Effect of Inhibition Treatment, Type of Inocula, and Incubation Temperature on Batch H2 Production From Organic Solid Waste Idania Valdez-Vazquez, Elvira Rı´os-Leal, Karla M. Mun˜oz-Pa´ez, Alessandro Carmona-Martı´nez, He´ctor M. Poggi-Varaldo CINVESTAV, Department of Biotechnology and Bioengineering, Environmental Biotechnology R & D Group, P.O. Box 14-740, Me´xico D. F., 07000, Me´xico; telephone: 5255 5061 3800 x 4324; fax: 5255 5061 3313; e-mail:
[email protected] Received 19 January 2005; accepted 28 November 2005 Published online 7 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20891
Abstract: Two types of induction treatments (heat-shock pretreatment, HSP, and acetylene, Ac), inocula (meso and thermophilic) and incubation temperatures (37 and 558C) were tested according to a full factorial design 23 with the aim of assessing their effects on cumulative H2 production (PH, mmol H2/mini-reactor), initial H2 production rate (Ri,H, mmol H2/(g VSi h)), lag time (Tlag, h), and metabolites distribution when fermenting organic solid waste with an undefined anerobic consortia in batch mini-reactors. Type of inocula did not have a significant effect on PH, Tlag, and Ri,H except for organic acids production: mini-reactors seeded with thermophilic inocula had the highest organic acid production. Concerning the induction treatment, it was found that on the average Ac only affected in a positive way the PH and Tlag. Thus, PH in Ac-inhibited units (6.97) was 20% larger than those in HSP-inhibited units (5.77). Also, Ac favored a shorter Tlag for PH in comparison with HSP (180 vs. 366). Additionally, a positive correlation was found between H2 and organic acid production. In contrast, solvent concentration in heat-shocked minireactors were slightly higher than in reactors spiked with Ac. Regarding the incubation temperature, on the average mesophilic temperature affected in a positive and very significant way PH (10.07 vs. 2.67) and Ri,H (2.43 vs. 0.76) with minimum Tlag (87 vs. 459). The positive correlation between H2 and organic acids production was found again. Yet, incubation temperature did not seem to affect solvent production. A strong interaction was observed between induction treatment and incubation temperature. Thus, Ac-inhibited units showed higher values of PH and Ri,H than that HSP-inhibited units only under thermophilic incubation. Contrary to this, HSP-inhibited units showed the highest values of PH and Ri,H only under mesophilic conditions. Therefore, the superiority of an induction treatment seems to strongly depend on the incubation temperature. ß 2006 Wiley Periodicals, Inc. Keywords: acetylene; batch fermentation; full factorial design; heat-shock pretreatment; hydrogen; solid waste; temperature
Correspondence to: Dr. H.M. Poggi-Varaldo Contract grant sponsors: CONACYT; CINVESTAV-IPN
ß 2006 Wiley Periodicals, Inc.
INTRODUCTION Fossil fuels, which meet most of the world’s energy demand today, are being rapidly depleted. Also, their combustion products are known to cause several environmental problems (Dickinson and Cicerone, 1986). Hydrogen is one of the most promising fuel candidates; it is a versatile, safe, renewable, environmentally compatible and economic fuel (Veziroglu and Barbir, 1992). Hydrogen can be produced by fermentative microorganisms from organic compounds (Hawkes et al., 2002). From an environmental and engineering points of view, the fermentative H2 production is attractive since it can utilize wastewater or solid wastes to produce H2 in nonsterile conditions (Valdez-Vazquez et al., 2004). Thus, H2 production from organic waste fermentation appears as a low-cost, reliable alternative that could provide cheap, clean energy while helping at the same time in the treatment and disposal of municipal/industrial solid wastes. Induction of H2 accumulation in fermentative consortia is related with inhibition of H2 consumers (especially methanogens) which is essential for its further scale-up and industrial application. Acidogenic operation, heat-shock pretreatment (HSP), and acetylene (Ac) have been used for this purpose (Valdez-Vazquez et al., 2004). Hydrogen production using anerobic consortia inhibited by acidogenic operation requires, however, an acclimatization time of the inocula which could be between 10 days to 30 days (Lin and Chang, 1999; Liu and Fang, 2002; Yu et al., 2002; ValdezVazquez et al., 2005a). Yet, it seems that HSP and Ac can be used without a long acclimatization time ( 0.05). This result could be related to the complexity of both microbial consortia (the mesoand the thermophilic), which consist of a variety of mesoand thermophilic microorganisms that respond in such a way that microbial differences could be compensated, probably leading to similar performances. However, it was found that volatile organic acids production was significantly higher in mini-reactors seeded with thermophilic inocula
Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit
Table II. Full factorial design matrix in coded and natural units and average results of H2 production, initial H2 production rate and total metabolites in batch mini-reactors fermenting organic fraction of municipal solid waste. A
B
Obs. No.
A
B
C
Type of inocula
Induction treatment
5,8 2,11 14,16 9,12 6,13 3,10 1,7 4,15
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
M T M T M T M T
tt tt tt tt Ac Ac Ac Ac
C Incubation Background temperature PH M M T T M M T T
Net PHa
0.86 0.35 10.17 0.22 1.49 0.15 11.89 0.38 0.00 0.00 0.67 0.28 0.00 0.00 0.34 0.14 0.24 0.10 8.01 0.45 1.16 0.06 10.19 0.81 0.50 0.10 5.82 0.84 0.19 0.08 3.85 0.17
Ri,Hb 2.30 0.33 3.4 10.18 0.41 0.17 0.21 0.09 1.98 0.02 2.01 0.23 0.95 0.31 1.48 0.36
r2c
Volatile organic acidsd
Solventse
HAcf HBu
0.95 0.97 0.89 0.89 0.90 0.97 0.96 0.92
3976 541 15324 1496 9389 235 4478 610 9880 372 20972 153 5936 310 11086 135
475 97 1727 11 1512 55 1557 318 1856 58 1039 32 565 115 606 124
0.56 4.45 1.05 1.66 1.08 3.84 1.33 2.47
a
Cumulative H2 production (mmol H2/mini-reactor). Initial H2 production rate (m mol H2/(g VSi h)). c Determination coefficient for the regression of the initial H2 production rate. d Sum of volatile organic acids: acetate, propionate, and butyrate (mg COD/kgwet mass). e Sum of solvents: acetone and ethanol (mg COD/kg wet mass). f Acetate/butyrate (HAc/HBu) ratio. b
compared to mesophilic inocula (12,965 and 7,295 mg COD/ kg wet mass, respectively, P < 0.0001). This could be due to the fact that volatile organic acids levels in thermophilic methanogenic process are generally higher than those in mesophilic regime (Gavala et al., 2003; Poggi-Varaldo and Oleszkiewicz, 1992; Song et al., 2004). It is likely that thermophilic inocula used in our work could be enriched with microorganisms that produce and/or tolerate volatile organic acids.
Effect of Induction Treatment Figure 2 shows that both HSP and Ac significantly affected PH, Tlag, Ri,H and metabolites production. In this way, PH in mini-reactors with Ac-inhibited consortia was 20% larger in the average than that in mini-reactors with HSP-inhibited consortia (Fig. 2a). Thus, Ac affected PH in a positive way (P < 0.05). The superiority of Ac-inhibited consortia could
Figure 2. Main effect of induction treatment on: (a) cumulative H2 production; (b) lag time; (c) initial H2 production rate; and (d) metabolites accumulation at the end of the incubation cycle. tt, heat-shock pretreatment; Ac, acetylene; VOA, volatile organic acids (sum of acetate, butyrate, and propionate); solvents sum of acetone and ethanol. The bars represent the standard deviation of the experiment for the corresponding response variable (Montgomery, 1991).
Valdez-Vazquez et al.: Effect of Inhibition Treatment, Type of Inocula and Incubation Temperature on H2 Production Biotechnology and Bioengineering. DOI 10.1002/bit
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be related to the fact that Ac is not as extremely aggressive to the present microorganisms as HSP is. Thus, it could be speculated that Ac might allow a major quantity/diversity of H2-producing fermentative microorganisms. In contrast, HSP only allows the survival of spore-forming microorganisms (Watanabe et al., 1997). This could cause the death of non-spore forming species such as Enterobacter sp., Thermoanaerobacterium sp., leading to a smaller H2producing capacity than that obtained with Ac-inhibited consortia. Mini-reactors treated with Ac had a significantly shorter Tlag than those treated with HSP (P < 0.001, Fig. 2b). Probably, the spores in the anerobic microflora inhibited by HSP took a long time to germinate. This was more evident in mini-reactors incubated at 558C since they had a lag time higher than 570 h, independently of type of inocula (mesoor thermophilic inocula, Fig. 1a). In consequence, on the average, the mini-reactors inhibited by HSP had the highest Tlag values (387 h). Other works producing H2 from solid organic waste by consortia inhibited by HSP have reported shorter lag times, c.a. 20 h under mesophilic conditions (Lay et al., 1999, 2003; Okamoto et al., 2000). In our work, HSP had a negative interaction with thermophilic incubation temperature which is discussed below in subsection effect of incubation temperature. HSP-inhibited units and incubated at 378C had the highest values of Ri,H (Fig. 2c). Contrary to this, HSP-inhibited units incubated at 558C showed the lowest Ri,H (Table II). Acinhibited units incubated at 378C displayed a moderate-tohigh Ri,H, of the same order of magnitude of Ri,H values obtained by Valdez-Vazquez et al. (2005b) for batch hydrogen generation from paper wastes (1.98 vs. 0.87 mmol H2/g VSi h, respectively). Ac-inhibited units incubated at 558C showed a moderate Ri,H. In this way, Ri,H of units inhibited by Ac and incubated at 558C was higher than that in units inhibited by HSP and incubated at the same temperature. Due to this interaction, on the average, Ri,H in units inhibited by HSP and Ac were not significantly different. Regarding metabolite accumulation, Figure 2d shows that Ac-inhibited mini-reactors contained higher organic acids concentration than those that were heat-shock treated (P < 0.0001). This is in agreement with higher PH in Acinhibited mini-reactors (Fig. 2a), that is, there was a positive correlation between H2 and organic acid production. This was an expected outcome, since according to the equations of H2 production (typical of Clostridia), organic acids such as acetate and butyrate are generated along with H2 (ValdezVazquez et al., 2005a). It has been demonstrated that solvent production negatively affects the H2 yield from carbohydrates fermentation (Grupe and Gottschalk, 1992). For this reason, it was important to know if the treatments with poor PH were associated to excessive solvent production. Figure 2d illustrates that solvent contents in HSP-inhibited units (P < 0.05) were slightly larger than in Ac-inhibited units. Therefore, it seems that HSP could select the inocula with solvent-producing clostridial strains (for instance, Clostridium acetobutylicum). Lower solvent concentrations
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in Ac-inhibited mini-reactors could be due to the induction treatment that allows the survival of other H2-producing bacteria, such as Enterobacter sp. whose H2 yield is not regulated by either oxidized (organic acids) or reduced (alcohols) products formed (Nakashimada et al., 2002). Effect of Incubation Temperature PH of mini-reactors incubated at 378C was significantly higher than that of mini-reactors incubated at 558C (P < 0.0001, Fig. 3a). At the moment, it was expected that the thermophilic mini-reactors would have the maximum PH. Yu et al. (2002) found that continuous H2 production from the anerobic acidogenesis of a high-strength rice winery wastewater by a mixed culture bacterial was higher under thermophilic operation. This result was corroborated by Morimoto et al. (2004) who found that the maximum H2 production was obtained by sludge compost degrading glucose in batch cultures incubated at 558C and ValdezVazquez et al. (2005a) who described the superiority of semicontinuous thermophilic acidogenic solid substrate anerobic H2 reactors over the corresponding mesophilic ones. Our results seem to contradict those reports that support increased PH under thermophilic conditions. However, the abovementioned authors utilized an inoculum enriched with H2producers microorganism by acidogenic operation. In our work, the induction procedures were different (HSP and Ac), and there are no previous reports of experiments with these treatments under thermophilic conditions. In consequence, it is likely that different induction treatments could generate different H2-producing microflora; this, in turn, could have resulted in a higher PH in mesophilic incubation. It is worth highlighting that those units incubated at 378C had a significantly higher PH for both induction treatments, thus contributing to the superiority of the mesophilic process over the thermophilic one in our work. Opposed to this, average PH in thermophilic incubation was lower because only Acinhibited mini-reactors exhibited a moderate PH while HSPinhibited mini-reactors showed the poorest PH (Table II). Therefore on the average, PH at 378C was significantly superior (P < 0.0001). Additionally to this, mini-reactors incubated at 378C experienced a shorter Tlag than those incubated at 558C (P < 0.0001, Fig. 3b). Also, Ri,H was another response variable that resulted significantly higher in mini-reactors incubated at 378C that those incubated at 558C (P < 0.0001, Fig. 3c). As it was mentioned above, there was a strong significant interaction between induction treatment and incubation temperature, therefore Ri,H was highest under mesophilic conditions when HSP was used, but when Ac was used, the highest values of Ri,H were found with Ac-inhibited units. This interaction is further discussed in the following subsection. Finally, it was found again a positive correlation between H2 and organic acids production: mini-reactors incubated at 378C had significantly higher organic acids that those incubated at 558C (Fig. 3d, P < 0.0001). Similar to this,
Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit
Figure 3. Main effect of incubation temperature on: (a) H2 production; (b) lag time; (c) initial H2 production rate; and (d) solvents accumulation at the end of the incubation cycle. M, mesophilic incubation; T, thermophilic incubation; VOA, volatile organic acids (sum of acetate, butyrate, and propionate); solvents sum of acetone and ethanol. The bars represent the standard deviation of the experiment for the corresponding response variable (Montgomery, 1991).
mesophilic mini-reactors had the highest PH as we discussed above (Fig. 3a). On the other hand, according to Figure 3d, it seems that incubation temperature did not affect solvent contents (P > 0.05). Interaction Between Induction Treatment and Incubation Temperature The results found in this work show that the superiority of an induction treatment type (HSP or Ac) depends greatly on the incubation temperature. Figure 4a shows performance of PH under both meso- and thermophilic incubation. Highest values of PH under mesophilic incubation were achieved in units inhibited by HSP; yet, under thermophilic incubation,
an opposed pattern was found (P < 0.001). Thus, there was a strong negative interaction between HSP and subsequent incubation at 558C. It is known that HSP selects for clostridial spores and the same time activate these spores (Gibbs, 1967). Later, these activated spores have to germinate to produce H2. Incubation temperature is one of the most important factors controlling the growth rate of clostridial species (Stringer et al., 1997). In our study, it seems that the best incubation temperature for spore germination was 378C, according to results of PH. Regarding this, the study carried out by Waites and Wyatt (1974) is of particular interest; they examined the effect of incubation temperature on the Clostridium bifermentans spores germination. The optimal temperature for the germination depended on the germinants used. They
Figure 4. Interaction between induction treatment and incubation temperature affecting hydrogen production and initial hydrogen production rate. The bars represent the standard deviation of the experiment for the corresponding response variable (Montgomery, 1991).
Valdez-Vazquez et al.: Effect of Inhibition Treatment, Type of Inocula and Incubation Temperature on H2 Production Biotechnology and Bioengineering. DOI 10.1002/bit
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found that the maximum germination rate was at 558C only in the presence of a mixture of amino acids such as L-arginine, L-phenylalanine, L-glycine; spores incubated at 558C in absence of these amino acids did not germinate completely. However, the spores with only L-alanine showed a rapid germination at 378C. It should be highlighted that the substrate utilized in our work was not supplement with specific germinants; in consequence, it is likely that this could contribute to poor performance of the HSP-inhibited mini-reactors and subsequent incubated at 558C. Other chemical species such as calcium and starch have long been known to promote recovery of bacterial spores from thermal injury (Labbe et al., 1981). Other researches have also reported that heat at 908C resulted in a thermal destruction of clostridial spores, starting from the first minutes of heating until the 60 min (Peck et al., 1995; Stringer et al., 1997). This is a condition very similar to our HSP which could negatively affect the survival of present spores. An interaction similar to that of the PH was observed for Ri,H (Fig. 4b). A possible explanation to the different Ri,Hs obtained between different induction treatments could be that HSP select for clostridial spores of the anerobic microflora which germinate in a fast way when the conditions become favorable (perhaps the mesophilic incubation). Several bacterial spores require some form of treatment before rapid germination; this treatment usually consisted of a short period of heating. The term in use at present for this process is activation, describing an increase in metabolic activity of activated spores (Gibbs, 1967). In this way, HSP not only could be effective for killing vegetative cells of H2 consumers (such as methanogens) and to select clostridial spores, but also to activate to the present spores (in combination with mesophilic incubation for H2 production). The average values of yields in individual treatments were (in mmol H2/g VS consumed): 23.8, 10.6, 1.7, 1.6, 13.9, 7.6, 14.3, 6.6 for M-tt-M, T-tt-M, M-tt-T, T-tt-T, M-Ac-M, T-AcM, M-Ac-T, and T-Ac-T, respectively (see Table I for treatment codes). As expected, yield values generally paralleled reported values of maximum hydrogen production PH of the treatments in this work. ANOVA of yields suggested a significant overall effect of incubation temperature (P < 0.01); units incubated at 358C displayed higher yields (14.0 average) than units at incubated at 558C (6.1 average). Interestingly, main effects of inhibitor type and type of inoculum were not significant (for instance, 10.6 and 9.4 mmol H2/g VS rem for acetylene and heat shock pretreatment, respectively; P > 0.20). However, the inhibitor type was involved in a significant interaction with incubation temperature (P < 0.01) similar to that shown by hydrogen production PH already discussed above. Units that received heat shock pretretment and were incubated at 358C had significantly higher yields (17.2 mmol H2/g VS rem average) than units that were heat-shocked and incubated at 558C (1.7 average) and units that were treated with acetylene and were incubated either at 358C or 558C (10.8 and 10.5 mmol H2/g VS rem average).
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The trend of yields with corresponding HAc/HBu ratios was also explored (Table I). It was found that HAc/HBu ratio was a poor descriptor. The association suggested lower yields for treatments with higher HAc/HBu ratios if units with heat treatment and thermophilic incubation were not taken into account (due to abnormally poor yields). Thus, the association yield-acids ratio can be approximated by the regression equation YH2/VS ¼ 19.39–2.88 (HAc/HBu) (Figure not shown) with a poor correlation coefficient 0.72 and P ¼ 0.103. CONCLUSIONS The main conclusions derived from this work are the following: Type of inocula did not have a significant effect on studied variable responses except for organic acids production: mini-reactors seeded with thermophilic inocula had the highest organic acid production. Regarding the other factors, it was found that on an average Ac only affected in a positive way PH and Tlag while mesophilic incubation affected in a positive and very significant way PH and Ri,H with minimum Tlag. It was found a strong interaction between induction treatment and incubation temperature. Thus, Ac-inhibited units showed higher values of PH and Ri,H (main response variables) than that HSP-inhibited units only under thermophilic incubation. Contrary to this, HSP-inhibited units showed the highest values of PH and Ri,H only under mesophilic conditions. Therefore, the superiority of an induction treatment seems to depend strongly on the incubation temperature.
NOMENCLATURE Ac COD HRT HSP or tt M OFMSW PH Ri,H T Tlag tt or HSP VOA YH2/VS
acetylene chemical oxygen demand hydraulic retention time heat-shock pretreatment mesophilic inocula or incubation organic fraction of municipal solid waste hydrogen production (mmol H2/mini-reactor) initial hydrogen production rate (m mol H2/(g VS i h)) thermophilic inocula or incubation lag time for hydrogen production onset (h) heat-shock pretreatment volatile organic acids hydrogen yield (mmol H2/g VS consumed) Sub indices initial
Sub indices I initial
The authors thank the positive input of the Editor and the anonymous referees of Biotechnology and Bioengineering that allowed the improvement of the manuscript. A graduate scholarship to IV-V from CONACYT is gratefully acknowledged. The authors appreciate partial financial aid from CINVESTAV-IPN and thank Stat-Ease, Inc. for a free license of Design Expert v.6.0.
Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit
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