using the ASBR with pH control at 5.5, the system should be operated to obtain an optimum total VFA of about 7500 mg/l as acetic acid, and this optimum total VFA can be used as a process indicator for hydrogen production for this type of wastewater.

3.2.

Effect of COD:N ratio

A proper COD:N ratio for mixed microora metabolism is necessary to maximize anaerobic hydrogen production from organic wastewater [35]. Therefore, the effect of COD:N ratio was further investigated in this work in order to maximize the biohydrogen production from the cassava wastewater. The effect of COD:N ratio on the COD removal efciency at a COD

loading rate of 30 kg/m3d and a cycle time of 6 cycles per day is shown in Fig. 5a. The COD removal efciency slightly decreased with increasing COD:N ratio. A COD removal efciency of 54% was obtained at the stoichiometric COD:N ratio of 100:2.2. It was somewhat lower than that of the system without nitrogen supplementation (raw cassava wastewater). A possible reason is that the excess nitrogen can alter the biodegradation mechanism as indicated by the increase in VFA, which will be discussed later. For the systems with nitrogen supplementation, the gas production rate slightly decreased with increasing COD:N ratio, from 2.3 l/h at a COD:N ratio of 100:2.2 to 2.1 l/h at a COD:N ratio of 100:4.4, as shown in Fig. 5b. A possible reason is the accumulation of excess ammonium derived from the added nitrogen source in the wastewater since a high ammonium concentration can cause toxicity problems [36]. Interestingly, the maximum gas production rate was found at the stoichiometric COD:N ratio of 100:2.2, which was much higher than that of the system without nitrogen supplementation. This clearly indicates that nutrient supplementation is necessary for acedogenic fermentation [37]. Fig. 5c shows the effect of nitrogen supplementation on produced gas composition. From the results, hydrogen and carbon dioxide were still the main components in the produced gas. Methane was detected at very low levels under the studied conditions. For the systems with nitrogen supplementation, the hydrogen content decreased signicantly with increasing COD:N ratio, from 40.6% at a COD:N ratio of 100:2.227.2% at a COD:N ratio of 100:4.4, whereas the carbon dioxide content increased almost proportionally. One possible reason is that the NH4HCO3 used as the nitrogen source has a carbonate group in the molecule, which can be converted to form carbon dioxide in the produced gas [36]. Another possible reason is that the metabolic pathway of microorganism shifts to the formation of more organic acids and ethanol that tend to induce more carbon dioxide production in the produced gas, as shown next in Fig. 6. The maximum hydrogen percentage was obtained at a COD:N ratio of 100:2.2, which was almost the same as that of the system without nitrogen supplementation. Fig. 5d shows the microbial concentration in the bioreactor in terms of MLVSS at different COD:N ratios. The MLVSS varied insignicantly in the range of 800012,000 mg/l under the studied conditions. The highest MLVSS was obtained at the condition with extremely excess nitrogen supplementation at the COD:N ratio of 100:4.4. This is possibly because the excess nitrogen concentration in the media shifts the microbial metabolism to the cell growth rather than to the gas production (Fig. 5b). The relationship between the total VFA concentration and the COD:N ratio is shown in Fig. 6a. The total VFA concentration increased signicantly with increasing COD:N ratio, from 5450 mg/l at a COD:N ratio of of 100:2.2 to 15,570 mg/l at a COD:N ratio of 100:4.4. The obtained results agree well with the results of Liu and Shen [38]. They reported that the increase in nitrogen concentration caused the increase in VFA production in the liquid media. The increase in total VFA concentration directly leads to the decrease in hydrogen production, as shown next in Fig. 7. The total VFA concentration at the optimum COD:N ratio of 100:2.2 was also found to be lower than that of the system without nitrogen

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a
CO D removal efficiency (%)

100 80 60 40 20 0 Without nitrogen 100:2.2 100:3.3 100:4.4

b
Gas production rate (l/h)

2.5 2 1.5 1 0.5 0 Without nitrogen 100:2.2 100:3.3 100:4.4

COD:N ratio

COD:N ratio

c
Gas composition (%)

100 H2 80 CO2 CH4

d
MLVSS (mg/l)

15000 12000 9000 6000 3000 0

60 40 20 0 Without nitrogen 100:2.2 100:3.3 100:4.4

Without nitrogen

100:2.2

100:3.3

100:4.4

COD:N ratio

COD:N Ratio

Fig. 5 Effect of COD:N ratio on (a) COD removal efciency, (b) gas production rate, (c) gas composition, and (d) MLVSS (COD loading rate [ 30 kg/m3d, cycle time [ 6 cycles per day, pH [ 5.5, and temperature [ 37 8C).

supplementation. Fig. 6b depicts the effect of nitrogen supplementation on VFA composition. When the COD:N ratio increased, the HPr, HBu, and HVa concentrations increased, while the HAc concentration decreased. The HBu was the predominant VFA product (11003850 mg/l), followed by HVa (1702580 mg/l), HAc (329544 mg/l), and HPr (46420 mg/l).

The increase in HBu production accompanying the decrease in HAc production when changing the COD:N ratio from 100:2.2 to 100:4.4 implies that the HAc fermentation via Eq. (2) gradually shifted to the HBu fermentation via Eq. (3) [39]. This leads to the observed decrease in hydrogen composition at a higher COD:N ratio (Fig. 5c).

a
Total VFA concentration (mg/l as acetic acid)

16000

b
Organic acid and alcohol concentration (mg/l)

5000 4000 3000 2000 1000 0

12000

8000

HAc HPr HBu HVa EtOH

4000

0 Without nitrogen 100:2.2 100:3.3 100:4.4

Without nitrogen

100:2.2

100:3.3

100:4.4

COD:N ratio

COD:N Ratio

Fig. 6 Effect of COD:N ratio on (a) total VFA concentration and (b) organic acid and alcohol composition (COD loading rate [ 30 kg/m3d, cycle time [ 6 cycles per day, pH [ 5.5, and temperature [ 37 8C).

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C6 H12 O6 2H2 O/ 2CH3 COOH 2CO2 4H2

Glucose Acetic acid

(2)

C6 H12 O6 / CH3 CH2 CH2 COOH 2CO2 2H2

Glucose Butyric acid

(3)

In addition, the increase in HPr production via Eq. (4) [39], the increase in HVa production via Eqs. (5) and (6) [40], and the increase in EtOH production via Eq. (7) [41] at a higher COD:N ratio resulted in the decrease in hydrogen composition and the increase in carbon dioxide composition (Fig. 5c). C6 H12 O6 2H2 / 2CH3 CH2 COOH 2H2 O
Glucose Propionic acid

(4)

CH3 CH2 COOH CH3 COOH2H2/CH3 CH2 CH2 CH2 COOH2H2 O

Propionic acid Acetic acid Valeric acid

(5) CH3 CH2 COOH 2CO2 6H2 / CH3 CH2 CH2 CH2 COOH 4H2 O
Propionic acid Valeric acid

(6) C6 H12 O6 / 2CH3 CH2 OH 2CO2

Glucose Ethanol

(7)

Therefore, for improving biohydrogen production, the production of HPr, HVa, and EtOH should be minimized or inhibited. At the COD:N ratio of 100:2.2, the lowest HPr and HVa concentrations with zero-level EtOH concentration were experimentally found (Fig. 6b), accompanying the maximum SHPR of 524 ml H2/g VSS d (5676 ml H2/l d) and the highest hydrogen yield of 438 ml H2/g COD removed (Fig. 7). Moreover, apart from the high carbon dioxide composition in the produced gas, partially via EtOH fermentation, the negative effect of EtOH, a kind of reduced product, is possibly because it consumes a large amount of free electrons that are required to form hydrogen [19]. The variation of hydrogen production rate at different COD:N ratios is shown in Fig. 7a. At a COD:N ratio of 100:2.2, the highest hydrogen production rate of 0.95 l/h was achieved since the highest gas production rate (Fig. 5b) and the highest hydrogen composition in the produced gas (Fig. 5c) were obtained. When the COD:N ratio increased, the hydrogen production rate decreased. A possible cause may be the same as explained above for the produced gas composition and the gas production rate. As compared to the system without nutrient supplementation, the hydrogen production rate was greatly enhanced, possibly because the nutrient supplementation increases the bacterial diversity in the bioreactor; in

a
Hydrogen production rate (l/h)

1 0.8

6000 5000 4000

z

ml H2/g VSS d ml H2/l d

SHPR
Without nitrogen 100:2.2 100:3.3 100:4.4

0.6 0.4 0.2 0

3000 2000 1000 0 Without nitrogen 100:2.2 100:3.3 100:4.4

COD:N ratio

COD:N ratio

c
Hydrogen yield (ml H2/g COD applied)

250 200 150 100 50 0 Without 100:2.2 nitrogen 100:3.3 100:4.4

d
Hydrogen yield (ml H2/g COD removed)

500 400 300 200 100 0 Without 100:2.2 nitrogen 100:3.3 100:4.4

COD:N ratio

COD:N ratio

Fig. 7 Effect of COD:N ratio on (a) hydrogen production rate, (b) SHPR, (c) hydrogen yield (ml H2/g COD applied), and (d) hydrogen yield (ml H2/g COD removed) (COD loading rate [ 30 kg/m3d, cycle time [ 6 cycles per day, pH [ 5.5, and temperature [ 37 8C).

international journal of hydrogen energy 35 (2010) 40924102

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particular, the growth of hydrogen-producing bacteria [42]. The effect of nitrogen supplementation on the SHPR is shown in Fig. 7b. The COD:N ratio had a signicant inuence on the SHPR. A maximum SHPR of 524 ml H2/g VSS d (5680 ml H2/l d) was obtained at a COD:N ratio of 100:2.2 because of the high hydrogen production rate (Fig. 7a). The nitrogen content also had a strong effect on the hydrogen yields. The hydrogen yield increased from 186 ml H2/g COD removed (125 ml H2/g COD applied) for the system without nitrogen supplementation to 438 ml H2/g COD removed (235 ml H2/g COD applied) for the system with nitrogen supplementation at a COD:N ratio of 100:2.2, as shown in Fig. 7c and d, respectively; and this was the maximum hydrogen yield obtained in this work.

[6]

[7]

[8]

[9] [10] [11]

4.

Conclusions

[12]

This work investigated the effects of operating parameters on biohydrogen production from cassava wastewater using anaerobic sequencing batch reactors. Without nitrogen supplementation, the systems operated under a COD loading rate of 30 kg/m3d and 6 cycles per day gave a maximum specic hydrogen production rate of 388 ml H2/g VSS d and a maximum hydrogen yield of 186 ml H2/g COD removed. However, when the wastewater was nitrogen-supplemented, the theoretical COD:N ratio of 100:2.2 was found to provide a maximum specic hydrogen production rate of 524 ml H2/ g VSS d and hydrogen yield of 438 ml H2/g COD removed. An excess nitrogen was found to promote higher organic acids and ethanol production, leading to a decreased hydrogen production efciency.

[13]

[14]

[15]

[16]

[17]

Acknowledgments
The authors would like to thank the Sustainable Petroleum and Petrochemicals Research Unit, Center for Petroleum, Petrochemicals, and Advanced Materials, Chulalongkorn University, Ministry of Education, Thailand. In addition, the authors would like to thank Sahamitr Tapioca Chonburi Ltd., Part, Chonburi, Thailand, for kindly providing the seed sludge and cassava wastewater.
[18]

[19]

[20]

references

[21]

[1] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001; 26:1328. [2] Yokoi H, Maki R, Hirose J, Hayashi S. Microbial production of hydrogen from starch-manufacturing wastes. Biomass Bioenerg 2002;22:38995. [3] Lin CY, Jo CH. Hydrogen production from sucrose using an anaerobic sequencing batch reactor process. J Chem Technol Biotechnol 2003;78:67884. [4] Khanal SK, Chen WH, Li L, Sung S. Biological hydrogen production: effects of pH and intermediate products. Int J Hydrogen Energy 2004;29:112331. [5] Argun H, Kargi F, Kapdan IK, Oztekin R. Biohydrogen production by dark fermentation of wheat powder solution:

[22]

[23]

[24]

[25]

effects of C/N and C/P ratio on hydrogen yield and formation rate. Int J Hydrogen Energy 2008;33:18139. Mohan SV, Babu VL, Sarma PN. Anaerobic biohydrogen production from dairy wastewater treatment in sequencing batch reactor (AnSBR): effect of organic loading rate. Enzyme Microb Technol 2007;41:50615. Vijayaraghavan K, Ahmad D, Ibrahim MKB. Biohydrogen generation from jackfruit peel using anaerobic contact lter. Int J Hydrogen Energy 2005;31:56979. International Starch Institute, Science Park Aarhus, Denmark, http://www.starch.dk/isi/prole/home.asp (accessed 5.02.10). Quantity and value of export tapioca starch statistics in 2009, http://www.thaitapiocastarch.org/ (accessed 5.02.10). Plevin R, Donnelly D. Converting waste to energy and prot, tapioca starch power in Thailand; 2004. Sriroth K. Outlook of biomass utilization as biofuel in Thailand; 2005. Production of organic acids from tapioca-starch wastewater. Thailand Institute of Scientic and Technological Research; 1988. Sangyoka S, Reungsang A, Moonamart S. Repeated-batch fermentative for bio-hydrogen production from cassava starch manufacturing wastewater. Pak J Biol Sci 2007;10:17829. Reungsang A, Sangyoka S, Chaiprasert P, Imai T. Factors affecting hydrogen production from cassava wastewater by a co-culture of anaerobic sludge and Rhodospirillum rubrum. Pak J Biol Sci 2007;10:35717. Wang CH, Lu WB, Chang JS. Feasibility study on fermentative conversion of raw and hydrolyzed starch to hydrogen using anaerobic mixed microora. Int J Hydrogen Energy 2007;32: 384959. Lee KS, Hsu YF, Lo YC, Lin PJ, Lin CY, Chang JS. Exploring optimal environmental factors for fermentative hydrogen production from starch using mixed anaerobic microora. Int J Hydrogen Energy 2008;33:156572. Chen SD, Lee KS, Lo YC, Chen WM, Wu JF, Lin CY, et al. Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species. Int J Hydrogen Energy 2008;33:180312. Cheng CH, Hung CH, Lee KS, Liau PY, Liang CM, Yang LH, et al. Microbial community structure of a starch-feeding fermentative hydrogen production reactor operated under different incubation conditions. Int J Hydrogen Energy 2008; 33:52429. Su H, Cheng J, Zhou J, Song W, Cen K. Improving hydrogen production from cassava starch by combination of dark and photo fermentation. Int J Hydrogen Energy 2009;34:17806. Argun H, Kargi F, Kapdan IK. Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation. Int J Hydrogen Energy 2009;34:2195200. Argun H, Kargi F, Kapdan IK. Effects of the substrate and cell concentration on bio-hydrogen production from ground wheat by combined dark and photo-fermentation. Int J Hydrogen Energy 2009;34:61818. Argun H, Kargi F. Effects of sludge pre-treatment method on bio-hydrogen production by dark fermentation of waste ground wheat. Int J Hydrogen Energy 2009;34:85438. Argun H, Kargi F. Effects of light source, intensity and lighting regime on bio-hydrogen production from ground wheat starch by combined dark and photo-fermentations. Int J Hydrogen Energy 2010;35:160412. Wei J, Liu ZT, Zhang X. Biohydrogen production from starch wastewater and application in fuel cell. Int J Hydrogen Energy, in press and corrected proof, doi:10.1016/j.ijhydene. 2009.05.035. Luo G, Xie L, Zhou Q. Enhanced treatment efciency of an anaerobic sequencing batch reactor (ASBR) for cassava

4102

international journal of hydrogen energy 35 (2010) 40924102

[26]

[27]

[28]

[29] [30]

[31]

[32]

[33] [34]

stillage with high solids content. J Biosci Bioeng 2009;107: 6415. Arooj MF, Han SK, Kim SH, Kim DH, Shin HS. Effect of HRT on ASBR converting starch into biological hydrogen. Int J Hydrogen Energy 2008;33:650914. Wang J, Wan W. Comparison of different pretreatment methods for enriching hydrogen-producing bacteria from digested sludge. Int J Hydrogen Energy 2008;33:293441. Hawkes FR, Dinsdale R, Hawkes DL, Hussy I. Sustainable fermentative hydrogen production: challenges for process optimisation. Int J Hydrogen Energy 2002;27:133947. Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresour Technol 2002;82:8793. Eaton AD, Clesceri LS, Rice EW, Greenberg AE. Standard methods for the examination of water and wastewater. Washington, DC: American Public Health Association (APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF); 2005. Chen WH, Sung S, Chen SY. Biological hydrogen production in an anaerobic sequencing batch reactor: pH and cyclic duration effects. Int J Hydrogen Energy 2009;34:22734. Hwang MH, Jang NJ, Hyun SH, Kim IS. Anaerobic biohydrogen production from ethanol fermentation: the role of pH. J Biotechnol 2004;111:297309. Hallenbeck PC. Fundamentals of the fermentative production of hydrogen. Water Sci Technol 2005;52:219. Yang H, Shao P, Lu T, Shen J, Wang D, Xu Z, et al. Continuous bio-hydrogen production from citric acid wastewater via facultative anaerobic bacteria. Int J Hydrogen Energy 2006;31: 130613.

[35] Lin CY, Lay CH. Carbon/nitrogen-ration effect on fermentative hydrogen production by mixed microora. Int J Hydrogen Energy 2004;29:414. [36] Lin CY, Lay CH. Effects of carbonate and phosphate concentrations on hydrogen production using anaerobic sewage sludge microora. Int J Hydrogen Energy 2004;29: 27581. [37] Lee K, Tremblay GH, Levy EM. Bioremediation application of slow-release fertilizers on low energy shorelines. In: Proceedings of the 1993 oil spill conference; 1993, p. 449454. [38] Liu G, Shen J. Effects of culture and medium conditions on hydrogen production from starch using anaerobic bacteria. J Biosci Bioeng 2004;98:2516. [39] Mohan SV, Mohanakrishna G, Sarma PN. Integration of acidogenic and methanogenic processes for simultaneous production of biohydrogen and methane from wastewater treatment. Int J Hydrogen Energy 2008;33:215666. [40] Zhao QB, Yu HQ. Fermentative H2 production in an upow anaerobic sludge blanket reactor at various pH values. Bioresour Technol 2008;99:13538. [41] Xu X, Cao L, Chen X. Elementary ux mode analysis for optimized ethanol yield in anaerobic fermentation of glucose with Saccharomyces cerevisiae. Chin J Chem Eng 2008;16: 13542. [42] O-Thong S, Prasertsan P, Intrasungkha N, Dhamwichukorn S, Birkeland NK. Improvement of biohydrogen production and treatment efciency on palm oil mill efuent with nutrient supplementation at thermophilic condition using an anaerobic sequencing batch reactor. Enzyme Microb Technol 2007;41:58390.