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Isolation of hydrogen generating microflora from cow dung for seeding anaerobic digester
International Journal of Hydrogen Energy 31 (2006) 708 – 720 www.elsevier.com/locate/ijhydene
Isolation of hydrogen generating microflora from cow dung for seeding anaerobic digester Krishnan Vijayaraghavan∗ , Desa Ahmad, Mohd Khairil Bin Ibrahim, Haryati Naemmah Binti Herman Department of Biological and Agricultural Engineering, UPM, 43400 Serdang, Selangor Malaysia Available online 19 September 2005
Abstract The effectiveness of using cow dung as a source for isolating hydrogen generating microflora was investigated under varying isolating conditions based on viz.: pH adjustment and pH adjustment coupled with heat treatment. The viability of the isolated microflora was tested in an anaerobic jar with respect to biogas generation, hydrogen content and pH. The results showed that for pH adjusted microflora isolated from cow dung with solids content at 10% resulted in a cumulative biogas generation of 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml during the anaerobic fermentation for 120 h at a pH of 4, 5 and 6, respectively. The biogas was free from methane when operated at pH 4 and 5, whereas at pH 6 methane generation was observed. In the case of microflora isolated from cow dung with 10% solids, by subjecting to pH adjustment coupled with heat treatment resulted in biogas free from methane content during the fermentation at pH 4, 5 and 6, respectively. At the end of 120 h of fermentation for a reactor pH at 4, 5 and 6 the cumulative biogas generation was 1685, 2610 and 2353 ml, whereas the cumulative hydrogen generation was 509, 1198 and 1165 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with heat treatment. The effect of initial solids content of the cow dung on the isolating efficiency of hydrogen generating microflora was also investigated at pH 5 and 6 coupled with heat treatment. The results revealed that with the increase in initial solids content of the cow dung the optimum heat treatment period also increased as the pH increased from 5 to 6. 䉷 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. Keywords: Biohydrogen; Anaerobic digestion; Cow dung; Isolation; Hydrogen generating microflora
1. Introduction Environmental concern over traditional fossil fuel use is the main driver in the move away from a carbon-based economy. The release of green house gases from the fossil fuel produces global warming and acid rain. In the last 150 years there has been a 1 ◦ C rise in global temperature and scientific consensus is that if left unchecked, the trend will ∗ Corresponding author. Tel.: +60 3 8946 6416; fax : +60 3 8946 6425. E-mail address: [email protected] (K. Vijayaraghavan).
accelerate leading to noticeable problems like flooding and global weather system in chaos. Hence, a transition to a less polluting source of energy will be required to meet these targets where hydrogen is seen as one of the ways to enable this transition [1,2]. Biological methods of hydrogen generation are based on biophotolysis of water by algae and cyanobacteria [3,4], photodecomposition of organic compounds by photosynthetic bacteria [5–8] fermentative hydrogen production from organic compounds and hybrid systems using photosynthetic and fermentative bacteria [9–17]. The advantage of biological hydrogen generation by anaerobic fermentation is that
0360-3199/$30.00 䉷 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2005.07.002
K. Vijayaraghavan et al. / International Journal of Hydrogen Energy 31 (2006) 708 – 720
they are capable of generating hydrogen independent of photoperiod [18]. Clostridial spore formers are selected from natural environments by heat treatment like pasteurizing the activated sludge for two consecutive periods of 20 min at 80 ◦ C [19] or boiling anaerobically digested sludge for 15 min. Both of the above methods resulted in a successful start-up of continuous laboratory-scale reactors feeding on glucose and starch, respectively [20]. The use of aerobically composted activated sludge as inoculum did not show a stable H2 production for 26 days at 2 day HRT and pH 6.8 [21]. Isolation of inoculum for hydrogen generation was successfully carried out by subjecting soil sample and heat-treated compost at 104 ◦ C for 1 h [22]. Gorman [18] isolated hydrogen generating bacteria from tomato plot soil by heating the soil above the boiling point of water for nearly 2 h. If the seed microflora is of predominantly clostridia species, then they may be unable to lower the redox potential to initiate the hydrogen production. Clostridia will be inhibited by oxygen present in the liquid medium or introduced from the headspace by mixing. The inhibition due to oxygen could be tackled by adding reducing agent in the growth medium to lower the redox potential, or by the introduction of the facultative H2 producing aerobe like Enterobacter aerogenes [23]. During the start-up of anaerobic reactor with mixed microflora the headspace was purged with argon, but the effect of this was not studied [24]. Microflora isolated from sewage exhibited hydrogen producing capability [9,11,20,22,25–29]. Sewage cultures exhibited a hydrogen yield of 1.15 mol H2 /mol glucose [30]. Heat-treated microflora resulted in sporulated bacteria. Spore germination into a fully active vegetative cell may require specific nutrients like amino acids. Addition of 0.1% polypeptone was found to be sufficient to break the sporulation of Clostridirum butyricum and mixture of C. butyricum/E. aerogenes which were feeding on sweet potato starch residue under batch operation. It was also found that batch method of reactor operation is not ideal for start-up due to the limitation in substrate [31]. Cohen et al. [19] also stated that interruption of feed could trigger anaerobic spore formers during the start-up phase. A lag period of 2 days occurred in the case of heat-treated sludge consisting of municipal solid waste/sludge mixture [32], where as it was 4 days for microcrystalline cellulose [33]. The effect of pH and intermediate products like volatile fatty acids concentration on the biological hydrogen production was studied using sucrose and starch as substrates. The results showed that specific hydrogen production was highest at an initial pH of 4.5, resulting in 214 ml H2 /g COD (sucrose) and 125 ml H2 /g COD (starch), respectively. The specific hydrogen production rate was found to be high in the pH range of 5.5–5.7 with HAc/HBu ratio 3–4 [34]. The aim of the article is to isolate the hydrogen generating microflora from cow dung. The efficiency of isolation was investigated in anaerobic jar using the isolated microflora
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from the cow dung based on the pH adjustment and pH adjustment coupled with heat treatment for varying initial solids content of the cow dung.
2. Methods and material 2.1. Anaerobic digester set-up The experimental set-up of the anaerobic jar is shown in Fig. 1. The anaerobic digestion experiments were conducted using six anaerobic jars in parallel. The anaerobic unit consists of cylindrical in shape with the following dimension (100 mm ID × 220 mm height). The reactor is of suspended growth type and was operated under continuous flow. 2.2. Substrate The feed composition of anaerobic agar medium (HiMedia Laboratories, India) is shown in Table 1, where as the characteristics of cow dung is shown in Table 2. 2.3. Pretreatment of cow dung The pretreatment of cow dung was carried out before subjecting the sample to isolation test by filtering through sieve having a pore opening of 2 mm. 2.4. Analytical process The organic strength of the cow dung was determined in terms of volatile solids. The total solids and volatile solids were determined by drying the sample at 105 ◦ C in oven and 550 ± 50 ◦ C in the furnace, respectively. The total nitrogen was determined by Kjeldhal method whereas volatile fatty acid (VFA) content was determined by distillation method [35]. The hydrogen and methane content in the biogas was determined by Drager Method [36]. 2.5. Isolation of seed microflora from cow dung The isolation experiments were carried out in order to kill the methanogenic organism and other hydrogen consuming bacteria present in the cow dung. The isolation tests were conducted in the following methods viz.: (1) pH adjustment and (2) pH adjustment coupled with heat treatment. The above experiments were conducted in duplicate. 2.6. Anaerobic jar The microflora isolated from two different conditions viz.: (a) pH corrected and (b) pH correction followed by heat treatment was tested for its ability to generate hydrogen in the anaerobic jar. The viability of the microflora was
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2.7. Isolation and investigation of seed microflora based on pH adjustment and pH adjustment coupled with heat treatment
1 2
90 mm
3 4
130 mm
5
100 mm 1. Inlet
4. Outlet
2. Gas outlet 3. Gas collection zone
5. Digestion zone
Fig. 1. Schematic diagram of anaerobic jar. 1. Inlet, 2. gas outlet, 3. gas collection zone, 4. outlet and 5. digestion zone. Table 1 Characteristics of anaerobic agar Parametersa
Concentration
pH Casein enzymic hydrolysate Dextrose Sodium chloride Sodium thioglycollate Sodium formaldehyde sulphoxylate Methylene blue Agar
7.0 ± 0.2 2 1 0.5 0.2 0.1 0.0002 2
a Except
pH all other parameter are in gram per litre.
Table 2 Characteristics of cow dung Parametersa
Concentration
pH Total solids Volatile solids Total nitrogen
7.1 ± 3 13 ± 2 68 ± 3 0.70 ± 0.04
a Except
pH all parameters are in percentage (%).
investigated with respect to digestion pH, VFA, biogas generated, hydrogen and methane content.
Isolation of hydrogen generating microflora was carried out by subjecting the 10% solids content of cow dung to varying initial pH viz. 4, 5 and 6 for a retention time 3 h. The microflora resulting from this treatment process was tested for its hydrogen generating capability in the anaerobic jar. In the case of isolating microflora based on pH adjustment and heat treatment, the pH of the cow dung was maintained at 4, 5 and 6, respectively, for a retention time of 3 h. Thereafter it was subjected to heat treatment in a water bath at 105 ◦ C for 1 h, for two consecutive times at an interval of 30 min. The microflora resulting after the heat treatment was tested for its hydrogen generating capability in anaerobic jar. The anaerobic fermentation was carried out by transferring 800 ml of microflora into the anaerobic jar to which 200 ml (6%) anaerobic agar is feed as substrate and maintained at their respective operating pH. 2.8. Isolation of hydrogen generating microflora from varying solids content of cow dung at pH 5 coupled with heat treatment The effect of initial solids content of cow dung in isolating hydrogen generating bacteria was investigated at pH 5 coupled with heat treatment at 2, 3 and 4 h, respectively. The solids content of cow dung was varied in the following concentration of 10%, 12% and 15%, respectively. The reason for choosing the pH 5 is that at this pH the biogas generation had a maximum value for microflora isolated based on pH adjustment coupled with heat treatment when compared at pH 4 and 6. 2.9. Isolation of hydrogen generating microflora from varying solids content of cow dung at pH 6 coupled with heat treatment The efficiency of isolating hydrogen generating microflora from cow dung was investigated at a solids content of 12% and 15% at pH 6 coupled with heat treatment. Since the hydrogen generation for a pH adjusted cow dung microflora at 5 and 6 had a marginal difference. The effect of initial solids content of cow dung in isolating hydrogen generating microflora was investigated at pH 6 coupled with heat treatment. 3. Results and discussion 3.1. Investigation of isolated seed microflora from cow dung based on pH adjustment and pH adjustment coupled with heat treatment The microflora isolated from cow dung after subjecting to pH adjustment and pH adjusted coupled with heat treatment was tested in the anaerobic jar having a capacity of 1 l.
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Cumulative biogas generation (ml/h)
3500 Heat treatment period & Fermentation pH
3000
4 pH 5 pH
2500
6 pH 2 h at 4 pH
2000
2 h at 5 pH 2 h at 6 pH
1500
Cow dung solids content : 10%
1000
500
0 0
20
40
60
80
100
120
140
Time (h) Fig. 2. Effect of cumulative biogas generation versus time (pH adjusted and pH adjusted coupled with heat-treated microflora from cow dung at 10% solids).
The anaerobic fermentation was commenced using 800 ml of isolated microflora from cow dung to which 200 ml of anaerobic agar medium was fed as the substrate. The substrate addition was kept in a continuous mode throughout the investigation at a flow rate of 200 ml/day. The reason for keeping the feed rate as a continuous flow is the substrate should not be limiting factor. Chen at al. [37] stated that hydrogen production in a batch culture seems to be limited when cell growth was thriving mainly due to rapid depletion of carbon substrate for the gain in biomass. Thus, continuous process could be favourable for hydrogen production due to a sufficient supply of carbon substrate. Hence continuous feed flow was used in this present investigation. The effect of biogas generation is shown in Fig. 2. In the case of pH adjusted cow dung microflora the biogas generation was found to be increasing in the following order of digester pH of 4, 5 and 6. At the end of 120 h of operation the cumulative biogas generation was found to be 1494, 2404 and 3327 ml during the anaerobic fermentation at pH 4, 5 and 6, respectively. In the case of pH adjusted coupled with heat-treated cow dung microflora, the cumulative biogas generation was found to be low at pH 4, whereas a maximum biogas generation was recorded at pH 5 followed by pH 6. At the end of 120 h of operation at pH 4, 5 and 6 the cumulative biogas production was found to be 1685, 2610 and 2353 ml, respectively. As shown in Fig. 2 the lag phase was longer for microflora isolated form cow dung which when subjected to pH adjustment coupled with heat treatment, whereas for the pH adjusted cow dung the lag phase was less. The pH ad-
justed microflora operating at 4, 5 and 6 pH resulted in a lag period of 24, 12 and 6 h, respectively. Whereas in the case of microflora isolated by subjecting the cow dung to pH adjustment followed by heat treatment at pH 4, 5 and 6 resulted in a lag period of 42, 24 and 30 h, respectively. The possible reason for the longer lag phase in the case of microflora isolated from pH adjustment followed by heat treatment than pH adjustment alone could be due to sporulation. The reason for carrying out the heat treatment on cow dung was to kill or suppress the methanogenic bacteria. Zoetemeyer et al. [44], Horiuchi et al. [45] as such stated that biohydrogen generation from anaerobic fermentation relies on the disruption of methanogenic archaea since they are significant hydrogen consumer microbial group in anaerobic consortia. Several methods are available for suppressing the growth of hydrogen consuming bacteria, for example, use of chemical inhibitors such as acetylene and bromoethane sulphonic acid [14]; heat shock pretreatment of inocula [44,38–40] and maintain the cultures at acidogenic range (5.8–6.5) [25,26]. The heat shock pretreatment relies on the killing or thermal suppression of methanogenic archaea and non-sporulating bacteria thereby enriching the culture with sporulating hydrogen producing bacteria such as Clostridia [41]. Fig. 3 shows that cumulative hydrogen generation during the anaerobic fermentation using microflora isolated from pH adjustment and pH adjustment coupled with heat treatment. Comparison of Figs. 2 and 3 shows that for pH adjusted sample even though the biogas generation is high, the corresponding hydrogen generation was found to be less. At the end of 120 h of anaerobic fermentation for a pH adjusted
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Cumulative hydrogen generation (ml/h)
1400 Heat treatment period & Fermentation pH
1200
4 pH 5 pH 6 pH 2 h at 4 pH 2 h at 5 pH 2 h at 6 pH
1000 800
Cow dung solids content : 10%
600 400 200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 3. Effect of cumulative hydrogen generation versus time (pH adjusted and pH adjusted coupled with heat-treated microflora from cow dung at 10% solids).
2000 Fermentation pH
Cumulative methane generation (ml/h)
1800 4 5 6
1600 1400
Cow dung solids content : 10%
1200 1000 800 600 400 200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 4. Effect of cumulative methane generation versus time (pH adjusted microflora from cow dung at 10% solids).
microflora at a pH 4, 5 and 6 the cumulative biogas generation was found to be 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with
heat treatment. Earlier researchers have shown that operating anaerobic fermentation process between pH 5 and 6 attained the highest total hydrogen production [10,22,28,29,42,43]. The possible reason for low hydrogen generation at pH 6 is due to generation of methane as shown in Fig. 4. In the case of pH adjusted microflora at 4 and 5 the methane
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1600 Heat treatment period & Fermentation pH
Volatile fatty acid (mg/l)
1400
4 pH 5 pH 6 pH 2 h at 4 pH 2 h at 5 pH 2 h at 6 pH
1200 1000 800 600 400 200
Cow dung solids content : 10%
0 0
20
40
60
80 Time (h)
100
120
140
Fig. 5. Volatile fatty acid content versus time (pH adjusted and pH adjusted coupled with heat-treated microflora from cow dung at 10% solids).
generation was found to be below detectable limit, whereas at pH 6 the cumulative methane generation was found to be 1840 ml at a fermentation period of 120 h. The optimum pH for methanogenic bacteria was found to be 6.0–7.5 [46–49]. Hence operating anaerobic fermentation at pH 6 using cow dung as the seed microorganism does not seem to be sound method for generating hydrogen as methanogenic bacteria can still dominate at this operating pH. Moreover operating at pH 6 revealed that hydrogen generation still occurred but the concentration was low which could be due to utilization of hydrogen as substrate by the methanogenic bacteria. In the case of operating the anaerobic fermentation at pH 4 and 5, using pH adjusted microflora resulted in a methane concentration below detectable limit, whereas the hydrogen concentration showed a gradual build-up as the fermentation proceeded. Hence it can be concluded that methanogenic population was being inhibited or killed at lower pH value. Similar observations were made by Ueno et al. [15] stating that acidogenic conditions were most suitable for hydrogen generation from anaerobic fermentative consortia, a fact corroborated later by Lin and Chang [25], Lay [20], Ueno et al. [24], Fang et al. [50]. The low pH could be attained either by pH adjustment or due to the overloading of the reactor which in turn led to the accumulation of organic acids resulting in drop in pH value [22,26]. In the case of pH adjusted coupled with heat-treated microflora isolated from cow dung, the methane content was found to be below detectable limit for pH 4, 5 and 6, respectively. The corresponding cumulative hydrogen generation at pH 4, 5 and 6 was found to be 509, 1198 and 1165 ml at
the end of 120 h of fermentation period. As shown in Fig. 3 the cumulative hydrogen generation for microflora isolated from pH adjusted cow dung was relatively less when compared to the microflora isolated from cow dung by subjecting them to pH adjustment coupled with heat treatment. The possible reason for the rise in the hydrogen generation could be due to destruction of hydrogen consuming bacteria during heat treatment. Lay et al. [51] and Oh et al. [52] investigation revealed that heat shock pretreatment inhibited or killed Methanogenic Archaea and lactic bacteria. Lactic bacteria can impair hydrogen production because they produce toxin that inhibit several subgroups of hydrogen generating consortium [53]. The volatile fatty acid content during the anaerobic fermentation for pH adjusted microflora and pH adjusted coupled with heat-treated microflora from cow dung is shown in Fig. 5. In the case of pH adjusted microflora at 4, 5 and 6 the VFA content during the fermentation process was found to be 870, 814 and 505 mg/l at 48 h, whereas at 120 h it was found to be 1404, 1203 and 733 mg/l, respectively. In the case of pH 4 even though the VFA content is high the biogas generation was found to be low. The possible reason for this type of behaviour could be due to the accumulation of organic acids leading to overall process inhibition [54] or a drop in reactor pH below 5.8 could switch over the reaction from acidogenesis to solventogenesis (a hydrogen consuming pathway) [55]. On comparison of anaerobic fermentation at pH 5 and 6 it can be seen that at pH 5 there is an accumulation of VFA when compared to pH 6. At pH 5 the methanogenic bacteria are subjected to inhibi-
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3000
Cumulative biogas generation (ml/h)
Heat treatment period & Cow dung solids content
2500
2 h at 10% 2 h at 12% 2 h at 15% 3 h at 10% 3 h at 12% 3 h at 15% 4 h at 10% 4 h at 12% 4 h at 15%
2000
1500
1000
Fermentation pH 5.1 ± 0.1
500
0 0
20
40
60
80
100
120
140
Time (h) Fig. 6. Effect of cumulative biogas generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 5.1 ± 0.1 coupled with heat treatment).
tion which could have lead to the accumulation of intermediate metabolic product, but that did not affect the biogas generation as shown in Fig. 2. In the pH range of 5.5–6 both methanogenesis and solventogenesis can be avoided or minimized [26,55]. At pH 6 there was a marginal increase in VFA content and this could be due to the utilization of metabolic intermediates in biogas generation thereby preventing their accumulation. In the case of pH adjusted coupled with heat-treated microflora at pH 4, 5 and 6 resulted in VFA content of 792, 698 and 720, respectively, at 48 h. Whereas at 120 h the VFA content was found to be 1289, 989 and 1117, respectively. The low biogas generation at pH 4 as shown in Fig. 2 could be due to the accumulation of VFA resulting in solventogenesis at pH 4. In the case of pH 5 even though there is a rise in VFA content in the reactor, there was a corresponding rise in biogas generation when compared with pH 6 which resulted in low VFA content and biogas generation. The rise in VFA content could be due to the existence of microflora in exponential growth phase, as the biogas generation showed a gradual increasing trend. Earlier researcher have stated a volatile fatty acid content of 1250 ± 87 mg/l while treating food waste under mesophilic condition in the pH range of 4.5–6.5 [56], whereas Chen et al. [37] stated a VFA content of 810 mg/l for a fermentation period of 33 h using sucrose as substrate. Isolating hydrogen generating microflora from cow dung having a solids content of 10%, by subjecting to pH adjustment at 5 and 6 coupled with heat treatment at 2, 3 and 4 h did not exhibit autotrophic acetogenesis as the hydrogen generation was found to be in increasing trend. The
autotrophic acetogenesis occurs during anaerobic fermentation at low pH, when the seed microflora is subjected to heat treatment at below the optimum level. In our present investigation since we did not experience loss in hydrogen content, it can be concluded that autotrophic acetogenesis did not occur in the present isolating conditions for the given solids content and heat treatment period. 3.2. Efficiency of isolating hydrogen generating microflora from cow dung based on pH adjustment at 5 coupled with heat treatment The efficiency of isolating hydrogen generating microflora from cow dung was investigated at pH 5 by varying the initial solids content (10%, 12% and 15%) with respect to heat treatment period (2, 3 and 4 h). Fig. 6 shows the biogas generation when the cow dung is pretreated by adjusting the initial pH to 5 and then subjecting them to 2, 3 and 4 h heat treatment. When the heat treatment was carried out 2, 3 and 4 h the lag phase was found to be 30, 36 and 60 h, respectively, irrespective of initial microflora solids content. The reason for this kind of behaviour could be due to sporulation when the heat treatment was carried out for longer period. The biogas generation was found be increasing with the increase in solids content of the cow dung, probably due to increase in biomass and substrate concentration. The cumulative biogas generation was found to be marginally high for heat-treated cow dung microflora at 3 h rather than 2 h for their corresponding solids content at 10%, 12% and 15%, respectively. In the case of heat-treated cow dung for a period of 4 h resulted in low cumulative
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1600 Heat treatment period & Cow dung solids content
Cumulative hydrogen generation (ml/h)
1400
2 h at 10% 2 h at 12% 2 h at 15% 3 h at 10% 3 h at 12% 3 h at 15% 4 h at 10% 4 h at 12% 4 h at 15%
1200 1000 800 600
Fermentation pH 5.1 ± 0.1
400 200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 7. Effect of cumulative hydrogen generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 5.1 ± 0.1 coupled with heat treatment).
biogas generation, which could be due to the longer lag phase. The cumulative hydrogen generation during the isolation of hydrogen generating microflora based on heat treatment duration for a constant pH of 5 is shown in Fig. 7. In the case of heat treatment at a duration of 2 and 3 h, the hydrogen generation was more or less the same for a given solids content of 10%, 12% and 15%, respectively. In the case of 4 h heat treatment the cumulative hydrogen generation was low in comparison with 2 and 3 h heat-treated cow dung. During a fermentation period of 120 h for a microflora isolated from at 15% solids content of cow dung using pH adjustment at 5 coupled with heat treatment for a period of 2, 3 and 4 h resulted in a cumulative hydrogen generation of 1305, 1400 and 734 ml, respectively. The results showed that biogas was free from methane, with a maximum hydrogen content of 63 ± 3% at a heat treatment period for 2 and 3 h, whereas at 4 h heat treatment it was 55 ± 2%. The low value of cumulative hydrogen generation for a heat treatment period of 4 h could be due to the longer lag phase as a result of sporulation. The volatile fatty acid content is shown in Fig. 8. At the end of 24 h fermentation period for a heat-treated microflora at 2 h the VFA content was found to have higher value as shown in Fig. 8, when compared to heat-treated microflora and 3 and 4 h, respectively. For example at a fermentation period of 24 h for 10% and 15% microflora when subjected to 2 h heat treatment resulted in a VFA content of 580 and 710 mg/l, whereas in the case of 4 h heat treatment it was 490 and 550 mg/l. The higher value of VFA for 2 h heat treatment
microflora could be due to shorter (24 h) lag phase when compared with 4 h heat treatment microflora, where the lag phase was found to be 60 h. In the case of fermentation period at 120 h for 10% and 15% microflora when subjected to 2 h heat treatment resulted in a VFA content of 820 and 950 mg/l, whereas in the case of 4 h heat treatment it was 917 and 1081 mg/l. In the case of 4 h heat-treated microflora there was a considerable rise in VFA content when compared between a fermentation period of 24 and 120 h. This could be due to the presence of microflora in the earlier exponential phase at 24 h whereas at 120 h it was in the exponential phase. Even though there was increase in VFA content with the fermentation period, it did not exhibit any inhibition to the biogas generation as shown in Fig. 6. Therefore it can be concluded that VFA even as high as 1081 mg/l was able to produce stable biogas generation. 3.3. Efficiency of isolating hydrogen generating microflora from cow dung based on pH adjustment at 6 coupled with heat treatment The efficiency of isolating hydrogen generating microflora from cow dung was investigated at an initial solids content of 12% and 15% by subjecting to pH adjustment at 6 coupled with heat treatment at 2, 3 and 4 h, respectively. The cumulative biogas generation during anaerobic fermentation using the isolated microflora is shown in Fig. 9. As the heat-treatment period increased the lag phase also increased, the cumulative biogas generation was high for 2 h heat-treated microflora than 3 and 4 h, which could be
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1600 2 h at 10% 2 h at 12% 2 h at 15% 3 h at 10% Heat treatment period & Cow dung 3 h at 12% solids content 3 h at 15% 4 h at 10% 4 h at 12% 4 h at 15%
1400
Volatile fatty acid (mg/l)
1200 1000 800 600 400
Fermentation pH 5.1 ± 0.1
200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 8. Volatile fatty acids content versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 5.1 ± 0.1 coupled with heat treatment).
3000
Cumulative biogas generation (ml/h)
Heat treatment period & Cow dung solids content
2500
2 h at 12% 2 h at 15% 3 h at 12%
2000
3 h at 15% 4 h at 12% 4 h at 15%
1500
Fermentation pH 6.1 ± 0.1
1000
500
0 20
30
40
50
60
70
80
90
100
110
120
Time (h)
Fig. 9. Effect of cumulative biogas generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
due to the sporulation of bacteria and spore breaking taking a longer time as reflected in the long lag phase when the heat treatment period increased to 3 and 4 h, respectively. In the case of initial microflora solids content of 12% and
15% when subjected to pH adjustment at 6 coupled with heat treatment for a period of 2, 3 and 4 h resulted in a lag period of 36, 48 and 60 h, respectively. In the case of isolating microflora from cow dung with an initial solid
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Cumulative methane generation (ml/h)
1600 Heat treatment period & Cow dung solids content
1400
2 h at 12% 2 h at 15%
1200
Fermentation pH 6.1 ± 0.1
1000 800 600 400 200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 10. Effect of cumulative methane generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
Cumulative hydrogen generation (ml/h)
1000 Heat treatment period & Cow dung solids content
900
2 h at 12% 2 h at 15% 3 h at 12% 3 h at 15% 4 h at 12% 4 h at 15%
800 700 600 500
Fermentation pH 6.1 ± 0.1
400 300 200 100 0 0
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Time (h) Fig. 11. Effect of cumulative hydrogen generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
content of 12% and 15% by subjecting to pH adjustment at 6 coupled with heat treatment for a period of 2, 3 and 4 h resulted in a lag period of 36, 28 and 60 h, respectively. The cumulative methane generation was investigated using isolated microflora from cow dung having a solids con-
tent of 12% and 15% at pH 6 by subjecting them to varying heat treatment period viz. 2, 3 and 4 h, respectively. In the case of microflora isolated from cow dung having a solids content of 12% resulted in biogas free from methane for varying heat treatment period as stated above. As the
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1000 900
Volatile fatty acid (mg/l)
800 700 600 Heat treatment period & Cow dung solids content
500
2 h at 12% 2 h at 15% 3 h at 12% 3 h at 15% 4 h at 12% 4 h at 15%
400 300 200 100
Fermentation pH 6.1 ± 0.1
0 0
20
40
60
80
100
120
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Time (h) Fig. 12. Effect of cumulative hydrogen generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
solids content of the microflora increased to 15%, coupled with heat treatment for 2 h resulted in biogas with methane (Fig. 10). In the case of microflora with 15% solids content when subjected to heat treatment period for 3 and 4 h, respectively, the biogas was free from methane. The possible reason for this type of behaviour could be due to the presence of high solids (15%), which might act as shield to protect the methanogenic bacteria from heat shock. Moreover at pH 6 the methanogenic bacteria were subjected to less stress, as this pH was well within the optimum level for the bacterial growth. The cumulative hydrogen generation is shown in Fig. 11. In the case of heat-treated microflora for 2 h at pH 6 with solids content of 12% and 15% resulted in a cumulative hydrogen generation 866 and 189 mg/l, respectively, for a fermentation period of 120 h. The maximum hydrogen content was found to be 49 ± 2% in the case of 12% solids content. Whereas during the initial stages of fermentation at pH 6 for the microflora isolated form cow dung subjected to 2 h heat treatment with a solids content of 15% resulted in a hydrogen content of 19 ± 1% till 72 h of fermentation, thereafter it decreased to 3 ± 1% hydrogen at 120 h. The low hydrogen generation at 15% solids content could be due to utilization of hydrogen as a substrate by the methanogenic bacteria for generating methane as reflected in Fig. 10. Therefore heat treatment for a period of 2 h at pH 6 was not sufficient to kill or inhibit the methanogenic bacteria. In the case of microflora when isolated at pH 6 by subjected to heat treatment
for a period of 3 and 4 h for a solids content at 12% and 15% cow dung, the biogas was free from methane. The maximum hydrogen content was found to be 47 ± 1% and 50 ± 2% in the case of 12% and 15% solids content irrespective of heat treatment period viz. 3 and 4 h, respectively. The rise in hydrogen generation could be due to the inhibitory effect of heat treatment on methanogenic bacteria, as reflected in the case of microflora isolated from cow dung having a solids content of 15% at pH 6 above a heat treatment period of 2 h. The volatile fatty acid content during the anaerobic fermentation using microflora isolated from cow dung when subjected to pretreatment at pH 6 coupled with heat treatment at 2, 3 and 4 is shown in Fig. 12. At a fermentation period of 24 h with a microflora having a solids content of 12% which when subjected to pH adjustment at 6 followed by heat treatment at 2, 3 and 4 h, resulted in a VFA content of 510, 528 and 520 mg/l. Whereas for a fermentation period of 120 h for the above condition resulted in a VFA content of 690, 710 and 780 mg/l, respectively. In the case of microflora isolated from cow dung having a solids content of 15% resulted in a VFA content of 530, 575 and 547 during 24 h fermentation period, whereas VFA content was found to be 802, 890 and 902 mg/l, respectively, at 120 h of fermentation period. Even though there was a rise in VFA content with the increase in fermentation period, increase in VFA content did not affect the fermentation process as the biogas generation and hydrogen content showed an increasing trend.
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4. Conclusions The hydrogen generating microflora isolated from cow dung based on pH adjustment coupled with heat treatment proved to be advantageous when compared with pH adjustment alone. The results showed that for pH adjusted microflora isolated from cow dung with solids content at 10% resulted in a cumulative biogas generation of 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml during the anaerobic fermentation for 120 h at a pH 4, 5 and 6, respectively. The biogas was free from methane when operated at pH 4 and 5, whereas at pH 6 methane generation was observed. For the above condition in the case of microflora isolated by subjecting to pH adjustment coupled with heat treatment was free from methane with a cumulative biogas generation of 1685, 2610 and 2353 ml, while the cumulative hydrogen generation was 509, 1198 and 1165 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with heat treatment. The effect of initial solids content of the cow dung on the isolating efficiency of hydrogen generating microflora was also investigated at pH 5 and 6 coupled with heat treatment. The results revealed that with the increase in initial solids content of the cow dung the optimum heat treatment period also increased as the pH increased from 5 to 6.
Acknowledgements This research was supported by the Fundamental Research Grant of Universiti Putra Malaysia, Project Number : 02-03-03-057J / 55180.
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Isolation of hydrogen generating microflora from cow dung for seeding anaerobic digester Krishnan Vijayaraghavan∗ , Desa Ahmad, Mohd Khairil Bin Ibrahim, Haryati Naemmah Binti Herman Department of Biological and Agricultural Engineering, UPM, 43400 Serdang, Selangor Malaysia Available online 19 September 2005
Abstract The effectiveness of using cow dung as a source for isolating hydrogen generating microflora was investigated under varying isolating conditions based on viz.: pH adjustment and pH adjustment coupled with heat treatment. The viability of the isolated microflora was tested in an anaerobic jar with respect to biogas generation, hydrogen content and pH. The results showed that for pH adjusted microflora isolated from cow dung with solids content at 10% resulted in a cumulative biogas generation of 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml during the anaerobic fermentation for 120 h at a pH of 4, 5 and 6, respectively. The biogas was free from methane when operated at pH 4 and 5, whereas at pH 6 methane generation was observed. In the case of microflora isolated from cow dung with 10% solids, by subjecting to pH adjustment coupled with heat treatment resulted in biogas free from methane content during the fermentation at pH 4, 5 and 6, respectively. At the end of 120 h of fermentation for a reactor pH at 4, 5 and 6 the cumulative biogas generation was 1685, 2610 and 2353 ml, whereas the cumulative hydrogen generation was 509, 1198 and 1165 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with heat treatment. The effect of initial solids content of the cow dung on the isolating efficiency of hydrogen generating microflora was also investigated at pH 5 and 6 coupled with heat treatment. The results revealed that with the increase in initial solids content of the cow dung the optimum heat treatment period also increased as the pH increased from 5 to 6. 䉷 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. Keywords: Biohydrogen; Anaerobic digestion; Cow dung; Isolation; Hydrogen generating microflora
1. Introduction Environmental concern over traditional fossil fuel use is the main driver in the move away from a carbon-based economy. The release of green house gases from the fossil fuel produces global warming and acid rain. In the last 150 years there has been a 1 ◦ C rise in global temperature and scientific consensus is that if left unchecked, the trend will ∗ Corresponding author. Tel.: +60 3 8946 6416; fax : +60 3 8946 6425. E-mail address: [email protected] (K. Vijayaraghavan).
accelerate leading to noticeable problems like flooding and global weather system in chaos. Hence, a transition to a less polluting source of energy will be required to meet these targets where hydrogen is seen as one of the ways to enable this transition [1,2]. Biological methods of hydrogen generation are based on biophotolysis of water by algae and cyanobacteria [3,4], photodecomposition of organic compounds by photosynthetic bacteria [5–8] fermentative hydrogen production from organic compounds and hybrid systems using photosynthetic and fermentative bacteria [9–17]. The advantage of biological hydrogen generation by anaerobic fermentation is that
0360-3199/$30.00 䉷 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2005.07.002
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they are capable of generating hydrogen independent of photoperiod [18]. Clostridial spore formers are selected from natural environments by heat treatment like pasteurizing the activated sludge for two consecutive periods of 20 min at 80 ◦ C [19] or boiling anaerobically digested sludge for 15 min. Both of the above methods resulted in a successful start-up of continuous laboratory-scale reactors feeding on glucose and starch, respectively [20]. The use of aerobically composted activated sludge as inoculum did not show a stable H2 production for 26 days at 2 day HRT and pH 6.8 [21]. Isolation of inoculum for hydrogen generation was successfully carried out by subjecting soil sample and heat-treated compost at 104 ◦ C for 1 h [22]. Gorman [18] isolated hydrogen generating bacteria from tomato plot soil by heating the soil above the boiling point of water for nearly 2 h. If the seed microflora is of predominantly clostridia species, then they may be unable to lower the redox potential to initiate the hydrogen production. Clostridia will be inhibited by oxygen present in the liquid medium or introduced from the headspace by mixing. The inhibition due to oxygen could be tackled by adding reducing agent in the growth medium to lower the redox potential, or by the introduction of the facultative H2 producing aerobe like Enterobacter aerogenes [23]. During the start-up of anaerobic reactor with mixed microflora the headspace was purged with argon, but the effect of this was not studied [24]. Microflora isolated from sewage exhibited hydrogen producing capability [9,11,20,22,25–29]. Sewage cultures exhibited a hydrogen yield of 1.15 mol H2 /mol glucose [30]. Heat-treated microflora resulted in sporulated bacteria. Spore germination into a fully active vegetative cell may require specific nutrients like amino acids. Addition of 0.1% polypeptone was found to be sufficient to break the sporulation of Clostridirum butyricum and mixture of C. butyricum/E. aerogenes which were feeding on sweet potato starch residue under batch operation. It was also found that batch method of reactor operation is not ideal for start-up due to the limitation in substrate [31]. Cohen et al. [19] also stated that interruption of feed could trigger anaerobic spore formers during the start-up phase. A lag period of 2 days occurred in the case of heat-treated sludge consisting of municipal solid waste/sludge mixture [32], where as it was 4 days for microcrystalline cellulose [33]. The effect of pH and intermediate products like volatile fatty acids concentration on the biological hydrogen production was studied using sucrose and starch as substrates. The results showed that specific hydrogen production was highest at an initial pH of 4.5, resulting in 214 ml H2 /g COD (sucrose) and 125 ml H2 /g COD (starch), respectively. The specific hydrogen production rate was found to be high in the pH range of 5.5–5.7 with HAc/HBu ratio 3–4 [34]. The aim of the article is to isolate the hydrogen generating microflora from cow dung. The efficiency of isolation was investigated in anaerobic jar using the isolated microflora
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from the cow dung based on the pH adjustment and pH adjustment coupled with heat treatment for varying initial solids content of the cow dung.
2. Methods and material 2.1. Anaerobic digester set-up The experimental set-up of the anaerobic jar is shown in Fig. 1. The anaerobic digestion experiments were conducted using six anaerobic jars in parallel. The anaerobic unit consists of cylindrical in shape with the following dimension (100 mm ID × 220 mm height). The reactor is of suspended growth type and was operated under continuous flow. 2.2. Substrate The feed composition of anaerobic agar medium (HiMedia Laboratories, India) is shown in Table 1, where as the characteristics of cow dung is shown in Table 2. 2.3. Pretreatment of cow dung The pretreatment of cow dung was carried out before subjecting the sample to isolation test by filtering through sieve having a pore opening of 2 mm. 2.4. Analytical process The organic strength of the cow dung was determined in terms of volatile solids. The total solids and volatile solids were determined by drying the sample at 105 ◦ C in oven and 550 ± 50 ◦ C in the furnace, respectively. The total nitrogen was determined by Kjeldhal method whereas volatile fatty acid (VFA) content was determined by distillation method [35]. The hydrogen and methane content in the biogas was determined by Drager Method [36]. 2.5. Isolation of seed microflora from cow dung The isolation experiments were carried out in order to kill the methanogenic organism and other hydrogen consuming bacteria present in the cow dung. The isolation tests were conducted in the following methods viz.: (1) pH adjustment and (2) pH adjustment coupled with heat treatment. The above experiments were conducted in duplicate. 2.6. Anaerobic jar The microflora isolated from two different conditions viz.: (a) pH corrected and (b) pH correction followed by heat treatment was tested for its ability to generate hydrogen in the anaerobic jar. The viability of the microflora was
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2.7. Isolation and investigation of seed microflora based on pH adjustment and pH adjustment coupled with heat treatment
1 2
90 mm
3 4
130 mm
5
100 mm 1. Inlet
4. Outlet
2. Gas outlet 3. Gas collection zone
5. Digestion zone
Fig. 1. Schematic diagram of anaerobic jar. 1. Inlet, 2. gas outlet, 3. gas collection zone, 4. outlet and 5. digestion zone. Table 1 Characteristics of anaerobic agar Parametersa
Concentration
pH Casein enzymic hydrolysate Dextrose Sodium chloride Sodium thioglycollate Sodium formaldehyde sulphoxylate Methylene blue Agar
7.0 ± 0.2 2 1 0.5 0.2 0.1 0.0002 2
a Except
pH all other parameter are in gram per litre.
Table 2 Characteristics of cow dung Parametersa
Concentration
pH Total solids Volatile solids Total nitrogen
7.1 ± 3 13 ± 2 68 ± 3 0.70 ± 0.04
a Except
pH all parameters are in percentage (%).
investigated with respect to digestion pH, VFA, biogas generated, hydrogen and methane content.
Isolation of hydrogen generating microflora was carried out by subjecting the 10% solids content of cow dung to varying initial pH viz. 4, 5 and 6 for a retention time 3 h. The microflora resulting from this treatment process was tested for its hydrogen generating capability in the anaerobic jar. In the case of isolating microflora based on pH adjustment and heat treatment, the pH of the cow dung was maintained at 4, 5 and 6, respectively, for a retention time of 3 h. Thereafter it was subjected to heat treatment in a water bath at 105 ◦ C for 1 h, for two consecutive times at an interval of 30 min. The microflora resulting after the heat treatment was tested for its hydrogen generating capability in anaerobic jar. The anaerobic fermentation was carried out by transferring 800 ml of microflora into the anaerobic jar to which 200 ml (6%) anaerobic agar is feed as substrate and maintained at their respective operating pH. 2.8. Isolation of hydrogen generating microflora from varying solids content of cow dung at pH 5 coupled with heat treatment The effect of initial solids content of cow dung in isolating hydrogen generating bacteria was investigated at pH 5 coupled with heat treatment at 2, 3 and 4 h, respectively. The solids content of cow dung was varied in the following concentration of 10%, 12% and 15%, respectively. The reason for choosing the pH 5 is that at this pH the biogas generation had a maximum value for microflora isolated based on pH adjustment coupled with heat treatment when compared at pH 4 and 6. 2.9. Isolation of hydrogen generating microflora from varying solids content of cow dung at pH 6 coupled with heat treatment The efficiency of isolating hydrogen generating microflora from cow dung was investigated at a solids content of 12% and 15% at pH 6 coupled with heat treatment. Since the hydrogen generation for a pH adjusted cow dung microflora at 5 and 6 had a marginal difference. The effect of initial solids content of cow dung in isolating hydrogen generating microflora was investigated at pH 6 coupled with heat treatment. 3. Results and discussion 3.1. Investigation of isolated seed microflora from cow dung based on pH adjustment and pH adjustment coupled with heat treatment The microflora isolated from cow dung after subjecting to pH adjustment and pH adjusted coupled with heat treatment was tested in the anaerobic jar having a capacity of 1 l.
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Cumulative biogas generation (ml/h)
3500 Heat treatment period & Fermentation pH
3000
4 pH 5 pH
2500
6 pH 2 h at 4 pH
2000
2 h at 5 pH 2 h at 6 pH
1500
Cow dung solids content : 10%
1000
500
0 0
20
40
60
80
100
120
140
Time (h) Fig. 2. Effect of cumulative biogas generation versus time (pH adjusted and pH adjusted coupled with heat-treated microflora from cow dung at 10% solids).
The anaerobic fermentation was commenced using 800 ml of isolated microflora from cow dung to which 200 ml of anaerobic agar medium was fed as the substrate. The substrate addition was kept in a continuous mode throughout the investigation at a flow rate of 200 ml/day. The reason for keeping the feed rate as a continuous flow is the substrate should not be limiting factor. Chen at al. [37] stated that hydrogen production in a batch culture seems to be limited when cell growth was thriving mainly due to rapid depletion of carbon substrate for the gain in biomass. Thus, continuous process could be favourable for hydrogen production due to a sufficient supply of carbon substrate. Hence continuous feed flow was used in this present investigation. The effect of biogas generation is shown in Fig. 2. In the case of pH adjusted cow dung microflora the biogas generation was found to be increasing in the following order of digester pH of 4, 5 and 6. At the end of 120 h of operation the cumulative biogas generation was found to be 1494, 2404 and 3327 ml during the anaerobic fermentation at pH 4, 5 and 6, respectively. In the case of pH adjusted coupled with heat-treated cow dung microflora, the cumulative biogas generation was found to be low at pH 4, whereas a maximum biogas generation was recorded at pH 5 followed by pH 6. At the end of 120 h of operation at pH 4, 5 and 6 the cumulative biogas production was found to be 1685, 2610 and 2353 ml, respectively. As shown in Fig. 2 the lag phase was longer for microflora isolated form cow dung which when subjected to pH adjustment coupled with heat treatment, whereas for the pH adjusted cow dung the lag phase was less. The pH ad-
justed microflora operating at 4, 5 and 6 pH resulted in a lag period of 24, 12 and 6 h, respectively. Whereas in the case of microflora isolated by subjecting the cow dung to pH adjustment followed by heat treatment at pH 4, 5 and 6 resulted in a lag period of 42, 24 and 30 h, respectively. The possible reason for the longer lag phase in the case of microflora isolated from pH adjustment followed by heat treatment than pH adjustment alone could be due to sporulation. The reason for carrying out the heat treatment on cow dung was to kill or suppress the methanogenic bacteria. Zoetemeyer et al. [44], Horiuchi et al. [45] as such stated that biohydrogen generation from anaerobic fermentation relies on the disruption of methanogenic archaea since they are significant hydrogen consumer microbial group in anaerobic consortia. Several methods are available for suppressing the growth of hydrogen consuming bacteria, for example, use of chemical inhibitors such as acetylene and bromoethane sulphonic acid [14]; heat shock pretreatment of inocula [44,38–40] and maintain the cultures at acidogenic range (5.8–6.5) [25,26]. The heat shock pretreatment relies on the killing or thermal suppression of methanogenic archaea and non-sporulating bacteria thereby enriching the culture with sporulating hydrogen producing bacteria such as Clostridia [41]. Fig. 3 shows that cumulative hydrogen generation during the anaerobic fermentation using microflora isolated from pH adjustment and pH adjustment coupled with heat treatment. Comparison of Figs. 2 and 3 shows that for pH adjusted sample even though the biogas generation is high, the corresponding hydrogen generation was found to be less. At the end of 120 h of anaerobic fermentation for a pH adjusted
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Cumulative hydrogen generation (ml/h)
1400 Heat treatment period & Fermentation pH
1200
4 pH 5 pH 6 pH 2 h at 4 pH 2 h at 5 pH 2 h at 6 pH
1000 800
Cow dung solids content : 10%
600 400 200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 3. Effect of cumulative hydrogen generation versus time (pH adjusted and pH adjusted coupled with heat-treated microflora from cow dung at 10% solids).
2000 Fermentation pH
Cumulative methane generation (ml/h)
1800 4 5 6
1600 1400
Cow dung solids content : 10%
1200 1000 800 600 400 200 0 0
20
40
60
80
100
120
140
Time (h) Fig. 4. Effect of cumulative methane generation versus time (pH adjusted microflora from cow dung at 10% solids).
microflora at a pH 4, 5 and 6 the cumulative biogas generation was found to be 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with
heat treatment. Earlier researchers have shown that operating anaerobic fermentation process between pH 5 and 6 attained the highest total hydrogen production [10,22,28,29,42,43]. The possible reason for low hydrogen generation at pH 6 is due to generation of methane as shown in Fig. 4. In the case of pH adjusted microflora at 4 and 5 the methane
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713
1600 Heat treatment period & Fermentation pH
Volatile fatty acid (mg/l)
1400
4 pH 5 pH 6 pH 2 h at 4 pH 2 h at 5 pH 2 h at 6 pH
1200 1000 800 600 400 200
Cow dung solids content : 10%
0 0
20
40
60
80 Time (h)
100
120
140
Fig. 5. Volatile fatty acid content versus time (pH adjusted and pH adjusted coupled with heat-treated microflora from cow dung at 10% solids).
generation was found to be below detectable limit, whereas at pH 6 the cumulative methane generation was found to be 1840 ml at a fermentation period of 120 h. The optimum pH for methanogenic bacteria was found to be 6.0–7.5 [46–49]. Hence operating anaerobic fermentation at pH 6 using cow dung as the seed microorganism does not seem to be sound method for generating hydrogen as methanogenic bacteria can still dominate at this operating pH. Moreover operating at pH 6 revealed that hydrogen generation still occurred but the concentration was low which could be due to utilization of hydrogen as substrate by the methanogenic bacteria. In the case of operating the anaerobic fermentation at pH 4 and 5, using pH adjusted microflora resulted in a methane concentration below detectable limit, whereas the hydrogen concentration showed a gradual build-up as the fermentation proceeded. Hence it can be concluded that methanogenic population was being inhibited or killed at lower pH value. Similar observations were made by Ueno et al. [15] stating that acidogenic conditions were most suitable for hydrogen generation from anaerobic fermentative consortia, a fact corroborated later by Lin and Chang [25], Lay [20], Ueno et al. [24], Fang et al. [50]. The low pH could be attained either by pH adjustment or due to the overloading of the reactor which in turn led to the accumulation of organic acids resulting in drop in pH value [22,26]. In the case of pH adjusted coupled with heat-treated microflora isolated from cow dung, the methane content was found to be below detectable limit for pH 4, 5 and 6, respectively. The corresponding cumulative hydrogen generation at pH 4, 5 and 6 was found to be 509, 1198 and 1165 ml at
the end of 120 h of fermentation period. As shown in Fig. 3 the cumulative hydrogen generation for microflora isolated from pH adjusted cow dung was relatively less when compared to the microflora isolated from cow dung by subjecting them to pH adjustment coupled with heat treatment. The possible reason for the rise in the hydrogen generation could be due to destruction of hydrogen consuming bacteria during heat treatment. Lay et al. [51] and Oh et al. [52] investigation revealed that heat shock pretreatment inhibited or killed Methanogenic Archaea and lactic bacteria. Lactic bacteria can impair hydrogen production because they produce toxin that inhibit several subgroups of hydrogen generating consortium [53]. The volatile fatty acid content during the anaerobic fermentation for pH adjusted microflora and pH adjusted coupled with heat-treated microflora from cow dung is shown in Fig. 5. In the case of pH adjusted microflora at 4, 5 and 6 the VFA content during the fermentation process was found to be 870, 814 and 505 mg/l at 48 h, whereas at 120 h it was found to be 1404, 1203 and 733 mg/l, respectively. In the case of pH 4 even though the VFA content is high the biogas generation was found to be low. The possible reason for this type of behaviour could be due to the accumulation of organic acids leading to overall process inhibition [54] or a drop in reactor pH below 5.8 could switch over the reaction from acidogenesis to solventogenesis (a hydrogen consuming pathway) [55]. On comparison of anaerobic fermentation at pH 5 and 6 it can be seen that at pH 5 there is an accumulation of VFA when compared to pH 6. At pH 5 the methanogenic bacteria are subjected to inhibi-
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Cumulative biogas generation (ml/h)
Heat treatment period & Cow dung solids content
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2 h at 10% 2 h at 12% 2 h at 15% 3 h at 10% 3 h at 12% 3 h at 15% 4 h at 10% 4 h at 12% 4 h at 15%
2000
1500
1000
Fermentation pH 5.1 ± 0.1
500
0 0
20
40
60
80
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120
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Time (h) Fig. 6. Effect of cumulative biogas generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 5.1 ± 0.1 coupled with heat treatment).
tion which could have lead to the accumulation of intermediate metabolic product, but that did not affect the biogas generation as shown in Fig. 2. In the pH range of 5.5–6 both methanogenesis and solventogenesis can be avoided or minimized [26,55]. At pH 6 there was a marginal increase in VFA content and this could be due to the utilization of metabolic intermediates in biogas generation thereby preventing their accumulation. In the case of pH adjusted coupled with heat-treated microflora at pH 4, 5 and 6 resulted in VFA content of 792, 698 and 720, respectively, at 48 h. Whereas at 120 h the VFA content was found to be 1289, 989 and 1117, respectively. The low biogas generation at pH 4 as shown in Fig. 2 could be due to the accumulation of VFA resulting in solventogenesis at pH 4. In the case of pH 5 even though there is a rise in VFA content in the reactor, there was a corresponding rise in biogas generation when compared with pH 6 which resulted in low VFA content and biogas generation. The rise in VFA content could be due to the existence of microflora in exponential growth phase, as the biogas generation showed a gradual increasing trend. Earlier researcher have stated a volatile fatty acid content of 1250 ± 87 mg/l while treating food waste under mesophilic condition in the pH range of 4.5–6.5 [56], whereas Chen et al. [37] stated a VFA content of 810 mg/l for a fermentation period of 33 h using sucrose as substrate. Isolating hydrogen generating microflora from cow dung having a solids content of 10%, by subjecting to pH adjustment at 5 and 6 coupled with heat treatment at 2, 3 and 4 h did not exhibit autotrophic acetogenesis as the hydrogen generation was found to be in increasing trend. The
autotrophic acetogenesis occurs during anaerobic fermentation at low pH, when the seed microflora is subjected to heat treatment at below the optimum level. In our present investigation since we did not experience loss in hydrogen content, it can be concluded that autotrophic acetogenesis did not occur in the present isolating conditions for the given solids content and heat treatment period. 3.2. Efficiency of isolating hydrogen generating microflora from cow dung based on pH adjustment at 5 coupled with heat treatment The efficiency of isolating hydrogen generating microflora from cow dung was investigated at pH 5 by varying the initial solids content (10%, 12% and 15%) with respect to heat treatment period (2, 3 and 4 h). Fig. 6 shows the biogas generation when the cow dung is pretreated by adjusting the initial pH to 5 and then subjecting them to 2, 3 and 4 h heat treatment. When the heat treatment was carried out 2, 3 and 4 h the lag phase was found to be 30, 36 and 60 h, respectively, irrespective of initial microflora solids content. The reason for this kind of behaviour could be due to sporulation when the heat treatment was carried out for longer period. The biogas generation was found be increasing with the increase in solids content of the cow dung, probably due to increase in biomass and substrate concentration. The cumulative biogas generation was found to be marginally high for heat-treated cow dung microflora at 3 h rather than 2 h for their corresponding solids content at 10%, 12% and 15%, respectively. In the case of heat-treated cow dung for a period of 4 h resulted in low cumulative
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1600 Heat treatment period & Cow dung solids content
Cumulative hydrogen generation (ml/h)
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2 h at 10% 2 h at 12% 2 h at 15% 3 h at 10% 3 h at 12% 3 h at 15% 4 h at 10% 4 h at 12% 4 h at 15%
1200 1000 800 600
Fermentation pH 5.1 ± 0.1
400 200 0 0
20
40
60
80
100
120
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Time (h) Fig. 7. Effect of cumulative hydrogen generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 5.1 ± 0.1 coupled with heat treatment).
biogas generation, which could be due to the longer lag phase. The cumulative hydrogen generation during the isolation of hydrogen generating microflora based on heat treatment duration for a constant pH of 5 is shown in Fig. 7. In the case of heat treatment at a duration of 2 and 3 h, the hydrogen generation was more or less the same for a given solids content of 10%, 12% and 15%, respectively. In the case of 4 h heat treatment the cumulative hydrogen generation was low in comparison with 2 and 3 h heat-treated cow dung. During a fermentation period of 120 h for a microflora isolated from at 15% solids content of cow dung using pH adjustment at 5 coupled with heat treatment for a period of 2, 3 and 4 h resulted in a cumulative hydrogen generation of 1305, 1400 and 734 ml, respectively. The results showed that biogas was free from methane, with a maximum hydrogen content of 63 ± 3% at a heat treatment period for 2 and 3 h, whereas at 4 h heat treatment it was 55 ± 2%. The low value of cumulative hydrogen generation for a heat treatment period of 4 h could be due to the longer lag phase as a result of sporulation. The volatile fatty acid content is shown in Fig. 8. At the end of 24 h fermentation period for a heat-treated microflora at 2 h the VFA content was found to have higher value as shown in Fig. 8, when compared to heat-treated microflora and 3 and 4 h, respectively. For example at a fermentation period of 24 h for 10% and 15% microflora when subjected to 2 h heat treatment resulted in a VFA content of 580 and 710 mg/l, whereas in the case of 4 h heat treatment it was 490 and 550 mg/l. The higher value of VFA for 2 h heat treatment
microflora could be due to shorter (24 h) lag phase when compared with 4 h heat treatment microflora, where the lag phase was found to be 60 h. In the case of fermentation period at 120 h for 10% and 15% microflora when subjected to 2 h heat treatment resulted in a VFA content of 820 and 950 mg/l, whereas in the case of 4 h heat treatment it was 917 and 1081 mg/l. In the case of 4 h heat-treated microflora there was a considerable rise in VFA content when compared between a fermentation period of 24 and 120 h. This could be due to the presence of microflora in the earlier exponential phase at 24 h whereas at 120 h it was in the exponential phase. Even though there was increase in VFA content with the fermentation period, it did not exhibit any inhibition to the biogas generation as shown in Fig. 6. Therefore it can be concluded that VFA even as high as 1081 mg/l was able to produce stable biogas generation. 3.3. Efficiency of isolating hydrogen generating microflora from cow dung based on pH adjustment at 6 coupled with heat treatment The efficiency of isolating hydrogen generating microflora from cow dung was investigated at an initial solids content of 12% and 15% by subjecting to pH adjustment at 6 coupled with heat treatment at 2, 3 and 4 h, respectively. The cumulative biogas generation during anaerobic fermentation using the isolated microflora is shown in Fig. 9. As the heat-treatment period increased the lag phase also increased, the cumulative biogas generation was high for 2 h heat-treated microflora than 3 and 4 h, which could be
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1600 2 h at 10% 2 h at 12% 2 h at 15% 3 h at 10% Heat treatment period & Cow dung 3 h at 12% solids content 3 h at 15% 4 h at 10% 4 h at 12% 4 h at 15%
1400
Volatile fatty acid (mg/l)
1200 1000 800 600 400
Fermentation pH 5.1 ± 0.1
200 0 0
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40
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Time (h) Fig. 8. Volatile fatty acids content versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 5.1 ± 0.1 coupled with heat treatment).
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Cumulative biogas generation (ml/h)
Heat treatment period & Cow dung solids content
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2 h at 12% 2 h at 15% 3 h at 12%
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3 h at 15% 4 h at 12% 4 h at 15%
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Fermentation pH 6.1 ± 0.1
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0 20
30
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Time (h)
Fig. 9. Effect of cumulative biogas generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
due to the sporulation of bacteria and spore breaking taking a longer time as reflected in the long lag phase when the heat treatment period increased to 3 and 4 h, respectively. In the case of initial microflora solids content of 12% and
15% when subjected to pH adjustment at 6 coupled with heat treatment for a period of 2, 3 and 4 h resulted in a lag period of 36, 48 and 60 h, respectively. In the case of isolating microflora from cow dung with an initial solid
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Cumulative methane generation (ml/h)
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2 h at 12% 2 h at 15%
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Fermentation pH 6.1 ± 0.1
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Time (h) Fig. 10. Effect of cumulative methane generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
Cumulative hydrogen generation (ml/h)
1000 Heat treatment period & Cow dung solids content
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2 h at 12% 2 h at 15% 3 h at 12% 3 h at 15% 4 h at 12% 4 h at 15%
800 700 600 500
Fermentation pH 6.1 ± 0.1
400 300 200 100 0 0
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Time (h) Fig. 11. Effect of cumulative hydrogen generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
content of 12% and 15% by subjecting to pH adjustment at 6 coupled with heat treatment for a period of 2, 3 and 4 h resulted in a lag period of 36, 28 and 60 h, respectively. The cumulative methane generation was investigated using isolated microflora from cow dung having a solids con-
tent of 12% and 15% at pH 6 by subjecting them to varying heat treatment period viz. 2, 3 and 4 h, respectively. In the case of microflora isolated from cow dung having a solids content of 12% resulted in biogas free from methane for varying heat treatment period as stated above. As the
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1000 900
Volatile fatty acid (mg/l)
800 700 600 Heat treatment period & Cow dung solids content
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2 h at 12% 2 h at 15% 3 h at 12% 3 h at 15% 4 h at 12% 4 h at 15%
400 300 200 100
Fermentation pH 6.1 ± 0.1
0 0
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Time (h) Fig. 12. Effect of cumulative hydrogen generation versus time (microflora isolated from cow dung with varying solids content by subjecting to pH adjustment at 6.1 ± 0.1 coupled with heat treatment).
solids content of the microflora increased to 15%, coupled with heat treatment for 2 h resulted in biogas with methane (Fig. 10). In the case of microflora with 15% solids content when subjected to heat treatment period for 3 and 4 h, respectively, the biogas was free from methane. The possible reason for this type of behaviour could be due to the presence of high solids (15%), which might act as shield to protect the methanogenic bacteria from heat shock. Moreover at pH 6 the methanogenic bacteria were subjected to less stress, as this pH was well within the optimum level for the bacterial growth. The cumulative hydrogen generation is shown in Fig. 11. In the case of heat-treated microflora for 2 h at pH 6 with solids content of 12% and 15% resulted in a cumulative hydrogen generation 866 and 189 mg/l, respectively, for a fermentation period of 120 h. The maximum hydrogen content was found to be 49 ± 2% in the case of 12% solids content. Whereas during the initial stages of fermentation at pH 6 for the microflora isolated form cow dung subjected to 2 h heat treatment with a solids content of 15% resulted in a hydrogen content of 19 ± 1% till 72 h of fermentation, thereafter it decreased to 3 ± 1% hydrogen at 120 h. The low hydrogen generation at 15% solids content could be due to utilization of hydrogen as a substrate by the methanogenic bacteria for generating methane as reflected in Fig. 10. Therefore heat treatment for a period of 2 h at pH 6 was not sufficient to kill or inhibit the methanogenic bacteria. In the case of microflora when isolated at pH 6 by subjected to heat treatment
for a period of 3 and 4 h for a solids content at 12% and 15% cow dung, the biogas was free from methane. The maximum hydrogen content was found to be 47 ± 1% and 50 ± 2% in the case of 12% and 15% solids content irrespective of heat treatment period viz. 3 and 4 h, respectively. The rise in hydrogen generation could be due to the inhibitory effect of heat treatment on methanogenic bacteria, as reflected in the case of microflora isolated from cow dung having a solids content of 15% at pH 6 above a heat treatment period of 2 h. The volatile fatty acid content during the anaerobic fermentation using microflora isolated from cow dung when subjected to pretreatment at pH 6 coupled with heat treatment at 2, 3 and 4 is shown in Fig. 12. At a fermentation period of 24 h with a microflora having a solids content of 12% which when subjected to pH adjustment at 6 followed by heat treatment at 2, 3 and 4 h, resulted in a VFA content of 510, 528 and 520 mg/l. Whereas for a fermentation period of 120 h for the above condition resulted in a VFA content of 690, 710 and 780 mg/l, respectively. In the case of microflora isolated from cow dung having a solids content of 15% resulted in a VFA content of 530, 575 and 547 during 24 h fermentation period, whereas VFA content was found to be 802, 890 and 902 mg/l, respectively, at 120 h of fermentation period. Even though there was a rise in VFA content with the increase in fermentation period, increase in VFA content did not affect the fermentation process as the biogas generation and hydrogen content showed an increasing trend.
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4. Conclusions The hydrogen generating microflora isolated from cow dung based on pH adjustment coupled with heat treatment proved to be advantageous when compared with pH adjustment alone. The results showed that for pH adjusted microflora isolated from cow dung with solids content at 10% resulted in a cumulative biogas generation of 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml during the anaerobic fermentation for 120 h at a pH 4, 5 and 6, respectively. The biogas was free from methane when operated at pH 4 and 5, whereas at pH 6 methane generation was observed. For the above condition in the case of microflora isolated by subjecting to pH adjustment coupled with heat treatment was free from methane with a cumulative biogas generation of 1685, 2610 and 2353 ml, while the cumulative hydrogen generation was 509, 1198 and 1165 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with heat treatment. The effect of initial solids content of the cow dung on the isolating efficiency of hydrogen generating microflora was also investigated at pH 5 and 6 coupled with heat treatment. The results revealed that with the increase in initial solids content of the cow dung the optimum heat treatment period also increased as the pH increased from 5 to 6.
Acknowledgements This research was supported by the Fundamental Research Grant of Universiti Putra Malaysia, Project Number : 02-03-03-057J / 55180.
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