Susceptibilidad a los deslizamientos

In order to increase H2 production, mixed cultures containing H2 consumers are treated using methods such as heat, chemicals, load-shock, and aeration. Pretreatment of the culture can delay H2 production and may consequently reduce the overall yield or the stability of the system (Hawkes et al., 2002; Minoda et al., 1983). A proper pretreatment method must be selected based on the treatment’s efficiency, the possibility of its application on a larger scale, its effect on the environment and cost-efficiency. Different pretreatment methods are described in the following sections.

2.6.1 Heat treatment

Among the available pretreatment methods, one of the most widely used for enrichment is heat treatment. This method destroy non-spore forming bacteria and enrich the acidogenic spore formers that produce H2 (Lay et al., 1999). During heat treatment, major non-spore forming organisms such as methanogens are destroyed and only the spore forming bacteria survive (Oh et al., 2003; Van Ginkel et al., 2001). However, not all H2 consumers belong to the non-spore forming group. For example, homo-acetogens (such as Clostridium aceticum, which are H2 consumers) are spore formers that can survive heat treatment (Oh et al., 2003; Ohwaki and Hungate, 1977). Hussy et al. (2003) reported that heat-treatment did not eliminate H2 consumers such as homoacetogens and HPr producing bacteria.

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Conditions for heat treatment can vary, i.e., the incubation temperature and residence time. The temperature range that is normally used in heat shock treatment is 80 to 105 oC and the retention time is 15 to 120 min (Chang and Lin, 2004; Lay et al., 1999; Zhu and Beland, 2006). However, Alibardi et al. (2012) and Lay et al. (2011) reported that optimum temperature and retention time for high H2 yield would be 100 oC for 4 h or 60 o

C for 40 minutes. Note, Lay et al. (2011) observed CH4 in addition to H2 at these conditions for the reactors operated at 55 ᴼC. Ren et al. (2008) studied the effect of heat shock using a sterilization temperature of 121 ᴼC for 20 min, and achieved a maximum H2 yield of 190 mL, corresponding to 1.65 mol mol-1 glucose.

Although these studies reported high H2 yields following applications of heat treatment, there are drawbacks to their use as a selective means for the enrichment of microorganisms. The use of heat shock may not only kill the H2 consuming methanogens, but also inactivate some of the H2 producing non-spore forming vegetative cells and also in addition, spore forming acetogens are not killed (Kraemer and Bagley, 2007). A lag in the initiation of H2 production was observed for the heat treated cultures in both batch and continuous systems (Duangmanee et al., 2007; Hawkes et al., 2002). Duangmanee et al. (2007) reported that repeated heat treatment was required to maintain H2 production in continuous systems. However, repeated heat treatment or prolonged heat treatment may affect the microbial granular structure in high rate systems such as an upflow anaerobic sludge blanket reactor (UASBR). In comparison, Liang et al. (2010) reported that heating followed by acid treatment of a granular culture resulted in low HPR and partial granulation; however, with subsequent heat shock treatment, the granulation and HPR improved. Heat treatment at a large scale is not economically viable as in case of reactor failure or revival of methanogens providing repeated heat treatment to the inoculum becomes inevitable.

2.6.2 Acid and alkali treatment

The most widely used pretreatment apart from heat is acid treatment. This is because the main H2 consuming organisms (methanogens) are active at pH ranging from 6.5 to 7.5 and most methanogens are inhibited at a lower pH (<5.5) (Fang and Liu, 2002; Fang et al., 2002b). Acid treatment is employed by adjusting the pH of the culture to 2.0 - 3.0 for an incubation period of 24 h, during which only the spore forming bacteria survive.

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Elimination of the non-spore forming methanogens thereby represses methanogenic activity (Chang et al., 2002; Lee et al., 2009b). However, the acid pretreatment may not be effective over sustained long periods of operation. Studies by Luo et al. (2011) suggest that the H2 production potential decreased over repeated batches. These authors reported that approximately 80% of the H2 was consumed by fifth generation acid pretreated inoculum compared to 10% in a freshly pretreated inoculum. Demirel et al. (2010) reported that H2 production increased by 80% with alkaline treatment (pH 11.0 for 24 h). However, Ren et al. (2008) reported low H2 yield and increased methane yield with alkali treatment of cultures with these parameters (inculbation of culture at pH 11.0 for 24 h). Mu et al. (2007) studied both acid and alkali treatments for enriching the microflora to enhance bio-H2 production and suppress methanogens. Variability in this process is a major concern and this has caused concerns related to implementing this technology in full-scale systems.

2.6.3 Chemical treatment

Chemical inhibitors include both synthetic and biochemicals. Some of the common synthetic chemical treatments include 2-bromoethanesulfonate (BES), iodopropane and acetylene (Sparling et al., 1997; Zhu and Beland, 2006). Among these, BES is a well known inhibitor for suppressing methanogenesis. The inhibitor binds to the co-enzyme M reductase complex, a prime component of the methanogenesis present in methanogens (Zhu and Beland, 2006). For example, in Methanobacterium thermoautotrophicium, when BES (an analog of co-enzyme M) was applied, reduction of methyl co-enzyme to CH4 was inhibited (Gunsalus et al., 1978). Sparling et al. (1997) reported that 25 mM of BES was effective in inhibiting methanogens and increasing the H2 production. However, studies by Cheong and Hansen (2006) have shown that the COD of a BES treated culture would be higher and may reduce the degrading efficiency of the feed or waste stream and hence cause environmental pollution problems at discharge. Kotsopoulos et al. (2006) reported that BES is not an efficient pretreatment method for long-term operation and may be toxic to H2 producers.

Sparling et al. (1997) reported that 1% (v/v) acetylene could be used for inhibiting methanogens and enhancing H2 production. Exposing methanogens to acetylene causes them lose the ability to maintain their transmembrane pH thus, resulting in reduced

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methanogenic activity. Ethylene, a similar compoundto acetylene, can also inhibit methanogens. The disadvantage of using ethylene is that, the amount of ethylelne required for the pretreatment is larger than that of acetylene due to their difference in solubility by a factor of 400 (Gordon and Ford, 1972; Sprott et al., 1982). Chloroform has been used to inhibit methanogens. Studies by Xu et al. (2010) have shown that inhibiting methanogens with chloroform increased H2 production. Chloroform or any methyl chlorinated compound are able to block corrinoid enzymes which leads to inhibition of methyl Co-enzyme A in methanogens (Oremland and Capone, 1988). However, the use of these chemicals could pose a threat to the environment if they are discharged in effluents from a bioreactor (Valdez-Vazquez and Poggi-Varaldo, 2009).

Another type of inhibitors used include biodegradable chemical that are able to inhibit methanogens. Long chain fatty acids (LCFAs) are a group of chemical inhibitor wich can act on both aceticlastic methanogens and hydrogenotrophic methanogens (Koster and Cramer, 1987; Lalman and Bagley, 2002). However, LCFAs may not inhibit H2 consuming-spore forming Clostridium aceticum and Desulfotomaculum geothermicum (Park et al., 2004b). The advantage of using LCFAs for pretreatment is that they are biodegradable compared to other synthetic chemicals and degrade to shorter chain fatty acids and HAc plus H2 (Weng and Jeris, 1976). The quantity of H2 produced from LCFAs is much lower than that of sugars such as glucose and xylose because they degrade very slowly (Chaganti et al., 2012a; Saady et al., 2012b).

2.6.4 Other treatment methods

Load shock is another form of pretreatment, in which no chemical treatment is involved. High loading of substrate is applied to the system, which makes the environment unsuitable for many microorganisms. Van Ginkel et al. (2001) reported that with high substrate loadings, the higher levels of volatile fatty acids produced reduced the survival of methanogens under acidic pH levels of 5.0-4.5. In continuous systems, increase in loading is an effective mechanism to eliminate a larger percent of methanogens and ultimately enhancing the H2 produced (Prasertsan et al., 2009).

Since methanogens are sensitive to oxygen, aeration could be used to inhibit methanogens. Ueno et al. (1996) reported complete inhibition of methanogenesis with no methane detected while achieving 65-70% of the theoretical maximum H2 yield in

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chemostat studies conducted with industrial wastewater. In comparison, Zhu and Beland (2006) reported no significant impact on the H2 yield with aeration. They observed similar levels of CH4 in aerated cultures as and control cultures.

Guo et al. (2008a), reported a shorter lag phase in culture treated with ultrasonication and microwaves. Studies conducted by Thungklin et al. (2011) revealed that microwave irradiation inhibited the H2-consuming activity and greater than 23 mL H2 L-1 culture was obtained from microwave inhibited culture containing slaughterhouse waste.

2.6.5 Summary

Although various pretreatment strategies have been employed for culture enrichment, there is no study demonstrating which of these methods is the most effective in a full- scale application. For example, Ren et al. (2008) and Luo et al. (2010) evaluated different pretreatment strategies for suppressing H2 consumption and enhancing H2 yield. Ren et al. (2008) concluded that the maximum H2 yield was obtained by repeated aeration and the lowest H2 yield was obtained with acidified culture. In comparison, Luo et al. (2010) reported that an untreated culture and a culture treated with load-shock performed the same and that the lowest yield was obtained with chloroform pretreated culture. Evaluation of acid, alkali and heat treatments by Mu et al. (2007) revealed that heat treatment may be considered as a potential treatment method for bio-H2 production. Recently, Pendyala et al. (2012) reported that the conflicting data was due to variation in the fermentation conditions and that the conclusions about the optimum pretreatment strategy had no statistical basis. The study by Pendyala et al. (2012) revealed that the treatment methods employed did not reveal any statistical difference between flocculated cultures whereas for granulated culture the effect of linoleic acid (LA, an unsaturated LCFA) and BES treatment was statistically the same, while other pretreatment methods showed lower yields. Based on the above discussion, the criteria for selecting the appropriate pretreatment technology involves not only efficiency in suppressing H2 consumption and enhancing H2 production but also in establishing diverse H2 producing microflora for long-term operation. Optimal pretreatment must result in efficient biological H2 production from a mixed anaerobic community. The selection of an appropriate technology should also consider the cost of implementation and addressing environmental concerns associated with them.

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2.7 Long chain fatty acids (LCFAs) and their role in hydrogen production