Inhibition of biohydrogen production by ammonia

Abstract

Ammonia inhibition of biohydrogen production was investigated in batch and continuous flow reactors with glucose as a substrate. In batch tests, biohydrogen production rate was highly dependent on pH and ammonia (defined as the sum of NH₃ of NH₄⁺ species) concentrations above 2 g N/L. At pH=6.2, the maximum production decreased from 56 mL/h at 2 g N/L to 16 mL/h at 10 g N/L. At pH=5.2, production decreased from 49 mL/h (2 g N/L) to 7 mL/h (16 g N/L). Hydrogen yield remained relatively constant in batch tests, varying from 0.96 to 1.17 mol-H₂/mol-glucose. In continuous flow tests, both hydrogen production rates and yields were adversely affected by ammonia. When the reactor (2.0 L) was first acclimated under batch conditions to a low nitrogen concentration (<0.8 g N/L), H₂ production and yields under continuous flow mode conditions were 170 mL/h and 1.9 mol-H₂/mol-glucose, but decreased with increased ammonia concentrations up to 7.8 g N/L to 105 mL/h and 1.1 mol-H₂/mol-glucose. There was no hydrogen production under continuous flow conditions if the reactor was initially operated under batch flow conditions at ammonia concentrations above 0.8 g N/L. It is concluded that the hydrogen production is possible at high concentrations (up to 7.8 g N/L) of ammonia in continuous flow systems as long as the reactor is initially acclimated to a lower ammonia concentration (<0.8 g N/L).

Introduction

Bacterial hydrogen production via fermentation has the potential to treat wastewater streams while producing a clean, renewable energy carrier in the process (Benemann, 1996; Logan, 2004). Different wastewaters that have been considered for biohydrogen production via “dark” fermentation (i.e. non-photobiological-based processes) include sewage sludge (Cai et al., 2004), food wastes (Kim et al., 2004; Zhu et al., 2002; Han and Shin, 2004), and animal (Angenent et al., 2004) wastewater streams. Hydrogen can be produced using a variety of reactor and operating conditions, including batch (Logan et al., 2002; Van Ginkel et al., 2001) and continuous flow stirred tanks (Fang and Liu, 2002; Iyer et al., 2004), membrane (Oh et al., 2004), and upflow anaerobic sludge blanket (UASB) reactors (Chang and Lin, 2004).

High hydrogen yields are needed to make the process economical (Logan, 2004). Obligate anaerobes such as Clostridia species are mainly responsible for fermenting sugars to hydrogen at high yields (Iyer et al., 2004), producing acetate, butyrate, and other fermentation end products as waste products (Gottschalk, 1986). For these bacteria, the maximum theoretical yield of hydrogen by known biochemical pathways is 4 mol-H₂/mol-glucose, when acetate is produced according to

C₆H₁₂O₆+2H₂O→4H₂+2CO₂+2C₂H₄O₂.

However, the need to regenerate NAD⁺ often results in the production of other volatile acids and solvents. For example, bacteria produce only 2 mol-H₂/mol-glucose when butyrate produced (Levin et al., 2004) via

C₆H₁₂O₆→2H₂+2CO₂+C₄H₈O₂.

Overall yields are lowered from these maximum values due to the production of cell biomass.

Several factors must be considered for maximizing hydrogen production in fermentation systems. It is important to avoid the loss of hydrogen to hydrogen-consuming anaerobes, such as methanogens (Lay, 2001). This can be done by heat-treating the inoculum to select for spore-formers, such as Clostridia, for glucose-fed reactors or even heat-treating the wastewater to kill methanogens (Levin et al., 2004; Lay, 2001; Oh et al., 2003; Ting et al., 2004). Low pH can also be used to minimize the growth of methanogens (Phelps and Zeikus, 1984). It has been shown that longer hydraulic retention times (HRT) and lower chemical oxygen demand (COD) levels (factored together as COD loading rate) contribute to greater overall efficiency of hydrogen production in continuously fed reactors (Dabrock et al., 1992; Van Ginkel and Logan, 2005). Other factors such as inoculum, substrate, temperature, nitrogen sparging, and initial start up have all been examined by various researchers in an effort to optimize the production of hydrogen (Dabrock et al., 1992; Hawkes et al., 2002; Iyer et al., 2004).

While animal wastewaters have been considered as potential wastewater sources for biohydrogen production, these wastewaters contain high (∼2 g/L) concentrations of ammonia (Ceccherini et al., 1998; Cruz et al., 2000). Ammonia is known to inhibit methanogenesis (Lay et al., 1998; Borja et al., 1996; Bhattacharya and Parkin, 1989; Koster and Koomen, 1988), but the effect of ammonia on hydrogen production has not been well examined. Zhu et al. (2001) found that NH₄⁺ concentrations of <0.14 g/L did not adversely affect hydrogen production by Clostridium butyricum. Lay et al. (1998) determined that it was the NH₃ species, as opposed to the NH₄⁺ ion, that was responsible for increased lag times in methanogenesis, but that NH₄⁺ ions had a larger effect than NH₃ on the overall methanogenic activity.

To investigate the inhibition to hydrogen-producing bacteria, various concentrations of ammonium chloride were added to a feed solution containing glucose as the energy source under both batch and continuous flow conditions. The effect of ammonia was examined in terms of hydrogen production rates and overall hydrogen yields. Ammonia is defined here as the sum of both NH₃+NH₄⁺ forms, with specific chemical formulas used to denote specific ammonia species.