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Pretreatment of methanogenic granules for immobilized hydrogen fermentation
International Journal of Hydrogen Energy 32 (2007) 3266 – 3273 www.elsevier.com/locate/ijhydene
Pretreatment of methanogenic granules for immobilized hydrogen fermentation Bo Hu, Shulin Chen ∗ Department of Biological Systems Engineering and Center for Multiphase Environmental Research, Washington State University, Pullman, WA 99164 6120, USA Received 19 December 2006; received in revised form 1 March 2007; accepted 1 March 2007 Available online 26 April 2007
Abstract Hydrogen can be produced through fermenting sugars in a mixed bacterial culture under anaerobic conditions. Anaerobic granular sludge was proposed as immobilized hydrogen producing bacteria to be used in hydrogen fermentation after methanogenic activity of the granule was eliminated in the pretreatment process. This paper reports an innovative treatment method to directly convert methanogenic granules to hydrogen producing granules using chloroform. Chloroform treatment was compared against acid and heat treatments of sewage sludge and methanogenic granules in terms of effectiveness to eliminate methanogenic activity. The results showed that chloroform treatment was the most effective among the three methods tested. Acid and heat treatments that were effective for sewage sludge treatment were observed not highly effective for application to granules because of the protection from the granular structure. In contrast, methanogens were very sensitive to the chloroform even at very low level. Methane production was almost completely inhibited in both sewage sludge and granules with the treatment with only 0.05% chloroform addition into the culture medium. If the chloroform concentration was controlled at low levels, chloroform selectively inhibited methanogenic activity while did not affect the hydrogen production. At high concentration range, chloroform also inhibited hydrogen production. Chloroform caused irreversible methanogenic activity elimination but hydrogen production recovered to normal after chloroform addition stopped. Chloroform showed desirable selectivity on inhibition of methanogens from hydrogen producing bacteria, nearly permanently eliminated the methane production, postponed the hydrogen consumption to acetic acid, while allowed recovery for normal hydrogen production. Chloroform treated granules were repeatedly cultured for eight time without noticeable damage. Continuous culture with chloroform treated granules showed that the granule structure could be kept for over 15 days and new granules started to form after 10 days operation. The hydrogen productivity reached 11.6 L/L/day at HRT 5.3 h, which all showed potential application of chloroform treatment of methanogenic granules in the immobilized hydrogen fermentation. 䉷 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. Keywords: Biological hydrogen production; Pretreatment; Granule; Chloroform
1. Introduction Fermentative hydrogen production from sugar has attracted increasing attention in recent years because of its ability to use biomass as well as addressing concerns on environment and fossil fuel limitations. The fermentative bacteria use glucose derived from a variety of sources as substrate to produce hydrogen and organic acids (e.g., acetate or butyrate) through
∗ Corresponding author. Tel.: +1 509 335 3743; fax: +1 509 335 2722. E-mail address: [email protected] (S. Chen).
anaerobic metabolic pathway, i.e., C6 H12 O6 + 2H2 O → 4H2 + 2CH3 COOH + 2CO2 , C6 H12 O6 → 2H2 + CH3 CH2 CH2 COOH + 2CO2 . These reactions, accomplished by acetogenic bacteria, are part of the anaerobic digestion process that is widely used in environmental engineering field. For the typical anaerobic methane production system, three groups of microorganisms coexist and work together to produce methane as following: acetogens degrade various carbon sources to produce volatile fatty acids (VFA) and hydrogen; acetogens degrade some VFA such as propionate and butyrate, and other solvents to produce acetate and hydrogen; finally, acetate and hydrogen are used to produce
0360-3199/$ - see front matter 䉷 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2007.03.005
B. Hu, S. Chen / International Journal of Hydrogen Energy 32 (2007) 3266 – 3273
methane by methanogens. Anaerobic fermentation has already been used for the production of methane and solvents such as acetone, butanol and ethanol (ABE fermentation). Anaerobic mixed fermentation can also be used to produce hydrogen as long as methanogenesis is blocked [1]. The major hydrogen producing microorganisms in anaerobic digestion are Clostridia, Gram-positive, spore-forming, rod-shaped bacteria. Clostridia are very resistant to heat or harmful chemicals, which provided important clues for treating the anaerobic sewage sludge to become hydrogen producing sludge. Many ways such as acid treatment [2], heat treatment [3,4], alkaline treatment [5], etc. have been demonstrated for turning methane production system into hydrogen production system because these treatment eliminated most of the methanogens. Heat treatment has been reported as being more effective than pH control for enhancing biological hydrogen production; however, substantial hydrogen lost occurs during the long time of batch culture due to homoacetogenic activity that consumes hydrogen to produce acetate [6]. Heat treatment seems unable to prevent acetogenesis, especially in most cases where some homoectogenic bacteria are also sporeforming bacteria. For the continuous fermentation, short hydraulic retention time (HRT) is also a method used to wash out the methanogens because Clostridia generally grow faster than methanogens [7]. Chloroform inhibited the production of CH4 from both H2 /CO2 and acetate [8]. It was reported that very low concentration (100 M) of CHCl3 showed strong inhibition to methanogens. Furthermore, chloroform fumigation, which applies high CHCl3 (vapor) concentrations, killed a large part of the soil microbial populations for subsequent extraction and determination of the microbial biomass carbon with preference to Gram-negative bacteria and thus might result in a shift towards a microbial community dominated by Gram-positive bacteria [9]. CHCl3 also inhibits acetate consumption by sulfate reducers. Furthermore, it was shown that the synthesis of acetate from CO2 by cells of Clostridium thermoaceticum fermenting glucose was inhibited by CHCl3 [10]. Most studies on microbial hydrogen production used suspended-cell systems, which are normally inefficient or difficult to control in continuous operations. Recycling the biomass back to the reactor has to be used to maintain sufficient cell density for high hydrogen production. Different immobilization methods can also be used to enhance the biomass concentration for both pure culture and mixed culture, such as calcium alginate entrapment, lignocellulosic materials supporting Enterobacter cloacae [11], and ethylene-vinyl acetate copolymer immobilized anaerobic sludge [12]. By using active carbon as a support matrix to allow the retention of the H2 producing bacteria within the reactor, fixed-bed bioreactors enhanced the rate of hydrogen production to 121 mmol L−1 h−1 [13]. Immobilized cells culture appears to be an more effective way to increase hydrogen productivity than suspended culture. Granulation, which immobilizes the bacteria by selfflocculation, is simple to use and easy to control at an industrial scale. This immobilization technique can also be used in biological hydrogen production. It was found that hydrogen pro-
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ducing biomass can develop into granules with high bioactivity [14]. Hydrogen producing sludge was firstly demonstrated to agglutinate into granules in a well-mixed reactor treating a synthetic sucrose-containing wastewater at 26 ◦ C, pH 5.5, with 6 h of hydraulic retention. The granules became visible by day 15 and the size stabilized at around 1.6 mm diameter after day 60 [14]. UASB systems, where granule formation is an indicator of their successful operation, were also used for the hydrogen fermentation. The sludge granulation became visible after 120 days of reactor operation at HRT 8–20 h and the average granular diameter peaked at day 173 at around 0.43 mm, which is smaller than general methane producing granular sludge [15]. Rapid and efficient granular sludge formation was found with carrier-induced granular sludge bed (CIGSB) bioreactor with addition of support carriers, especially activated carbon. Packing of a small quantity of carrier on the bottom of the upflow reactor significantly stimulated the granulation within 100 h [16]. The mechanism of the granule formation, however, has not been fully understood yet. Most researchers concluded that Methanosaeta concilii is a key organism in methanogenic granulation [17] and it is contradictory to recent reports [14–16] that showed granule formed directly by hydrogen producing system, from which methanogen should be mostly eliminated. Possible explanation of these results is the extracellular polymer (ECP) excreted by bacteria during the granulation process. Although it has been found [14] that there are slightly difference of ECP composition between methanogenic granules and hydrogen producing granules, ECP plays the key role in agglutinating bacteria into granules and also in maintaining structural integrity of granules. The straightforward way to obtain hydrogen producing granules is to treat the methanogenic granules by heat or chemicals, so that methanogens can be eliminated while hydrogen producing bacteria in the granular structure still remain. The purpose of this study was to investigate the feasibility treating methanogenic granules by different methods and to compare the treated granules for immobilized hydrogen production. 2. Materials and methods 2.1. Seed sewage sludge and methanogenic granules The anaerobic sewage sludge was taken from the wastewater treatment plant in Pullman, WA. The methanogenic granules were taken from a UASB treating starch wastewater, and the average settling velocity was 29.5 m h−1 (measured with 1000 ml graduated cylinder full of DI water, 100 samples). 2.2. Glucose culture medium The medium used for H2 fermentation contained 20 g glucose COD L−1 (i.e., 18.75 g glucose L−1 ) as the carbon source and sufficient amounts of inorganic supplements [18], including: NH4 HCO3 (5.24 g L−1 ), NaHCO3 (6.72 g L−1 ), K2 HPO4 (0.125 g L−1 ), MgCl2 · 6H2 O (0.1 g/L−1 ), MnSO4 · 6H2 O
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(0.015 g L−1 ), FeSO4 · 7H2 O (0.025 g L−1 ), CuSO4 · 5H2 O (0.005 g L−1 ), and CoCl2 · 5H2 O (1.25 × 10−4 g L−1 ). 2.3. Acid pretreatment The acidic pretreatment involved decreasing the pH of the sludge or granule solution to 3.0 using 0.1 N HCl solution for 24 h and a readjustment of pH back to 7.0 by 0.1 N NaOH solution. 2.4. Heat pretreatment In the heat pretreatment, the sludge or granules was heated in boiling water bath for a short period of time (10–30 min) first, then cooled down. 2.5. Batch culture Culture medium of 20 ml was placed in a serum bottle with 3 ml pretreated sewage sludge or pretreated granules. Nitrogen gas was pumped into the serum bottle for 2 min before the fermentation to eliminate the oxygen inside the bottle. The culture temperature was 35 ◦ C and the shaking speed was 100–150 rpm (Incubator IC 600, Yamato). 2.6. Chloroform pretreatment Chloroform was added into the culture medium at the first batch culture for the chloroform pretreatments. Six levels of chloroform were used separately at 5%, 2.5%, 1%, 0.5%, 0.25% and 0.1%.
order to collect the biogas and measure the biogas volume. The reactor was maintained at batch mode for three days, and then began continuous mode at HRT = 13 h. The HRT was adjusted quickly to 5.3 h and kept operation for three days until it ran constantly (constantly here means that the pH of the effluent, the biogas and hydrogen production, the glucose conversion and VFA production are constant for over 12 h). 2.9. Analysis Total biogas volume produced in the headspace of the serum bottle was measured using Owen’s method [19]. The biogas was released at the end of the fermentation into a U-tube with water. The biogas volume produced during the fermentation was measured through the water pressured out of the U-tube. The composition of biogas (H2 , CO2 , CH4 and H2 S) in the headspace of the reactor was measured using a gas chromatograph (GC, CP-3800, Varian, Walnut Creek, CA) equipped with detectors including a thermal conductivity detector for H2 and CO2 , a flame ionization detector for CH4 , a Valco Instrument Pulsed Discharge Detector run in Helium Ionization Mode D2 for H2 S, an 18 × 1/8 HayeSep Q 80/100 Mesh Silcosteel column for CO2 , H2 and CH4 with nitrogen as the carrier gas, and a 50 m × 0.53 mm × 4 m SilicaPLOT column for H2 S with helium as the carrier gas. VFA were analyzed using a Dionex DX-500 system (Sunnyvale, CA, USA) containing an AS11-HC (4 mm 10–32) column, a quaternary gradient pump (GP40), a CD20 conductivity detector, and an AS3500 auto-sampler. Settling velocity of granules was measured in water.
2.7. Repeat culture
3. Results and discussion
After batch culture of granules with chloroform addition, another batch culture was carried out without chloroform addition to see whether chloroform pretreatment caused permanent damage to hydrogen producing bacteria and methanogen. The granules were taken out, washed with water and filtered. Then, these granules were put into a serum bottle with the same amount of original culture medium to start another batch that was cultured with the same conditions as described above. Also, this procedure were repeated for several times to see whether the chloroform treated granule can be reused.
3.1. Acid pretreatment
2.8. Continuous culture An upflow reactor was set up with a volume of 450 ml and height of 18 cm. Methanogenic granule of 50 ml was treated with 0.1% chloroform following the method mentioned previously. Then chloroform treated granules was packed in the upflow reactor as expanded bed. Twenty grams COD L−1 glucose culture medium was fed into the reactor at the bottom with the pump. Biogas and fermentation broth flowed out the reactor at the top and were separated in the separation bottle. Water replacement bottles were connected with separation bottle in
The methanogenic activity of granule was much higher than the sewage sludge (Table 1). The batch culture with granules as seeds (granule culture) produced 75.76 ml CH4 g−1 glucose, while sewage culture only produced about 0.74 ml CH4 g−1 glucose. After acid pretreatment, the methane production for sewage culture decreased close to zero, while the methane production for granule culture decreased slightly from 75.76 glucose to 61.00 ml CH4 g−1 glucose. Granules showed their advantage in methane production not only in terms of their higher methanogenic activity, but also by providing a protective niche for methanogens from the harsh environmental change, in this case the acid shot. Acid treatment has already been proved be an effective way to eliminate methanogens from sewage sludge and turn sewage sludge to hydrogen producing sludge as most of hydrogen producing bacteria in the sewage sludge are spore-forming Clostridia that can tolerate harsh environment. However, acid treatment of granules did not eliminate the methane production dramatically and could not turn methanogenic granule to be hydrogen producing granules because methanogens were protected by granular structure.
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Table 1 Cumulative gas production for acid treatment of granule and sewage sludge H2 ml g−1 glucose
Acid pretreatment
Granule Sewage
Control Acid Control Acid
CH4 ml g−1 glucose
Average
Standard dev
Average
Standard dev
0.42 0.00 124.99 89.01
0.50 0.00 26.42 14.45
75.76 61.00 0.74 0.26
24.75 24.21 0.16 0.14
Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml. Data was the average of triplicates with standard deviations (n = 3) at = 0.05. Table 2 Cumulative gas production for heat treatment of granule and sewage sludge H2 ml g−1 glucose
Heat pretreatment
Granule
Sewage
Control Heat (10 min) Heat (30 min) Control Heat (10 min) Heat (30 min)
CH4 ml g−1 glucose
Average
Standard dev
Average
Standard dev
0.42 0.34 118.29 124.99 121.29 134.12
0.50 0.48 0.00 26.42 25.70 5.77
75.76 59.97 0.06 0.74 0.01 0.00
24.75 12.45 0.00 0.16 0.01 0.00
Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml. Data was the average of triplicates with standard deviations (n = 3) at = 0.05. Table 3 Cumulative gas production for chloroform addition to granule and sewage sludge culture Chloroform added into culture medium (%)
Granule
Sewage
5 2.5 1 0.5 0.25 0.05 Control 5 2.5 1 0.5 0.25 0.1 Control
H2 ml g−1 glucose
CH4 ml g−1 glucose
Average
Standard dev
Average
Standard dev
0.30 0.00 29.13 94.15 121.41 135.09 0.42 0.61 0.00 32.87 45.57 113.14 145.32 124.99
0.51 0.00 5.28 27.87 14.59 4.43 0.50 1.06 0.00 36.13 17.48 11.09 27.91 26.42
0.14 0.00 0.04 0.03 0.10 0.04 75.76 0.53 0.56 0.16 0.14 0.17 0.12 0.74
0.12 0.00 0.03 0.02 0.18 0.04 24.75 0.06 0.13 0.03 0.02 0.00 0.02 0.16
Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml. Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
3.2. Heat pretreatment Methanogenic activity was almost eliminated from sewage sludge culture after 10 min heat treatment of sewage sludge (Table 2). This method has already been proved to be effective and the results of this study confirm that. For granules on the other hand, methanogen was still very active with massive methane production after the granules were treated by heat for 10 min. Longer treatment time was needed to eliminate all methanogenic activity as shown in Table 2. After 30 min heat treatment, granule culture could be able to produce hydrogen instead of methane. Similar reason to acid treatment, granu-
lar structure provided protection for methanogens against harsh environment, leading to longer time required for heat treatment to eliminate methanogens. 3.3. Chloroform pretreatment Chloroform showed strong inhibitory effects when added into both granules and sewage sludge culture systems (Table 3). More than 2.5% of chloroform addition severally inhibited both hydrogen and methane production for both the granule culture and sewage sludge culture. However, the toxicity of chloroform showed difference between hydrogen
B. Hu, S. Chen / International Journal of Hydrogen Energy 32 (2007) 3266 – 3273
Hydrogen ml/g glucose
160
Gas production ml/g glucose
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y = -117.11x + 148.75
140
R2 = 0.9873
120 100 80 60 40 20 0 0
0.2
0.4
0.6
0.8
1
1.2
Chloroform % Fig. 1. Chloroform inhibition on hydrogen production (20 ml glucose culture medium, three days each batch, initial pH = 8.0, inoculums 3 ml). Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
producing bacteria and methanogens at low level of chloroform addition into the culture medium. Methanogens were so sensitive to the chloroform that only 0.05% addition into the culture medium almost completely inhibited methane production even for granulized methanogens. Chloroform has been used to inhibit methanogenic activity in different fields [8] because chloroform can block the function of corrinoid enzymes and inhibit methyl-coenzyme M reductase. Weathers et al. [20] found that the loss of methanogenic activity was strongly correlated with the mass of chloroform transformed, rather than with the timeintegrated chloroform exposure which methanogens received. Meanwhile, with the decrease of chloroform addition from 2.5% to 0.05%, hydrogen production increased dramatically for both granule and sewage sludge culture. When only 0.1% chloroform added into the culture mediums, hydrogen production was even higher than the sewage sludge control culture. The inhibition of chloroform to hydrogen production also showed strong correlation to the mass of chloroform added into the culture system (Fig. 1). Hydrogen production was totally inhibited with the chloroform at a treatment level of above 2.5%, meanwhile, the system was transferred to methane production with no chloroform addition. The data of chloroform addition from 1% to 0.05% were fit to a linear equation: y = −117.11x + 148.75,
(1)
where, y was the volume of hydrogen production from pretreatment batch culture per gram glucose and x was the percent of chloroform concentration added into the culture medium. Based on this model, it can be estimated that the hydrogen production would be totally inhibited if the chloroform addition is higher than 1.27% of the culture medium. In addition to eliminating the methanogenic activity for hydrogen production system, chloroform can also inhibit the activity of hydrogen producing bacteria, mostly Clostridia, but if the concentration was controlled at low level, chloroform selectively inhibited methanogenic activity while did not affect the hydrogen production activity. It was also reported that chloroform can inhibit the activity of other hydrogen consuming bacteria such as homoacetogens and acetate-consuming sulfate-
180.00 Hydrogen
160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00 5
2.5
1 0.5 Chloroform %
0.25
0.05
Fig. 2. H2 production from culture of chloroform treated granule (20 ml glucose culture medium, three days each batch, initial pH = 8.0, inoculums 3 ml). Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
reducing bacteria [21]. This might be part of the reason why hydrogen production at 0.1% chloroform addition for sewage sludge culture was even higher than any other sewage sludge from other treatment methods. 3.4. Batch culture of chloroform pretreated granule Fig. 2 showed that all the chloroform treated granule cultures produced hydrogen and nearly no methane. For chloroform level below 2.5%, batch cultures produced nearly same amount of hydrogen no matter whether pretreated chloroform concentration was different, which means that the hydrogen production ability was not affected by the chloroform inhibition in the pretreatment process. For the pretreatment level of 2.5%, the average hydrogen production of the batch culture reached 166.15 ml g−1 glucose, which accounts the yield of 1.34 ± 0.11 mol H2 mol−1 glucose calculated as ideal gas at room temperature. The yield of the hydrogen production can be influenced by several factors, such as initial glucose concentration, fermentation time, reactor type, hydrogen partial pressure, etc. This yield is within the range summarized by Nath et al. [22] on reported fermentative hydrogen production by different organisms. To date, several approaches have been reported in the reference to increase the hydrogen production yield such as lowering the hydrogen partial pressure, or removing carbon dioxide in the headspace. Techniques for increasing the hydrogen yield will be a topic of research priority. For 5% chloroform treated granule culture, data showed significant fluctuation of hydrogen production, which might be caused by the inhibition of the remaining chloroform from previous pretreatment batch. Table 4 showed the comparison of cumulative gas production for untreated and treated granule inoculums. For the batch culture inoculated with methanogenic granules, acetic acid was accumulated in the beginning of the fermentation and was converted completely to methane at 18 days of fermentation. It can also be noticed that little hydrogen was accumulated in the beginning of the fermentation process, which means high homoacetogenic activity converting the hydrogen to acetate. For the batch culture inoculated with 0.1% chloroform treated
B. Hu, S. Chen / International Journal of Hydrogen Energy 32 (2007) 3266 – 3273
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Table 4 Comparison of cumulative gas production for untreated and treated granule inoculums Time (Days) Untreated granule
Treated granule
LA (g L−1 )
AC (g L−1 )
PR (g L−1 )
BU (g L−1 )
H2 (ml g−1 glucose)
CO2 (ml g−1 glucose)
CH4 (ml g−1 glucose)
3 18 3
0.00 0.00 1.91
3.13 0.00 1.82
1.65 0.00 0.00
0.00 0.00 2.65
7.79 1.04 109.87
292.66 453.93 290.12
80.88 374.37 0.01
3(18) 6(18) 18
0.00 0.00 0.00
0.79 0.67 2.00
0.35 0.71 0.88
1.58 2.71 1.79
180.64 176.68 0.06
317.91 364.50 284.01
1.44 1.49 13.71
(Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml, chloroform treatment 0.1%. Data was the average of triplicates with standard deviations (n = 3). LA: lactic acid; AC: acetic acid; PR: propionic acid; BU: butyric acid. Time 3(18) means that gas was released at day 3 and broth was measured at day 18, same as Time 6(18).
granules, VFA was also accumulated because methane production was inhibited. However, after gas release at three days fermentation or six days fermentation, detectable methane was recorded at 18 days when the fermentation was continued. In the same time, the acetic acid concentration of the broth at 18 days was significantly lower than that at three days, which means that there should be methanogenic activity recovery. It might have some residue methanogens after chloroform treatment, and still very little methane could be detected after 18 days of recovery because methanogens grow very slowly. There is no significant difference of cumulative hydrogen production between gas release at day 3 and at day 6, however, for the batch culture without gas release in the beginning of the fermentation, nearly no hydrogen was detected at day 18 with little amount of methane. CO2 production was also decreased. Homoacetogenic activity showed significant effect at long fermentation time, which means that chloroform treatment postponed or increased the lag time of the homoacetogenic activity, avoided fast conversion of hydrogen and CO2 to acetic acid. Little or no methane was produced from the chloroform treated cultures, which means that chloroform caused irreversible damage to methanogens, and the methanogenic activity did not recover even without chloroform existence. Meanwhile, hydrogen production ability recovered to normal even though chloroform partly inhibited hydrogen production at certain level in the pretreatment batch. Chloroform showed good selectivity on inhibition of hydrogen producing bacteria and methanogens, nearly permanently eliminated the methane production, postponed the hydrogen consumption to acetic acid, while allowed for normal hydrogen production recovery. Chloroform is required only in pretreatment process and the required concentration can be as low as 0.05%. 3.5. Comparison of acid treatment, heat treatment and chloroform treatment Acid pretreatment and heat pretreatment have already been proved effective for obtaining hydrogen producing sludge from sewage sludge. Their effectiveness was also confirmed in the this study. Chloroform addition showed same effectiveness for treating sewage sludge if the concentration was
controlled properly. Compare to the acid treatment or heat treatment of sewage sludge, chloroform treatment is also a simple step that can be easily implemented. Chloroform treatment might cause some chloroform pollution, but the concentration required is low (0.05%) without need for repeated treatment. From the experiment results, chloroform treatment showed many advantages over acid and heat treatments when dealing with granules because granular structure forms a protective environment. Chloroform resulted in more completely methanogens elimination. Furthermore, chloroform also postponed the activity of homoacetogens, another hydrogen consuming bacteria, while no reports about acid treatment and heat treatment mentioning about this possibility. Actually, some homoacetogens are also spore-forming bacteria, which are hardly eliminated by the heat treatment method [6]. 3.6. Repeat culture of chloroform treated granule There was no significant difference of hydrogen production for three repeated batch cultures with granules pretreated by 0.25% and 0.05% of chloroform. This result also confirmed that the chloroform treatment concentration can be lowered to 0.05%. Further repeated batch experiments only focused on 0.25% chloroform treated granules. Fig. 3 showed that chloroform treated granules can last for at least 8 batches (24 days) with nearly constant hydrogen production yet without obvious morphology damage. Chloroform treated granules can be the immobilized hydrogen producing bacteria for the immobilized hydrogen culture. 3.7. Continuous hydrogen production in the upflow reactor packed with chloroform treated granule Fig. 4 showed the performance of continuous hydrogen production in the upflow reactor packed with chloroform treated granules. The hydrogen productivity reached 11.6 L L−1 day−1 . After the HRT adjusted to below 4 h, the overall yield decreased, so the initial test for the continuous operation stopped. The granules changed its color gradually from deep black to dark gray, which might be caused by the
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160.00
0.25% 0.05%
Hydrogen ml/g glucose
140.00 120.00 100.00 80.00
irreversible damage to methanogens while hydrogen production ability recovered to normal after the pretreatment batch. Chloroform treated granule could be repeatedly cultured for eight time without obvious damage. The treated granule can be kept their structure over 15 days and new granule started to form after 10 days operation. Acknowledgments
60.00
Funding for this project was from the Washington Statue University Agriculture Research Center. We also thank Simon Smith for contributing thoughts to this study.
40.00 20.00
References
0.00 1
2
3
4
5 Batch
6
7
8
16
14
14
12
12
10
10 8
Hydrogen
8
HRT
6
6 4
4
2
2
0
HRT h
Hydrogen L /L /d
Fig. 3. H2 production from repeat culture of chloroform treated granule (20 ml glucose culture medium, three days each batch, initial pH = 8.0, inoculums 3 ml). Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
0 0
2
4
6 8 Time days
10
12
Fig. 4. Continuous hydrogen production in the up-flow reactor packed with chloroform treated granule (20 g COD L−1 glucose culture medium, initial pH = 8.0, 0.1% chloroform treatment).
washing out of the sulfur reducing bacteria. The granular structure was mostly kept after 15 days operation. New granules, which have shallow gray color, could be seen after 10 days of operation. 4. Conclusion Acid treatment and heat treatment were proved to be effective to sewage sludge while not completely effective to methanogenic microorganisms from granules because of the protective environment from the granular structure. Chloroform treatment can be an effective way for eliminating methanogenic activity. Hydrogen production also was inhibited by chloroform. If the concentration was controlled at low level, chloroform selectively inhibited methanogenic activity while did not influence the hydrogen production activity. Chloroform caused
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methanogenic and sulfate-reducing populations in freshwater sediment. FEMS Microbiol Ecol 2000;32(1):35–42. [22] Nath K, Das D. Improvement of fermentative hydrogen production: various approaches. Appl Microbiol Biotechnol 2004;65(5):520–9.
Pretreatment of methanogenic granules for immobilized hydrogen fermentation Bo Hu, Shulin Chen ∗ Department of Biological Systems Engineering and Center for Multiphase Environmental Research, Washington State University, Pullman, WA 99164 6120, USA Received 19 December 2006; received in revised form 1 March 2007; accepted 1 March 2007 Available online 26 April 2007
Abstract Hydrogen can be produced through fermenting sugars in a mixed bacterial culture under anaerobic conditions. Anaerobic granular sludge was proposed as immobilized hydrogen producing bacteria to be used in hydrogen fermentation after methanogenic activity of the granule was eliminated in the pretreatment process. This paper reports an innovative treatment method to directly convert methanogenic granules to hydrogen producing granules using chloroform. Chloroform treatment was compared against acid and heat treatments of sewage sludge and methanogenic granules in terms of effectiveness to eliminate methanogenic activity. The results showed that chloroform treatment was the most effective among the three methods tested. Acid and heat treatments that were effective for sewage sludge treatment were observed not highly effective for application to granules because of the protection from the granular structure. In contrast, methanogens were very sensitive to the chloroform even at very low level. Methane production was almost completely inhibited in both sewage sludge and granules with the treatment with only 0.05% chloroform addition into the culture medium. If the chloroform concentration was controlled at low levels, chloroform selectively inhibited methanogenic activity while did not affect the hydrogen production. At high concentration range, chloroform also inhibited hydrogen production. Chloroform caused irreversible methanogenic activity elimination but hydrogen production recovered to normal after chloroform addition stopped. Chloroform showed desirable selectivity on inhibition of methanogens from hydrogen producing bacteria, nearly permanently eliminated the methane production, postponed the hydrogen consumption to acetic acid, while allowed recovery for normal hydrogen production. Chloroform treated granules were repeatedly cultured for eight time without noticeable damage. Continuous culture with chloroform treated granules showed that the granule structure could be kept for over 15 days and new granules started to form after 10 days operation. The hydrogen productivity reached 11.6 L/L/day at HRT 5.3 h, which all showed potential application of chloroform treatment of methanogenic granules in the immobilized hydrogen fermentation. 䉷 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. Keywords: Biological hydrogen production; Pretreatment; Granule; Chloroform
1. Introduction Fermentative hydrogen production from sugar has attracted increasing attention in recent years because of its ability to use biomass as well as addressing concerns on environment and fossil fuel limitations. The fermentative bacteria use glucose derived from a variety of sources as substrate to produce hydrogen and organic acids (e.g., acetate or butyrate) through
∗ Corresponding author. Tel.: +1 509 335 3743; fax: +1 509 335 2722. E-mail address: [email protected] (S. Chen).
anaerobic metabolic pathway, i.e., C6 H12 O6 + 2H2 O → 4H2 + 2CH3 COOH + 2CO2 , C6 H12 O6 → 2H2 + CH3 CH2 CH2 COOH + 2CO2 . These reactions, accomplished by acetogenic bacteria, are part of the anaerobic digestion process that is widely used in environmental engineering field. For the typical anaerobic methane production system, three groups of microorganisms coexist and work together to produce methane as following: acetogens degrade various carbon sources to produce volatile fatty acids (VFA) and hydrogen; acetogens degrade some VFA such as propionate and butyrate, and other solvents to produce acetate and hydrogen; finally, acetate and hydrogen are used to produce
0360-3199/$ - see front matter 䉷 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2007.03.005
B. Hu, S. Chen / International Journal of Hydrogen Energy 32 (2007) 3266 – 3273
methane by methanogens. Anaerobic fermentation has already been used for the production of methane and solvents such as acetone, butanol and ethanol (ABE fermentation). Anaerobic mixed fermentation can also be used to produce hydrogen as long as methanogenesis is blocked [1]. The major hydrogen producing microorganisms in anaerobic digestion are Clostridia, Gram-positive, spore-forming, rod-shaped bacteria. Clostridia are very resistant to heat or harmful chemicals, which provided important clues for treating the anaerobic sewage sludge to become hydrogen producing sludge. Many ways such as acid treatment [2], heat treatment [3,4], alkaline treatment [5], etc. have been demonstrated for turning methane production system into hydrogen production system because these treatment eliminated most of the methanogens. Heat treatment has been reported as being more effective than pH control for enhancing biological hydrogen production; however, substantial hydrogen lost occurs during the long time of batch culture due to homoacetogenic activity that consumes hydrogen to produce acetate [6]. Heat treatment seems unable to prevent acetogenesis, especially in most cases where some homoectogenic bacteria are also sporeforming bacteria. For the continuous fermentation, short hydraulic retention time (HRT) is also a method used to wash out the methanogens because Clostridia generally grow faster than methanogens [7]. Chloroform inhibited the production of CH4 from both H2 /CO2 and acetate [8]. It was reported that very low concentration (100 M) of CHCl3 showed strong inhibition to methanogens. Furthermore, chloroform fumigation, which applies high CHCl3 (vapor) concentrations, killed a large part of the soil microbial populations for subsequent extraction and determination of the microbial biomass carbon with preference to Gram-negative bacteria and thus might result in a shift towards a microbial community dominated by Gram-positive bacteria [9]. CHCl3 also inhibits acetate consumption by sulfate reducers. Furthermore, it was shown that the synthesis of acetate from CO2 by cells of Clostridium thermoaceticum fermenting glucose was inhibited by CHCl3 [10]. Most studies on microbial hydrogen production used suspended-cell systems, which are normally inefficient or difficult to control in continuous operations. Recycling the biomass back to the reactor has to be used to maintain sufficient cell density for high hydrogen production. Different immobilization methods can also be used to enhance the biomass concentration for both pure culture and mixed culture, such as calcium alginate entrapment, lignocellulosic materials supporting Enterobacter cloacae [11], and ethylene-vinyl acetate copolymer immobilized anaerobic sludge [12]. By using active carbon as a support matrix to allow the retention of the H2 producing bacteria within the reactor, fixed-bed bioreactors enhanced the rate of hydrogen production to 121 mmol L−1 h−1 [13]. Immobilized cells culture appears to be an more effective way to increase hydrogen productivity than suspended culture. Granulation, which immobilizes the bacteria by selfflocculation, is simple to use and easy to control at an industrial scale. This immobilization technique can also be used in biological hydrogen production. It was found that hydrogen pro-
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ducing biomass can develop into granules with high bioactivity [14]. Hydrogen producing sludge was firstly demonstrated to agglutinate into granules in a well-mixed reactor treating a synthetic sucrose-containing wastewater at 26 ◦ C, pH 5.5, with 6 h of hydraulic retention. The granules became visible by day 15 and the size stabilized at around 1.6 mm diameter after day 60 [14]. UASB systems, where granule formation is an indicator of their successful operation, were also used for the hydrogen fermentation. The sludge granulation became visible after 120 days of reactor operation at HRT 8–20 h and the average granular diameter peaked at day 173 at around 0.43 mm, which is smaller than general methane producing granular sludge [15]. Rapid and efficient granular sludge formation was found with carrier-induced granular sludge bed (CIGSB) bioreactor with addition of support carriers, especially activated carbon. Packing of a small quantity of carrier on the bottom of the upflow reactor significantly stimulated the granulation within 100 h [16]. The mechanism of the granule formation, however, has not been fully understood yet. Most researchers concluded that Methanosaeta concilii is a key organism in methanogenic granulation [17] and it is contradictory to recent reports [14–16] that showed granule formed directly by hydrogen producing system, from which methanogen should be mostly eliminated. Possible explanation of these results is the extracellular polymer (ECP) excreted by bacteria during the granulation process. Although it has been found [14] that there are slightly difference of ECP composition between methanogenic granules and hydrogen producing granules, ECP plays the key role in agglutinating bacteria into granules and also in maintaining structural integrity of granules. The straightforward way to obtain hydrogen producing granules is to treat the methanogenic granules by heat or chemicals, so that methanogens can be eliminated while hydrogen producing bacteria in the granular structure still remain. The purpose of this study was to investigate the feasibility treating methanogenic granules by different methods and to compare the treated granules for immobilized hydrogen production. 2. Materials and methods 2.1. Seed sewage sludge and methanogenic granules The anaerobic sewage sludge was taken from the wastewater treatment plant in Pullman, WA. The methanogenic granules were taken from a UASB treating starch wastewater, and the average settling velocity was 29.5 m h−1 (measured with 1000 ml graduated cylinder full of DI water, 100 samples). 2.2. Glucose culture medium The medium used for H2 fermentation contained 20 g glucose COD L−1 (i.e., 18.75 g glucose L−1 ) as the carbon source and sufficient amounts of inorganic supplements [18], including: NH4 HCO3 (5.24 g L−1 ), NaHCO3 (6.72 g L−1 ), K2 HPO4 (0.125 g L−1 ), MgCl2 · 6H2 O (0.1 g/L−1 ), MnSO4 · 6H2 O
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(0.015 g L−1 ), FeSO4 · 7H2 O (0.025 g L−1 ), CuSO4 · 5H2 O (0.005 g L−1 ), and CoCl2 · 5H2 O (1.25 × 10−4 g L−1 ). 2.3. Acid pretreatment The acidic pretreatment involved decreasing the pH of the sludge or granule solution to 3.0 using 0.1 N HCl solution for 24 h and a readjustment of pH back to 7.0 by 0.1 N NaOH solution. 2.4. Heat pretreatment In the heat pretreatment, the sludge or granules was heated in boiling water bath for a short period of time (10–30 min) first, then cooled down. 2.5. Batch culture Culture medium of 20 ml was placed in a serum bottle with 3 ml pretreated sewage sludge or pretreated granules. Nitrogen gas was pumped into the serum bottle for 2 min before the fermentation to eliminate the oxygen inside the bottle. The culture temperature was 35 ◦ C and the shaking speed was 100–150 rpm (Incubator IC 600, Yamato). 2.6. Chloroform pretreatment Chloroform was added into the culture medium at the first batch culture for the chloroform pretreatments. Six levels of chloroform were used separately at 5%, 2.5%, 1%, 0.5%, 0.25% and 0.1%.
order to collect the biogas and measure the biogas volume. The reactor was maintained at batch mode for three days, and then began continuous mode at HRT = 13 h. The HRT was adjusted quickly to 5.3 h and kept operation for three days until it ran constantly (constantly here means that the pH of the effluent, the biogas and hydrogen production, the glucose conversion and VFA production are constant for over 12 h). 2.9. Analysis Total biogas volume produced in the headspace of the serum bottle was measured using Owen’s method [19]. The biogas was released at the end of the fermentation into a U-tube with water. The biogas volume produced during the fermentation was measured through the water pressured out of the U-tube. The composition of biogas (H2 , CO2 , CH4 and H2 S) in the headspace of the reactor was measured using a gas chromatograph (GC, CP-3800, Varian, Walnut Creek, CA) equipped with detectors including a thermal conductivity detector for H2 and CO2 , a flame ionization detector for CH4 , a Valco Instrument Pulsed Discharge Detector run in Helium Ionization Mode D2 for H2 S, an 18 × 1/8 HayeSep Q 80/100 Mesh Silcosteel column for CO2 , H2 and CH4 with nitrogen as the carrier gas, and a 50 m × 0.53 mm × 4 m SilicaPLOT column for H2 S with helium as the carrier gas. VFA were analyzed using a Dionex DX-500 system (Sunnyvale, CA, USA) containing an AS11-HC (4 mm 10–32) column, a quaternary gradient pump (GP40), a CD20 conductivity detector, and an AS3500 auto-sampler. Settling velocity of granules was measured in water.
2.7. Repeat culture
3. Results and discussion
After batch culture of granules with chloroform addition, another batch culture was carried out without chloroform addition to see whether chloroform pretreatment caused permanent damage to hydrogen producing bacteria and methanogen. The granules were taken out, washed with water and filtered. Then, these granules were put into a serum bottle with the same amount of original culture medium to start another batch that was cultured with the same conditions as described above. Also, this procedure were repeated for several times to see whether the chloroform treated granule can be reused.
3.1. Acid pretreatment
2.8. Continuous culture An upflow reactor was set up with a volume of 450 ml and height of 18 cm. Methanogenic granule of 50 ml was treated with 0.1% chloroform following the method mentioned previously. Then chloroform treated granules was packed in the upflow reactor as expanded bed. Twenty grams COD L−1 glucose culture medium was fed into the reactor at the bottom with the pump. Biogas and fermentation broth flowed out the reactor at the top and were separated in the separation bottle. Water replacement bottles were connected with separation bottle in
The methanogenic activity of granule was much higher than the sewage sludge (Table 1). The batch culture with granules as seeds (granule culture) produced 75.76 ml CH4 g−1 glucose, while sewage culture only produced about 0.74 ml CH4 g−1 glucose. After acid pretreatment, the methane production for sewage culture decreased close to zero, while the methane production for granule culture decreased slightly from 75.76 glucose to 61.00 ml CH4 g−1 glucose. Granules showed their advantage in methane production not only in terms of their higher methanogenic activity, but also by providing a protective niche for methanogens from the harsh environmental change, in this case the acid shot. Acid treatment has already been proved be an effective way to eliminate methanogens from sewage sludge and turn sewage sludge to hydrogen producing sludge as most of hydrogen producing bacteria in the sewage sludge are spore-forming Clostridia that can tolerate harsh environment. However, acid treatment of granules did not eliminate the methane production dramatically and could not turn methanogenic granule to be hydrogen producing granules because methanogens were protected by granular structure.
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Table 1 Cumulative gas production for acid treatment of granule and sewage sludge H2 ml g−1 glucose
Acid pretreatment
Granule Sewage
Control Acid Control Acid
CH4 ml g−1 glucose
Average
Standard dev
Average
Standard dev
0.42 0.00 124.99 89.01
0.50 0.00 26.42 14.45
75.76 61.00 0.74 0.26
24.75 24.21 0.16 0.14
Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml. Data was the average of triplicates with standard deviations (n = 3) at = 0.05. Table 2 Cumulative gas production for heat treatment of granule and sewage sludge H2 ml g−1 glucose
Heat pretreatment
Granule
Sewage
Control Heat (10 min) Heat (30 min) Control Heat (10 min) Heat (30 min)
CH4 ml g−1 glucose
Average
Standard dev
Average
Standard dev
0.42 0.34 118.29 124.99 121.29 134.12
0.50 0.48 0.00 26.42 25.70 5.77
75.76 59.97 0.06 0.74 0.01 0.00
24.75 12.45 0.00 0.16 0.01 0.00
Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml. Data was the average of triplicates with standard deviations (n = 3) at = 0.05. Table 3 Cumulative gas production for chloroform addition to granule and sewage sludge culture Chloroform added into culture medium (%)
Granule
Sewage
5 2.5 1 0.5 0.25 0.05 Control 5 2.5 1 0.5 0.25 0.1 Control
H2 ml g−1 glucose
CH4 ml g−1 glucose
Average
Standard dev
Average
Standard dev
0.30 0.00 29.13 94.15 121.41 135.09 0.42 0.61 0.00 32.87 45.57 113.14 145.32 124.99
0.51 0.00 5.28 27.87 14.59 4.43 0.50 1.06 0.00 36.13 17.48 11.09 27.91 26.42
0.14 0.00 0.04 0.03 0.10 0.04 75.76 0.53 0.56 0.16 0.14 0.17 0.12 0.74
0.12 0.00 0.03 0.02 0.18 0.04 24.75 0.06 0.13 0.03 0.02 0.00 0.02 0.16
Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml. Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
3.2. Heat pretreatment Methanogenic activity was almost eliminated from sewage sludge culture after 10 min heat treatment of sewage sludge (Table 2). This method has already been proved to be effective and the results of this study confirm that. For granules on the other hand, methanogen was still very active with massive methane production after the granules were treated by heat for 10 min. Longer treatment time was needed to eliminate all methanogenic activity as shown in Table 2. After 30 min heat treatment, granule culture could be able to produce hydrogen instead of methane. Similar reason to acid treatment, granu-
lar structure provided protection for methanogens against harsh environment, leading to longer time required for heat treatment to eliminate methanogens. 3.3. Chloroform pretreatment Chloroform showed strong inhibitory effects when added into both granules and sewage sludge culture systems (Table 3). More than 2.5% of chloroform addition severally inhibited both hydrogen and methane production for both the granule culture and sewage sludge culture. However, the toxicity of chloroform showed difference between hydrogen
B. Hu, S. Chen / International Journal of Hydrogen Energy 32 (2007) 3266 – 3273
Hydrogen ml/g glucose
160
Gas production ml/g glucose
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y = -117.11x + 148.75
140
R2 = 0.9873
120 100 80 60 40 20 0 0
0.2
0.4
0.6
0.8
1
1.2
Chloroform % Fig. 1. Chloroform inhibition on hydrogen production (20 ml glucose culture medium, three days each batch, initial pH = 8.0, inoculums 3 ml). Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
producing bacteria and methanogens at low level of chloroform addition into the culture medium. Methanogens were so sensitive to the chloroform that only 0.05% addition into the culture medium almost completely inhibited methane production even for granulized methanogens. Chloroform has been used to inhibit methanogenic activity in different fields [8] because chloroform can block the function of corrinoid enzymes and inhibit methyl-coenzyme M reductase. Weathers et al. [20] found that the loss of methanogenic activity was strongly correlated with the mass of chloroform transformed, rather than with the timeintegrated chloroform exposure which methanogens received. Meanwhile, with the decrease of chloroform addition from 2.5% to 0.05%, hydrogen production increased dramatically for both granule and sewage sludge culture. When only 0.1% chloroform added into the culture mediums, hydrogen production was even higher than the sewage sludge control culture. The inhibition of chloroform to hydrogen production also showed strong correlation to the mass of chloroform added into the culture system (Fig. 1). Hydrogen production was totally inhibited with the chloroform at a treatment level of above 2.5%, meanwhile, the system was transferred to methane production with no chloroform addition. The data of chloroform addition from 1% to 0.05% were fit to a linear equation: y = −117.11x + 148.75,
(1)
where, y was the volume of hydrogen production from pretreatment batch culture per gram glucose and x was the percent of chloroform concentration added into the culture medium. Based on this model, it can be estimated that the hydrogen production would be totally inhibited if the chloroform addition is higher than 1.27% of the culture medium. In addition to eliminating the methanogenic activity for hydrogen production system, chloroform can also inhibit the activity of hydrogen producing bacteria, mostly Clostridia, but if the concentration was controlled at low level, chloroform selectively inhibited methanogenic activity while did not affect the hydrogen production activity. It was also reported that chloroform can inhibit the activity of other hydrogen consuming bacteria such as homoacetogens and acetate-consuming sulfate-
180.00 Hydrogen
160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00 5
2.5
1 0.5 Chloroform %
0.25
0.05
Fig. 2. H2 production from culture of chloroform treated granule (20 ml glucose culture medium, three days each batch, initial pH = 8.0, inoculums 3 ml). Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
reducing bacteria [21]. This might be part of the reason why hydrogen production at 0.1% chloroform addition for sewage sludge culture was even higher than any other sewage sludge from other treatment methods. 3.4. Batch culture of chloroform pretreated granule Fig. 2 showed that all the chloroform treated granule cultures produced hydrogen and nearly no methane. For chloroform level below 2.5%, batch cultures produced nearly same amount of hydrogen no matter whether pretreated chloroform concentration was different, which means that the hydrogen production ability was not affected by the chloroform inhibition in the pretreatment process. For the pretreatment level of 2.5%, the average hydrogen production of the batch culture reached 166.15 ml g−1 glucose, which accounts the yield of 1.34 ± 0.11 mol H2 mol−1 glucose calculated as ideal gas at room temperature. The yield of the hydrogen production can be influenced by several factors, such as initial glucose concentration, fermentation time, reactor type, hydrogen partial pressure, etc. This yield is within the range summarized by Nath et al. [22] on reported fermentative hydrogen production by different organisms. To date, several approaches have been reported in the reference to increase the hydrogen production yield such as lowering the hydrogen partial pressure, or removing carbon dioxide in the headspace. Techniques for increasing the hydrogen yield will be a topic of research priority. For 5% chloroform treated granule culture, data showed significant fluctuation of hydrogen production, which might be caused by the inhibition of the remaining chloroform from previous pretreatment batch. Table 4 showed the comparison of cumulative gas production for untreated and treated granule inoculums. For the batch culture inoculated with methanogenic granules, acetic acid was accumulated in the beginning of the fermentation and was converted completely to methane at 18 days of fermentation. It can also be noticed that little hydrogen was accumulated in the beginning of the fermentation process, which means high homoacetogenic activity converting the hydrogen to acetate. For the batch culture inoculated with 0.1% chloroform treated
B. Hu, S. Chen / International Journal of Hydrogen Energy 32 (2007) 3266 – 3273
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Table 4 Comparison of cumulative gas production for untreated and treated granule inoculums Time (Days) Untreated granule
Treated granule
LA (g L−1 )
AC (g L−1 )
PR (g L−1 )
BU (g L−1 )
H2 (ml g−1 glucose)
CO2 (ml g−1 glucose)
CH4 (ml g−1 glucose)
3 18 3
0.00 0.00 1.91
3.13 0.00 1.82
1.65 0.00 0.00
0.00 0.00 2.65
7.79 1.04 109.87
292.66 453.93 290.12
80.88 374.37 0.01
3(18) 6(18) 18
0.00 0.00 0.00
0.79 0.67 2.00
0.35 0.71 0.88
1.58 2.71 1.79
180.64 176.68 0.06
317.91 364.50 284.01
1.44 1.49 13.71
(Batch culture, 20 ml glucose culture medium, three days, inoculums 3 ml, chloroform treatment 0.1%. Data was the average of triplicates with standard deviations (n = 3). LA: lactic acid; AC: acetic acid; PR: propionic acid; BU: butyric acid. Time 3(18) means that gas was released at day 3 and broth was measured at day 18, same as Time 6(18).
granules, VFA was also accumulated because methane production was inhibited. However, after gas release at three days fermentation or six days fermentation, detectable methane was recorded at 18 days when the fermentation was continued. In the same time, the acetic acid concentration of the broth at 18 days was significantly lower than that at three days, which means that there should be methanogenic activity recovery. It might have some residue methanogens after chloroform treatment, and still very little methane could be detected after 18 days of recovery because methanogens grow very slowly. There is no significant difference of cumulative hydrogen production between gas release at day 3 and at day 6, however, for the batch culture without gas release in the beginning of the fermentation, nearly no hydrogen was detected at day 18 with little amount of methane. CO2 production was also decreased. Homoacetogenic activity showed significant effect at long fermentation time, which means that chloroform treatment postponed or increased the lag time of the homoacetogenic activity, avoided fast conversion of hydrogen and CO2 to acetic acid. Little or no methane was produced from the chloroform treated cultures, which means that chloroform caused irreversible damage to methanogens, and the methanogenic activity did not recover even without chloroform existence. Meanwhile, hydrogen production ability recovered to normal even though chloroform partly inhibited hydrogen production at certain level in the pretreatment batch. Chloroform showed good selectivity on inhibition of hydrogen producing bacteria and methanogens, nearly permanently eliminated the methane production, postponed the hydrogen consumption to acetic acid, while allowed for normal hydrogen production recovery. Chloroform is required only in pretreatment process and the required concentration can be as low as 0.05%. 3.5. Comparison of acid treatment, heat treatment and chloroform treatment Acid pretreatment and heat pretreatment have already been proved effective for obtaining hydrogen producing sludge from sewage sludge. Their effectiveness was also confirmed in the this study. Chloroform addition showed same effectiveness for treating sewage sludge if the concentration was
controlled properly. Compare to the acid treatment or heat treatment of sewage sludge, chloroform treatment is also a simple step that can be easily implemented. Chloroform treatment might cause some chloroform pollution, but the concentration required is low (0.05%) without need for repeated treatment. From the experiment results, chloroform treatment showed many advantages over acid and heat treatments when dealing with granules because granular structure forms a protective environment. Chloroform resulted in more completely methanogens elimination. Furthermore, chloroform also postponed the activity of homoacetogens, another hydrogen consuming bacteria, while no reports about acid treatment and heat treatment mentioning about this possibility. Actually, some homoacetogens are also spore-forming bacteria, which are hardly eliminated by the heat treatment method [6]. 3.6. Repeat culture of chloroform treated granule There was no significant difference of hydrogen production for three repeated batch cultures with granules pretreated by 0.25% and 0.05% of chloroform. This result also confirmed that the chloroform treatment concentration can be lowered to 0.05%. Further repeated batch experiments only focused on 0.25% chloroform treated granules. Fig. 3 showed that chloroform treated granules can last for at least 8 batches (24 days) with nearly constant hydrogen production yet without obvious morphology damage. Chloroform treated granules can be the immobilized hydrogen producing bacteria for the immobilized hydrogen culture. 3.7. Continuous hydrogen production in the upflow reactor packed with chloroform treated granule Fig. 4 showed the performance of continuous hydrogen production in the upflow reactor packed with chloroform treated granules. The hydrogen productivity reached 11.6 L L−1 day−1 . After the HRT adjusted to below 4 h, the overall yield decreased, so the initial test for the continuous operation stopped. The granules changed its color gradually from deep black to dark gray, which might be caused by the
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160.00
0.25% 0.05%
Hydrogen ml/g glucose
140.00 120.00 100.00 80.00
irreversible damage to methanogens while hydrogen production ability recovered to normal after the pretreatment batch. Chloroform treated granule could be repeatedly cultured for eight time without obvious damage. The treated granule can be kept their structure over 15 days and new granule started to form after 10 days operation. Acknowledgments
60.00
Funding for this project was from the Washington Statue University Agriculture Research Center. We also thank Simon Smith for contributing thoughts to this study.
40.00 20.00
References
0.00 1
2
3
4
5 Batch
6
7
8
16
14
14
12
12
10
10 8
Hydrogen
8
HRT
6
6 4
4
2
2
0
HRT h
Hydrogen L /L /d
Fig. 3. H2 production from repeat culture of chloroform treated granule (20 ml glucose culture medium, three days each batch, initial pH = 8.0, inoculums 3 ml). Data was the average of triplicates with standard deviations (n = 3) at = 0.05.
0 0
2
4
6 8 Time days
10
12
Fig. 4. Continuous hydrogen production in the up-flow reactor packed with chloroform treated granule (20 g COD L−1 glucose culture medium, initial pH = 8.0, 0.1% chloroform treatment).
washing out of the sulfur reducing bacteria. The granular structure was mostly kept after 15 days operation. New granules, which have shallow gray color, could be seen after 10 days of operation. 4. Conclusion Acid treatment and heat treatment were proved to be effective to sewage sludge while not completely effective to methanogenic microorganisms from granules because of the protective environment from the granular structure. Chloroform treatment can be an effective way for eliminating methanogenic activity. Hydrogen production also was inhibited by chloroform. If the concentration was controlled at low level, chloroform selectively inhibited methanogenic activity while did not influence the hydrogen production activity. Chloroform caused
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