Improvement of Biohydrogen Production from Solid Wastes by

Environ. Sci. Technol. 2006, 40, 3409-3415

Improvement of Biohydrogen Production from Solid Wastes by Intermittent Venting and Gas Flushing of Batch Reactors Headspace

consortia are used (3). This process could be applied full scale if some operational problems, such as inhibition byproducts, are overcome. Hydrogen production (PH) is linked with organic acid formation according to the following typical equations based on carbohydrate fermentation (4):

IDANIA VALDEZ-VAZQUEZ, E L V I R A R IÄ O S - L E A L , A L E S S A N D R O C A R M O N A - M A R T IÄ N E Z , KARLA M. MUN ˜ O Z - P AÄ E Z , A N D H EÄ C T O R M . P O G G I - V A R A L D O * CINVESTAV-IPN, Department of Biotechnology and Bioengineering, Environmental Biotechnology R&D Group, P.O. Box 14-740, Me´xico D. F., 07000, Me´xico

Yet, under certain environmental conditions, acidogenic fermentation shifts to solventogenic fermentation (production of end products such as acetone, butanol and ethanol, among others) where H2 productivity decreases or stops (5, 6). Solventogenesis initiation is related to high partial pressures of hydrogen (pH2), high organic acids concentrations, and/or low pH. These environmental conditions may alter the electron flow in the biochemical pathway of the microorganisms causing PH inhibition and changes in the composition of the end products (7-10). Most of the studies on solventogenesis have used pure, liquid cultures, where works with Clostridium prevail. In this way, Clostridium has been identified as the microorganism with the highest tolerance to high pH2 before suffering a metabolic shift (11-15). Also, PH inhibition and metabolite distribution changes, due to high pH2 and/or high acid concentration, have been observed by some authors using anaerobic mixed cultures degrading complex substrates (16, 17). Only a few works have used strategies for overcoming the negative effect of high pH2 in studies of hydrogen production by consortia. It was reported that continuous release of biogas could enhance hydrogen production with respect to the conventional release Owen method in one cycle incubation of glucose (18). Other researchers continuously sparged with an inert gas to continuous mesophilic reactors fermenting soluble substrates by anaerobic consortia; however, this procedure seemed to dilute the obtained biogas (5-7% v/v), and the cost for H2 purification and concentration could be very high. (19, 20). On the other hand, intermittent venting of solid substrate for anaerobic H2 production (IV-SSAH) plus one time flushing was utilized in mesophilic batch reactors loaded with paper mill waste and seeded with an anaerobic consortium (5). This procedure was effective for eliminating the inhibitory effect of high pH2 allowing at the same time a biogas rich in H2, which probably requires less purification. Also, in situ removal of H2 by means of membranes has been used to reduce the inhibitory effect of high pH2; however, reported H2 production improvement was very poor (21). PH by anaerobic consortia commonly involves the application of treatments to induce H2 accumulation by means of elimination/inhibition of H2-consuming microorganisms; heat-shock pretreatment and acetylene have been used with this purpose (3). However, there are no comparative works of these induction treatments under meso- and thermophilic conditions. Thus, the objective of this work was to study the effects of (a) type of inocula, (b) induction treatment, and (c) incubation temperature on H2 production (PH), initial H2 production rate (Ri,H), lag time (Tlag), and metabolite distribution in batch reactors degrading organic solid wastes by anaerobic consortia. Reactors headspace was intermittently vented and gas flushed when PH inhibition was observed.

Headspace of batch minireactors was intermittently vented and gas flushed with N2 in order to enhance H2 production (PH) by anaerobic consortia degrading organic solid wastes. Type of inocula (meso and thermophilic), induction treatment (heat-shock pretreatment, HSP, and acetylene, Ac), and incubation temperature (37 and 55 °C) were studied by means of a factorial design. On average, it was found that mesophilic incubation had the most significant positive effect on PH followed by treatment with Ac, although the units with the best performance (high values of PH, initial hydrogen production rate, and short lag time) were those HSP-induced units incubated at 37°C (type of inocula was not significant). In this way, after 720 h of incubation PH was inhibited in those units by H2 partial pressure (pH2) of 0.54 atm. Venting and gas flushing with N2 was efficient to eliminate that inhibition achieving additional hydrogen generation in subsequent incubation cycles although smaller than the first one. Thus, four cycles of PH were obtained from the same substrate with neither addition of inocula nor application of induction treatment obtaining an increment of 100% in the generated H2. In those subsequent cycles there was a positive correlation between PH and organic acids/solvent ratio; maximum values were found in the first cycle. Solventogenesis could be clearly distinguished in third and fourth production cycles, probably due to a metabolic shift originated by high organic acid concentrations.

Introduction The use of fossil fuels has generated strong environmental problems, whereas production of high quality fuels is more and more expensive (1). Experts have suggested that H2 could substitute for fossil fuels in a near future since it fulfills diverse energetic, economic, and environmental requirements (2). Hydrogen can be produced by chemical, thermal, and biological processes. The latter are the most interesting since H2 can be generated from renewable sources (i.e., nonsterile organic wastes) with low energy supply when fermentative *Corresponding author phone 5255 5061 3800 x 4324; fax: 5255 5061 3313; e-mail:[email protected]. 10.1021/es052119j CCC: $33.50 Published on Web 04/08/2006

 2006 American Chemical Society

Hexose f 2 H2 + Butyrate + 2CO2

(1)

Hexose + 2H2O f 4 H2 + 2 Acetate + 2CO2

(2)

Materials and Methods Feedstock and Seed Microorganisms. Paper (40% wet basis) and food (60% wet basis) wastes were mixed to obtain a VOL. 40, NO. 10, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Cumulative Hydrogen Production from Solid Waste Fermentation in Each Incubation Cycle According to Full Factorial Design in Natural Units factor A

B

C

M T M T M T M T

HSP HSP HSP HSP Ac Ac Ac Ac

M M T T M M T T

a

PH aper incubation cycle

background PH 0.86 ( 0.35 1.49 ( 0.15 0.00 ( 0.00 0.00 ( 0.00 0.24 ( 0.10 1.16 ( 0.06 0.50 ( 0.10 0.19 ( 0.08

1

2

3

4

10.17 ( 0.22 11.89 ( 0.38 0.67 ( 0.28 0.34 ( 0.14 8.01 ( 0.45 10.19 ( 0.81 5.82 ( 0.84 3.85 ( 0.17

5.21 ( 0.21 4.95 ( 0.11 2.23 ( 0.42 3.05 ( 1.24 5.39 ( 0.54 7.24 ( 0.36 3.76 ( 0.34 5.88 ( 0.50

3.07 ( 0.42 2.46 ( 0.23 1.80 ( 0.24 1.21 ( 0.49 2.25 ( 0.09 3.02 ( 0.21 2.37 ( 0.45 1.62 ( 0.15

2.58 ( 0.25 1.89 ( 0.66 0.01 ( 0.00 0.11 ( 0.05 1.77 ( 0.72 1.08 ( 0.20 0.10 ( 0.04 0.00 ( 0.00

Net H2 production average in each incubation cycle (mmoles H2 / unit).

model organic fraction of municipal solid waste and prepared as previously described (4, 22). The main characteristics of feedstock were as follows: TS 25%; VS 18%; pH 9.0, total Kjedahl nitrogen (% TS) 1.9%, cellulose (% TS) 26.2%, and lignin (% TS) 19.4%. Seed microorganisms were obtained from methanogenic solid substrate anaerobic digesters degrading a mixture of organic solid wastes (23). Experimental Design. The experiment was a full factorial 23 with two replicates (Table 1A) to describe the main effects of type of inocula (factor A) at two levels (meso- and thermophilic), induction treatment (factor B) at two levels (heat-shock pretreatment, HSP, and acetylene, Ac) and incubation temperature (factor C) at two levels (37 and 55 °C) on PH, Ri,H, Tlag, and metabolite distribution in subsequent incubations. The results were subjected to analysis of variance using Design-Expert 6.0.0 (24). Experimental Minireactors and Procedure. Glass bottles of 250 mL volume were loaded with 20 g inoculum and 80 g of wet substrate according to the experimental design above and to procedures described elsewhere (5). When HSP was used, just before the inoculation, the inocula were heatshock treated at 93 °C for 60 min. in a boiling water bath. When Ac was used, it was injected into the anaerobic gas phase to a final concentration of 1% v/v (25). The bottles were incubated statically in the dark at 37 and 55 °C. During the incubation the biogas in headspace was frequently released (intermittently vented) to maintain atmospheric pressure of 0.77 atm. When a maximum H2 cumulative production was observed, the bottles’ headspace was vented and then one-time flushed with N2 to wash-out the accumulated H2. After this, the bottles were reincubated; neither fresh inoculum nor substrate addition nor induction treatment was applied again (5). Two control bottles of each treatment (blank) were also prepared without substrate addition. Analytical Methods. Biogas composition in the headspace was determined by GC (4, 5). Total solids, volatile solids, total Kjeldahl nitrogen, cellulose, and lignin were determined in the feedstock as reported elsewhere (26). The pH and volatile organic acids were determined according to procedures described by Valdez-Vazquez et al. (4).

Results and Discussion In the first cycle, it was found that induction treatment and incubation temperature had a significant effect on response variables. Thus, on average Ac-treated units had 20% more PH and a shorter Tlag than HSP-induced units (first cycle of Figure 1a,b). Yet, on an individual basis, HSP-induced units incubated at 37 °C had highest PH and Ri,H. Lowest values of PH and Ri,H were registered for HSP units incubated at 55 °C (Tables 1 and 2A, Supporting Information). As a consequence, average Ri,H in HSP-induced units was similar to average Ri,H in Ac-induced units (first cycle of Figure 1c). 3410

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total PH (∑14PH)b

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b

21.03 ( 3.10 21.20 ( 0.02 4.71 ( 0.65 4.71 ( 2.32 17.42 ( 3.96 21.53 ( 2.34 12.05 ( 3.77 11.35 ( 2.57

Cumulative sum of H2 produced in the four incubation cycles.

Regarding the effect of incubation temperature, units incubated at 37 °C had the highest values of PH with the lowest Tlag. Also, average Ri,H was positively affected by mesophilic incubation (first cycle, Figure 1d,e,f). However, under thermophilic incubation, only Ac-induced units had significant to moderate PH (Table 1). Thus, there was a strong negative two-factor interaction between induction treatment and incubation temperature (24). It seems that the superiority of induction treatment depends on incubation temperature. Thus, HSP-induced units were superior to Ac-induced units only under mesophilic incubation; this is probably because this incubation temperature was the best for germination of spores selected by HSP. Contrary to this, Ac-induced units had similar performance under meso- and thermophilic incubation. This could be due to the fact that this treatment permits a superior quantity/diversity of H2-producing microorganisms, while HSP only selects spore-forming microorganisms, but those H2 producers that cannot sporulate are not able to survive to this pretreatment. The PH and Tlag obtained in our work were similar to those reported by other authors under mesophilic conditions using microbial consortia degrading OFMSW (27-29). However, the negative interaction between HSP and subsequent thermophilic incubation had not been reported before in studies of hydrogen production. Regarding this, other works focused on bacterial spore germination have determined that some germinants (amino acids, carbohydrates, salts) are required for optimal and complete germination of spores under thermophilic conditions (30). It should be highlighted that the substrate utilized in our work was not supplemented with specific germinants; in consequence, this fact could contribute to poor performance of the HSP-induced units incubated at 55 °C. In addition, it is possible that HSP could negatively affect the survival of present spores in the inoculum since it has been reported that prolonged heat treatment at 90 °C resulted in a thermal destruction of spores (31, 32). For that reason, it is likely that HSP duration should be limited to the necessary time for eliminating or killing the H2consuming microorganisms. The pH2 increased in most mesophilic units around 720 h of incubation reaching a first H2 plateau at which H2 accumulation stopped (Figure 1A, Supporting Infomation). At that time, the IV-SSAH procedure was applied in all units (including those with slight H2 accumulation). It was found that PH was resumed obtaining another H2 plateau although smaller than the first one and lower Ri,H than in those units that produced H2 in the first cycle. Therefore, it was observed that high pH2 was an important factor affecting PH in batch reactors degrading OFMSW by anaerobic microflora. As a general tendency, cumulative PH and Ri,H in subsequent incubation cycles were lower than those obtained in the first cycle (Table 1 and Table 2A, Supporting Information). Also, the effects of induction treatment and incubation

FIGURE 1. I. Effect of induction treatment on (a), cumulative H2 production; (b), lag time, and (c), initial H2 production rate in each incubation cycle. HSP, heat-shock treatment; Ac, acetylene; p, level of significance; ns, not significant. II. Effect of incubation temperature on (d), cumulative H2 production; (e), lag time, and (f), initial H2 production rate in each incubation cycle. M: mesophilic incubation; T: thermophilic incubation; p, level of significance; ns, not significant. temperature on response variables were less significant in subsequent production cycles (Figure 1). This is probably due to consumption of the most easily degradable substrate in the first cycle and also a presumably secondary inhibitory effect due to accumulation of metabolites such as organic acids and solvents in the solid digestates. The intermittent venting, plus one time flushing with inert gas, seemed to avoid the inhibitory effect of high pH2 (and, likely, it also decreased the CO2 concentration in biogas, minimizing a possible loss of H2 via acetogenesis) (33), and three more cycles with PH were obtained from the same substrate with neither addition of new inocula nor application of induction treatment. Thus, in all units the overall produced H2 in the four incubation cycles was double or higher than that of the first cycle alone (Table 1). Other researchers have used continuous sparging to avoid the inhibitory effect by high pH2. Mizuno et al. (19) found that N2 sparging at a flow

rate approximately 15 times the H2 generation rate resulted in a 68% increase in H2 yield (0.85 to 1.43 mol/mol hexose) and an increase of 59% of H2 production in a continuous liquid culture at 35 °C fermenting glucose by a microbial consortium. However, H2 percentage decreased from 53.4% to 5.3% in the biogas. Hussy et al. (20) utilized a mesophilic chemostat reactor at 15 h hydraulic retention time fermenting soluble wheat starch by a mixed microflora, which was continuously sparged with N2 (59.1 ( 2.8 mL/min), to investigate the effect of pH2 decrease on H2 yield. In that work, H2 yield increased from 1.26 to 1.87 mol/mol hexose consumed (an increase of 48%). Yet, H2 concentration in the biogas was very low; H2 percentage decreased from 57 to 7.0%. So, sparging at high flow rate dilutes the obtained biogas, and the cost for H2 purification and concentration could be very high. Contrary to this fact, the intermittent venting followed by one time flushing with inert gas at the VOL. 40, NO. 10, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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end of each cycle of incubation exhibits several advantages over continuous sparging. For example, H2 percentage in the biogas is not diminished during venting (5 to 75% v/v hydrogen, average 50%), whereas H2 concentration in the last portion of harvested biogas after headspace flushing is nearly 20-30%. Flushing headspace with N2 might have minimized inhibition of hydrogen production by high pH2 (18), and it is likely that CO2 percentage in headspace after flushing also decreased so any eventual hydrogen sink by acetogenesis could be minimized (33); the substrate utilization at the end of the fourth production cycle was augmented, and eventually lower costs for used N2 and for H2 purification and concentration could be achieved. These facts were corroborated by Valdez-Vazquez et al. (5) who utilized the intermittent venting and one time gas flushing in mesophilic batch reactors with paper mill waste as substrate and acetylene-inhibited mesophilic consortia as seed. Their results also showed that this procedure was effective for eliminating the inhibitory effect by H2 accumulation obtaining, at the same time, a biogas rich in H2, which probably requires less purification. An improvement of PH from 17 to 34 mmol H2/ reactor was also found in that report, in agreement with an increase of 100% in current work (Table 1) Clostridium incubated under pH2 approximately between 0.5 and 2.5 atm exhibited metabolic shift where the major change occurred at pH2 lower than 2.5 atm (12, 13, 15). Other works with thermophilic microorganisms observed inhibition of PH along with a metabolic shift from very low pH2 of 0.016 to 0.75 atm (14, 33-37). In our work, pH2 at which the first PH plateau was observed (0.45 to 0.54 atm), was superior or similar to most values found in earlier works, except for Clostridium (12, 13). Despite relatively significant pH2 reached in our batch reactors, no shift to solventogenesis was found at the end of first cycle. This result suggests an advantage when using consortia instead of pure strains; in a consortium it is possible to have microorganisms that are inhibited at low pH2 but it is also likely to find microorganisms with higher tolerance to high pH2; in consequence, the average capacity of the consortium to tolerate high pH2 would increase without the added drawback of a metabolic shift to solventogenesis. Figure 2 summarizes the distribution of organic acids (acetate, butyrate, and propionate) and solvents (acetone and ethanol) at the end of each production cycle in our batch minireactors. On average, it was observed that only in the first cycle did the incubation temperature and inhibition treatment have significant effects on metabolites distribution. Highest organic acid concentrations (88.5% on basis of mmoles/kg wet basis) were observed in units incubated at 37 °C (p < 0.001, which correlated with highest H2 values) and inhibited with acetylene (p < 0.01). Yet, a generalized H2: acetate ratio (2:1), according to eq 1, was not found, as would have been expected. This could be because our study used a microbial consortium where probably the metabolism was directed toward several fermentation pathways. In subsequent cycles the main factors of our experiment did not seem to have significant effects on metabolite distribution. However, at the end of the second cycle the metabolites distribution slightly changed showing that organic acids proportion declined (constituting ca. 65.6% of the accumulated metabolites) while solvent proportion augmented. So, in this cycle a metabolic shift to solvent production started. In the third and fourth incubation cycles solventogenic fermentation could be clearly distinguished since approximately 60-70% of the total of accumulated metabolites were constituted by solvents such as ethanol which, according to eq 3, are not linked with PH (6):

Hexose f 2 Ethanol + 2 CO2 3412

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(3)


Also, an interconversion of organic acids plus hydrogen yielding solvents could contribute to solvent increase (38). In general, solvent percentage increased from 11.5 to up 70% at the end of the fourth production cycle while organic acids decreased from 88.5 to less than 30%. The fact that solvent concentration increased in subsequent production cycles would be expected due to different physiological events such as high pH2 and/or high organic acids concentration, which are favorable conditions for inducing the shift from acidogenic to solventogenic fermentation in Clostridia (7, 10, 15). In our work at the beginning of the second cycle, pH2 was very low or zero due to IV-SSAH application and pH in our units was between 6.69 and 8.39; however, the total organic acids concentration (dissociated plus undissociated forms) was very high in some units. Previous reports have suggested that both dissociated and undissociated acids can exert inhibitory effects on H2-producing microorganisms (39), although it is generally accepted that the cell is far more sensitive to the undissociated acid than the corresponding anions (40, 41). Undissociated acid concentration (UAC) was calculated on the basis of pKa for each acid using HendersonHasselbalch equation as a function of pH and was considered to be equal inside and outside the cells since undissociated acids permeates freely across cell membranes (9). In general, the highest UAC were found in units incubated at 37 °C (1.59 and 3.32 mM). It has been reported that a threshold UAC is necessary for the initiation of solventogenesis along with PH inhibition using pure cultures of Clostridium and others, although the threshold can range from as low as 0.3 up to 40 mM (Table 3A, Supporting Information) (9, 10, 39, 42, 43). From this comparison, it seems that inhibitory levels of free acids and anions on H2-producing microorganisms widely differ among species (44). In our work, the maximum UAC (acetic acid, propionic acid plus butyric acid) achieved was 3.3 mM. These results indicate that, probably, the metabolic shift to solventogenesis using anaerobic consortia in cycles 2, 3, and 4 of our work could be induced mainly by organic acid concentration rather than high pH2. Decrease of PH in subsequent production cycles was in agreement with increased solvent generation. This pattern has been observed during solventogenic fermentation. Decline of organic acids concentration results in pH increase and is due to uptake and reutilization of these acids by the cell in order to avoid the inhibitory effect by low pH (38). In our study, this pattern was observed since pH decreased at the end of the first cycle and later increased at the end of fourth cycle (Table 4A, Supporting Information). Jones and Woods (38) reported that solventogenesis is coupled to acid uptake; in our work, a pH increase along with organic acids decrease were observed in the last incubation cycles (Table 4A and Figure 2A, Supporting Information). Also, it seems pH change was well correlated with hydrogen production, implying the pH changed in proportion to volatile acids produced. In most of the units there was a strong relationship between H2 and generated organic acids with the exception of HSP-induced units incubated at 55 °C in the first cycle which probably suffered a strong shock. The relationship shown in Figure 2A was expected since several authors using clostridia genera have reported that solvent production inhibits PH as it was discussed above. Consequently, high organic acids concentrations found at the end of first incubation cycles seemed to induce the shift to solventogenic fermentation which, in turn, lowered H2 production in subsequent cycles of incubation. Overall it was apparent that batch IV-SSAH of OFMSW led to a 100% improvement of hydrogen generation compared to one cycle batch fermentation process, despite a shift to solventogenesis in the last incubation cycles. Our research demonstrates that inhibited methanogenic consortia can be

FIGURE 2. Effect of intermittent venting and gas flushing on organic acids and solvents distribution at the end of each incubation cycle. I. Bottles inhibited with heat-shock pretreatment; II. Bottles inhibited with acetylene; M, mesophilic; T, thermophilic. used to produce significant amounts of hydrogen from organic wastes at high total solids contents. Hydrogen in biogas can be purified and concentrated in new generation

membrane separation processes (45) and burned in fuel cells for in situ or ad situ electric energy generation as suggested by Logan et al. (18). Byproducts of this process are organic VOL. 40, NO. 10, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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acids and solvents, which can be extracted from the spent solids and further purified for use as organic chemicals by the chemical industry or biofuels (46, 47), or further methanized as extracts (18, 48) or as whole spent solids in solid substrate anaerobic digestion processes (23). Washed spent solids, in turn, can be transformed into soil amenders for remediation of eroded and saline soils (49). Our approach could significantly influence municipal and industrial solid waste management and disposal practices, as they are known today, and it can be considered a first step toward new, more cost-effective and sustainable strategies for dealing with the solid waste problem in modern societies.

Supporting Information Available Additional details about this experiment. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review October 24, 2005. Revised manuscript received February 7, 2006. Accepted February 10, 2006. ES052119J

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