Hydrogen generation via anaerobic fermentation of paper mill wastes

Hydrogen generation via anaerobic fermentation of paper mill wastes

Bioresource Technology 96 (2005) 1907–1913 Hydrogen generation via anaerobic fermentation of paper mill wastes Idania Valdez-Vazquez a, Richard Sparl...

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Bioresource Technology 96 (2005) 1907–1913

Hydrogen generation via anaerobic fermentation of paper mill wastes Idania Valdez-Vazquez a, Richard Sparling b, Derek Risbey b, Noemi Rinderknecht-Seijas c, He´ctor M. Poggi-Varaldo a,* a

CINVESTAV-IPN, Department of Biotechnology and Bioengineering, Environmental Biotechnology R&D Group, P.O. Box 14-740, Me´xico D.F., 07000, Me´xico b University of Manitoba, Department of Microbiology, Winnipeg, Man., Canada c ESIQUIE-IPN, Division Basic Sciences, Me´xico D.F., Me´xico Received 17 December 2003; received in revised form 27 January 2005; accepted 27 January 2005 Available online 18 April 2005

Abstract The objective of this work was to determine the hydrogen production from paper mill wastes using microbial consortia of solid substrate anaerobic digesters. Inocula from mesophilic, continuous solid substrate anaerobic digestion (SSAD) reactors were transferred to small lab scale, batch reactors. Milled paper (used as a surrogate paper waste) was added as substrate and acetylene or 2bromoethanesulfonate (BES) was spiked for methanogenesis inhibition. In the first phase of experiments it was found that acetylene at 1% v/v in the headspace was as effective as BES in inhibiting methanogenic activity. Hydrogen gas accumulated in the headspace of the bottles, reaching a plateau. Similar final hydrogen concentrations were obtained for reactors spiked with acetylene and BES. In the second phase of tests the headspace of the batch reactors was flushed with nitrogen gas after the first plateau of hydrogen was reached, and subsequently incubated, with no further addition of inhibitor nor substrate. It was found that hydrogen production resumed and reached a second plateau, although somewhat lower than the first one. This procedure was repeated a third time and an additional amount of hydrogen was obtained. The plateaux and initial rates of hydrogen accumulation decreased in each subsequent incubation cycle. The total cumulative hydrogen harvested in the three cycles was much higher (approx. double) than in the first cycle alone. We coined this procedure as IV-SSAH (intermittently vented solid substrate anaerobic hydrogen generation). Our results point out to a feasible strategy for obtaining higher hydrogen yields from the fermentation of industrial solid wastes, and a possible combination of waste treatment processes consisting of a first stage IV-SSAH followed by a second SSAD stage. Useful products of this approach would be hydrogen, organic acids or methane, and anaerobic digestates that could be used as soil amenders after post-treatment.  2005 Elsevier Ltd. All rights reserved. Keywords: Anaerobic digestion; Anaerobic fermentation; Hydrogen; Paper wastes; Solid substrate

1. Introduction Fossil fuels (i.e., petroleum, natural gas and coal), which meet most of the worlds energy demand today, are being depleted rapidly (Das and Veziroglu, 2001). Also, their combustion products are causing global * Corresponding author. Tel.: +5255 5061 3800x4324; fax: +5255 5061 7002. E-mail address: [email protected] (H.M. Poggi-Varaldo).

0960-8524/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.01.036

problems, such as the greenhouse effect, ozone layer depletion, acid rain and pollution. The search for renewable energy sources is an important issue for attaining both sustainable development and a higher efficiency of the industry performance in modern societies. There are quite a number of primary energy sources available, such as thermonuclear energy, nuclear breeders, solar energy, wind energy, hydropower, geothermal energy, ocean currents, tidal and wave energy. In contrast with the fossil fuels, none of the new primary energy sources can be used directly as a fuel, e.g., for

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air transportation, land transportation. Consequently, they must be used to manufacture a fuel or fuels, as well as to generate electricity. There are many candidates, such as synthetic gasoline, synthetic natural gas (methane), methanol, ethanol and hydrogen. The fuel of choice must satisfy the following conditions (Veziroglu and Barbir, 1992): ease of transportation; it should be versatile or convert with ease to other energy forms at the user end; it must have high utilization efficiency; and its handling and use should be safe. In addition, the resulting energy system must be environmentally compatible and economical. Hydrogen appears to be one of the best transportation fuels, the most versatile, the most efficient and the safest fuel (Veziroglu, 1987; Barbir et al., 1990; Veziroglu and Barbir, 1992). Burning hydrogen produces only water with no CO, CO2, hydrocarbons or fine particles, and since it can be produced without causing any ecological disorder, hydrogen as a future fuel has been drawing greater attention (Yamin et al., 2000). Hydrogen can be produced chemically, electrochemically, as a by-product of oil/coal processing, or by the use of microorganisms. There are mainly, two systems to obtain microbial hydrogen production, namely photochemical and fermentative. The former consists in producing by means of photosynthetic microorganisms such as algae and photosynthetic bacteria (Ike et al., 1997; Melis and Happe, 2001). The second one is carried out by fermentative hydrogen-producing microorganisms, such as facultative anaerobes and obligate anaerobes (Joyner and Winter, 1977; Nandi and Sengupta, 1998). Studies on microbial hydrogen production have been conducted mostly using pure cultures, either natural or genetically modified (Asada et al., 2000; Evvyernie et al., 2000, 2001; Fabiano and Perego, 2002). Hydrogen is a key intermediate in the anaerobic degradation of organic compounds and may be recovered from wastewater (Ueno et al., 1996) or solid waste (Sparling et al., 1997; Lay et al., 1999; Mizuno et al., 2000) using mixed cultures. In these studies, hydrogen production resulted from the inhibition of methane fermentation. On the other hand, solid substrate anaerobic digestion (SSAD) has been shown to be an effective way of reclaiming paper mill sludge as well as obtaining methane as a fuel and soil amender or protein enrichments from the digested solids (Poggi-Varaldo et al., 1997a,b, 1999, 2002). Yet, methane or carbon dioxide as its combustion products are known to be greenhouse gases (Dickinson and Cicerone, 1986). Hydrogen might be produced from paper wastes using microorganisms from SSAD, suppressing the activity of hydrogenotrophic methanogens with specific and non-specific inhibitors such as 2-bromoethanesulfonate (BES) or acetylene (Sparling and Daniels, 1987; Sparling et al., 1997). In Me´xico, a total of 423,000 tonne/day of municipal solid wastes and industrial solid wastes are generated.

The main component of the MSW is organic putrescible wastes, making 50–60% of the waste stream. The Mexican pulp and paper industry (PPI) alone generates a conservatively low estimate of 110,000 dry tonne/yr of paper mill sludge and other solid wastes, and it ranks as second in the list of industrial polluters (Poggi-Varaldo, 1994; Poggi-Varaldo et al., 1999). Currently, an important proportion of the solid waste stream is dumped in sites which do not meet actual sanitary landfill design standards and environmental regulations. Therefore, the objective of this work was to determine the hydrogen production from paper mill wastes using microbial consortia of solid substrate anaerobic digesters. If feasible, in the future it could alleviate the waste treatment and disposal and at the same time it would contribute to the sustainable development of the PPI.

2. Methods Inocula were drawn from mesophilic, continuous methanogenic solid substrate anaerobic digesters MSSAD degrading a mixture of municipal solid wastes operated at 21 day mass retention time. More details regarding the reactor start-up, operation and monitoring can be found elsewhere (Poggi-Varaldo et al., 1997a, 1999; Valdez-Va´zquez, 2003). In short, M-SSAD bioreactors were loaded with a 330 g of cattle manure, 330 g of screened garden soil, and 330 mL of waste activated sludge. 10 mL of a stock of concentrated sucrose and sodium bicarbonate was added to each reactor to give a concentration of 2 g sucrose/kg reactor mass content and 4 g CaCO3 alkalinity/kg reactor mass content and manually shaken. Headspace of reactor was flushed with inert N2 gas and subsequently stoppered with a rubber stopper fitted with a nipple, tee, and tubing for biogas exit. Reactors were connected to acidic brine biogas meters and incubated in a mesophilic walk-in room in batch mode for two days. After incipient biogas production, they were fed with 10 mL of sucrose/bicarbonate concentrated stock for a second round of biogas production. This procedure was repeated (usually once more) until at least a concentration of 60% of methane in biogas was reached. Then, reactors were fed twice a week with solid substrate (a mixture of 66% food and 33% paper wastes) at 35% total solids content in such amount to give an average mass retention time of 21 days. Milled and mashed waste office paper was used as model substrate for batch reactors. Dry waste paper was cut in thin threads in an office shredder and further milled in a home blender for ca. 5 min. The milled paper had an average diameter corresponding to mesh 40 ASTM (Perry et al., 1963; p. 21–51). Approximately 20 g of the milled paper was mixed with 60 ml of med-

ium to even moisture distribution and give 25% (w/w) dry matter content. Once mixed, 80 g (wet basis) was added in recycled 250 mL glass juice bottles which were stoppered using green butyl-rubber stoppers and a metallic harness. The bottles were made anaerobic under N2 using an evacuation-gas replacement method. These were subsequently autoclaved and stored until used. The medium used for wetting the office paper was modified from Roychowdhury et al. (1988), and contained per liter: 20 g KH2PO4, 30 g K2HPO4, 12.5 g NH4Cl, 5 g NaCl, 2 g MgSO4, 0.43 g Na2S. In the anaerobic glove chamber, 20 g of inoculum from SSAD reactor was weighed and transferred into fresh bottles which were restoppered. These were subsequently vented and flushed with N2. The bottles were statically incubated at 37 C. When acetylene was used, it was injected into the anaerobic gas phase to a final concentration of 1% (v/v). BES was added from a 2.5 M anaerobic stock solution to a final concentration of 25 mM (Sparling and Daniels, 1987). Experiments were performed twice, in duplicate. Biogas production of M-SSAD was measured on a daily basis by acid brine displacement. Biogas volumes were reported as the daily average for the feed cycle, at 0 C and 1 ata (NmL) and corrected for moisture. Hydrogen production in batch reactors was determined by gas chromatography of headspace and doing mass balances taking into account the volume of the chromatographic syringe and the volume of reactor headspace. Hydrogen and methane contents were determined by gas chromatography in a GOW-MAC chromatograph model 350 fitted with a thermal conductivity detector (TCD) and Molecular Sieve 5A packed column (injector, detector and column temperatures were 25, 100 and 25 C, respectively) with He as carrier gas (PoggiVaraldo et al., 1997a).

3. Results and discussion In the first phase of experiments with mashed paper as substrate, it was found that acetylene at 1% v/v in the headspace was very effective in inhibiting methanogenic activity (Fig. 1). Hydrogen gas accumulated in the headspace of the bottles, reaching a plateau. In contrast, a control reactor seeded with SSAD inocula and substrate continued methane production and hydrogen accumulation was none. It can be seen that the exposure of the batch minireactors to air (that is, 20% oxygen in air) had a slight inhibitory effect on the methanogenic microflora, which reflected as a small peak of hydrogen accumulation and negligible methane production in the first 80 h of incubation (Fig. 1). Yet, the inhibitorial episode was overcome and the consortia consumed the hydrogen and

H2 or CH4 accumulation (mmole/reactor)

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C2H2

CH4

10

8

H2 6

4

CH4

2

H2 0 0

500

1000

1500

Time (h) Fig. 1. The effect of 1% acetylene on methane and hydrogen production from an anaerobic, methanogenic consortium in a batch minireactor degrading paper. (s) methane production and (d) hydrogen accumulation in an non-exposed, non-inhibited minireactor (inoculum plus mashed paper). (h) methane production and (j) hydrogen accumulation of a batch minireactor exposed to air at the start-up and spiked with acetylene at 420 h. The arrow represents the time at which acetylene was added.

started to produce methane (period 180–400 h). It has been reported that methanogenic archaea in anaerobic, methanogenic consortia can tolerate exposure to molecular oxygen (Estrada-Va´zquez et al., 2001, 2002). Estrada-Va´zquez et al. (in press) also demonstrated that the protective effect increased with increasing concentrations of sucrose. Since our paper-to-hydrogen minireactors posses abundant degradable organic matter and are rich in facultative microorganisms, we could expect a strong protective effect (biochemical shield) of the methanogenic bacteria from oxygen via aerobic respiration. Similar hydrogen concentrations were obtained for reactors spiked with acetylene and BES, although a slightly lower for BES at the end of the first cycle of incubation (Table 1, Fig. 2). In the second phase of tests using paper mill primary sludge as substrate, the headspace of the batch reactors was flushed with N2 after the first plateau of hydrogen was reached, and subsequently incubated, with no further addition of inhibitor nor substrate (Fig. 2). It was found that hydrogen production resumed and reached a second plateau, although somewhat lower than the first one (Fig. 2). This procedure was repeated a third time and an additional amount of hydrogen was obtained. The plateaux and initial rates of hydrogen accumulation decreased in each subsequent cycle of incubation. Yet, the total cumulative hydrogen harvested in the three cycles was nearly double than that in the first cycle alone (Table 2). After the 3rd cycle, the H2 accumulation was negligible in the conditions of our experiment (Fig. 2). The total hydrogen accumulated in the batch minireactors spiked with acetylene was

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Table 1 Hydrogen and methane accumulation and initial rates of hydrogen and methane production in batch minireactors loaded with anaerobic solid inoculum plus paper waste substrate Parameter

Cycle of incubation 1

2

C2H2 PHc Ri,H · 103d [r, P(F)]f Pmg Ri,m · 103i [r, P(F)]j

a

b

BES

17 ± 1.2 14.4 ± 1.4 10.6 ± 0.8 9.6 ± 0.9 [0.96; 105] [0.95; 104] 12.1 ± 0.8h 17.3 ± 1.2h [0.99; 2 · 104]

3

Total

C2H2

BES

C2H2

BES

C2H2

BES

11.1 ± 1.0 9.1 ± 0.4 [0.98; 106] <0.2 –

9.6 ± 0.8 8.0 ± 0.3 [0.98; 106]

5.9 ± 0.7 6. ± 0.2 [0.99; 106] <0.2 –

5.2 ± 0.4 5.4 ± 0.4 [0.98; 105]

34.0 ± 1.7 NAe NA 12.1 ± 0.8h NA NA

29.2 ± 1.6 NA NA NA NA

a

Acetylene. Bromoethanesulfonate. c Maximum amount of hydrogen accumulated at the end of the incubation period (mmole H2/reactor) in inhibited minireactors, the methane production was null. d Initial rate of hydrogen accumulation (mmole H2/(reactor Æ h)). e Not applicable. f Correlation coefficient and probability of the F statistics for the regression of the initial rate of H2 (significance level of the regression). g Maximum amount of methane accumulated at the end of the incubation period (mmole CH4/reactor) in non-inhibited minireactors, the hydrogen production was null. h The methanogenic batch reactor was not spiked with acetylene or BES (that is, it was not inhibited). i Initial rate of methane accumulation (mmole CH4/(reactor Æ h)). j Correlation coefficient and probability of the statistics F for the regression of the initial rate of CH4 (significance level of the regression).

CH4 or H2 (mmole/reactor)

b

18 15 12 9 6 3 0

0

1000

2000

3000

4000

5000

Time (h) Fig. 2. Effect of venting and flushing with N2 the headspace of the batch minireactors and subsequent incubation on gas accumulation. (s) methane from inhibited batch minireactors; (d) hydrogen from minireactors spiked with BES; (m) hydrogen from minireactors spiked with acetylene. The arrows indicate the venting and flushing with N2.

slightly higher than that corresponding to the minireactors spiked with BES. This indicates a clear advantage of the use of acetylene over BES, since the second is more expensive and it will remain in the solid phase as a potential pollutant. Control minireactors with inoculum, no substrate and spiked with acetylene produced 0.71 mmole H2/reactor and no methane. It is likely that the venting and flushing the headspace with the inert gas N2 would have released the product inhibition effected by hydrogen accumulation on the activity of some fermentative microorganisms in the consortia (for instance, the syntrophic bacteria, Brock and Madigan, 1991), allowing for an increase in actual hydrogen generation. It is known that anaerobic syntrophic bacteria grow on butyric or propionic acid producing hydrogen, CO2 and acetate, according to Eqs. (1)

and (2) in Table 2. They are inhibited by the product hydrogen; in fact the biochemical reactions are endergonic at high partial pressures of H2 (Brock and Madigan, 1991). Another plausible cause contributing to the biochemical inhibition might be the accumulation of organic acids and a possible drop of pH in the solid cultures, in spite of the buffer added before incubation. We did not determine the concentration of organic acids in the solid materials of the bottles at the end of the incubation. Yet, a conservatively low estimate of butyric (HBu) and acetic acid (HAc) final concentrations can be made based on the biochemical Eqs. (3) and (4) in Table 2 for the carbohydrate fermentation by Clostridia (Brock and Madigan, 1991; Nandi and Sengupta, 1998) and the final amount of hydrogen harvested in the batch minireactor. We made the following simplifying assumptions: (i) 95% of the paper is degradable cellulose; (ii) cellulose is approximately equal to glucose; (iii) half of the consumed substrate is fermented according to Eq. (3) and the other half to Eq. (4); (iv) the hydrogen harvested is directly related to consumed substrate. This underestimates the latter, since part of the H2 produced might not be harvested due to cell biomass synthesis and losses to acetogenesis and sulfate reducing processes (see Eqs. (6) and (7), Table 2). In this way, a final concentration of short chain volatile organic acids ca. 6800 mg HAc/kg wet basis (34,000 mg HAc/kg dry initial substrate) and 6000 mg HBu/kg wet basis (25,000 HBu/kg dry initial substrate) can be expected inside the minireactors at the end of the 3rd cycle. It would be worth determining whether the extraction of the organic acids from the spent substrate could open the pos-

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Table 2 Important biochemical processes in anaerobic, methanogenic consortia (adapted from Brock and Madigan, 1991) No.

1. 2. 3. 4. 5. 6. 7. 8.

Type of reaction

Fermentation of carbohydrates to butyrate Fermentation of carbohydrates to acetate Anaerobic oxidation of butyrate (syntrophy)c Anaerobic oxidation of propionate (syntrophy)d Hydrogenotrophic methanogenesis Acetogenesis from CO2 and hydrogen Sulfate reduction Propionate from glucosef

Reaction

Gibbs free energy (kJ/reaction) Butyrate þ 2HCO 3 2Acetate þ 2HCO 3 +

þ

Glucose þ 2H2 O ! 2H2 þ þ 3H Glucose þ 4H2 O ! 4H2 þ þ 4Hþ Butyrate + 2H2O ! 2H2 + 2Acetate + H þ Propionate þ 3H2 O ! 3H2 þ Acetate þ HCO 3 þH þ 4H2 þ HCO 3 þ H ! CH4 þ 3H2 O þ 4H2 þ 2HCO 3 þ H ! Acetate þ 4H2 O   4H2 þ SO2 4 ! HS þ 3H2 O þ OH þ 3Glucose ! 4Propionate þ 2Acetate þ 2HCO 3 þ 8H

DG0a

DG 0 b

135 207 +48.2 +76.2 136 105 NAe NA

284 319 17.6 5.5 3.2 7.1 165 1016

a

Standard conditions: soluble species 1 M, gas species 1 atm pressure. Conditions prevailing in anaerobic ecosystems, i.e., [organic acids] = 1 mM, pH = 7, ½HCO 3  ¼ 20 mM, [Glucose] = 10 mM, and the partial pressures of H2 and CH4 are 104 and 0.6 atm, respectively. Negative values of the Gibbs free energy indicate a spontaneous, feasible thermodynamic process (exergonic) whereas positive values indicate an impossible process (endergonic). c Typically effected by Syntrophomonas genera. d Typically carried out by Syntrophobacter species. e Not available. f It was assumed homofermentative lactic pathway from glucose (Brock and Madigan, 1991; Fig. 19–82) and subsequent fermentation of lactic acid to propionic typically effected by Propionibacterium (Brock and Madigan, 1991; Fig. 19–97, Section 19–30). b

sibility for its further fermenting and consequently increasing the hydrogen yield. Comparing the hydrogen harvested with the maximum potential hydrogen that could be produced according to the assumptions (i)–(iv) above, it can be shown that a maximum of 320 mmole H2/reactor should be expected. This implies a hydrogen production efficiency of approximately 11% ((34/320) · 100, Table 1). On the other hand, if a maximum 6 moles of H2 per mole of glucose is assumed (although debatable), it can be shown that the maximum hydrogen per reactor would be 630 mmole H2/reactor and the hydrogen production efficiency would be 5.4% ((34/630) · 100). A preliminary appraisal of the IV-SSAH process would shed light on its relative merits and disadvantages with respect to other biological alternatives for hydrogen production: (i) it may be more cost-effective than processes working with pure microbial strains because no sterile conditions are required; (ii) it could be more attractive than processes that ferment soluble carbohydrates to hydrogen, since our carbon source consists of free or cheap organic wastes. Moreover, some additional credits for savings in waste treatment could be assigned to the IV-SSAH, (iii) no light is needed as compared to photobiological hydrogen production; (iv) the inhibitor used (acetylene) is a cheap gas that will exit the bioreactor with the hydrogen-rich gas stream. However, at least three stumbling blocks can be recognized: (i) the kinetics of hydrogen accumulation is slower than reported rates for liquid fermentation processes; (ii) there is a potential for lower hydrogen yields than those of processes using pure cultures, since the anaerobic, methanogenic microbial consortia may contain acetogenic and sulfate-reducing bacteria that could consume a fraction of the

hydrogen generated (see Eqs. (6) and (7), Table 2); and (iii) there is an upper limit for the amount of hydrogen that can be obtained via anaerobic fermentation of cellulose and other carbohydrates (see Eqs. (1) and (2), Table 2). Our results point out to a feasible strategy for obtaining higher hydrogen yields from the fermentation of industrial solid wastes, and a possible combination of waste treatment processes consisting of a first stage IV-SSAH followed by a second M-SSAD stage. Useful products of this approach would be hydrogen, organic acids or methane, and anaerobic digestates that could be used as soil amenders or protein enrichments after a suitable post-treatment.

4. Conclusions In the first phase of experiments it was found that acetylene at 1% v/v in the headspace was as effective as BES in inhibiting methanogenic activity. Similar final hydrogen contents were obtained for reactors spiked with acetylene and BES. Exposure of the consortia to the atmospheric oxygen as a way to inhibit the methanogenesis and promote the hydrogen production was not successful. In the second phase of tests the headspace of the batch reactors was flushed with Nitrogen after the first plateau of hydrogen was reached, and subsequently incubated, with no further addition of inhibitor nor substrate. It was found that hydrogen production resumed and reached a second plateau. This procedure was repeated a third time. We coined this procedure as IVSSAH (intermittently vented solid substrate anaerobic

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hydrogen generation). The total cumulative hydrogen harvested in the three-cycle incubation was nearly double than that in the first cycle alone (17 and 34 mmole/ bottle, for one cycle and three cycles respectively).

initial rate of hydrogen accumulation (mmole H2/(reactor Æ h)); Ri,m initial rate of methane accumulation (mmole CH4/(reactor Æ h)); SSAD solid substrate anaerobic digestion. Ri,H

Acknowledgements The authors wish to thank the positive input of the Editor and the anonymous referees of Bioresource Technology that allowed the improvement of the manuscript. A graduate scholarship to IV–V from CONACYT is gratefully acknowledged. The authors wish to thank Mr. Rafael Herna´ndez-Vera (Environmental Biotechnology R&D Group, Department of Biotechnol. and Bioeng., CINVESTAV-IPN) for his help with the start-up of the M-SSAD reactors. The first part of this research was supported by the University of Manitoba Research Foundation with a grant to RS and HMP-V who were Assistant Professors at the University of Manitoba at that time. The authors appreciate partial financial aid from CINVESTAV-IPN and a grant license of Design Expert v. 6.0 from Design-Ease, Inc. NR-S also appreciates support from COFAA-IPN, Mexico.

Appendix A. Notation BES DG0

bromoethanesulfonate; Gibbs free energy at standard conditions: soluble species 1 M, gas species 1 atm pressure; DG 0 Gibbs free energy at conditions prevailing in anaerobic ecosystems, i.e., conditions prevailing [organic acids] = 1 mM, pH = 7, ½HCO 3 ¼ 20 mM, [Glucose] = 10 mM, and the partial pressures of H2 and CH4 are 104 and 0.6 atm, respectively. Negative values of the Gibbs free energy indicate a spontaneous, feasible thermodynamic process (exergonic) whereas positive values indicate an impossible process (endergonic); HAc acetic acid; HBu butyric acid; IV-SSAH intermittently vented and flushed, solid substrate anaerobic hydrogen generation; M-SSAD methanogenic solid substrate anaerobic digestion; PPI pulp and paper industry; PH maximum amount of hydrogen accumulated at the end of the incubation period (mmole H2/ reactor) in inhibited minireactors; Pm maximum amount of methane accumulated at the end of the incubation period (mmole CH4/ reactor) in non-inhibited minireactors; r correlation coefficient of the regression for estimating the initial rates of gas accumulation;

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