The hypochlorite ion (OCl⁻) is a widely used disinfecting agent to sterilize pig pens and to prevent animal diseases spreading in Korea. The hypochlorite ion is typically sprayed onto the pig pen floor and could be retained in the pig slurry when it is discharged from the pig farmhouse. The residual hypochlorite ion in the collected pig slurry may inhibit the anaerobic microbial activity during the anaerobic fermentation for the biogas production.

Hypochlorite ion is a non-selective, highly reactive oxidant for a wide range of cellular and subcellular compounds, and inactivation of enzymes and interference of DNA synthesis were reported (Albrich et al., 1981; McKenna and Davies, 1988; Rakita et al., 1990; Ducan and Daniele, 1996). But its actual mechanism of action is not fully known (McDonnell and Russell, 1999). No microorganism is known to possess a specific enzymatic mechanism for the detoxification of hypochlorite ion (Leyer and Johnson, 1997). Hypochlorous acid is dissociated as H⁺ and OCl⁻, and the pK_a of HOCl is 7.53. Metcalf (2002) reported that the protonated form (HOCl) has a more toxic effect than the deprotonated form (OCl⁻). When hypochlorite ions react with ammonium in aqueous solution, chloramine (NH₂Cl) is formed. Shih and Lederberg (1976) reported that NH₂Cl induces lesions in DNA and cause a disinfection effect. The non-selective and highly oxidative characteristics of hypochlorite ions result in reactions with various types of organic matter. Thus the disinfecting power of hypochlorite ion could be reduced by oxidation of organic matter other than microorganisms (Metcalf, 2002).

Biogas is generally produced through anaerobic biological processes that convert biodegradable organic materials into methane (CH₄) and carbon dioxide (CO₂). The biogas production is carried out by the sequential microbial reactions of hydrolysis of biodegradable organic materials (Hydrolysis), fermentation of the hydrolyzed products (Acidogenesis), formation of substrates (acetic acid, CO₂, H₂) for CH₄ fermentation (Acetogenesis), and CH₄ production (Methanogenesis). The CH₄ producing microbial community consists of different types of bacteria within the sequential mechanism of CH₄ production as described above. Therefore, stability of anaerobic microbial communities and balance between microbial populations within the sequential reactions are very important in the operation of an anaerobic digester (Yu et al., 2005).

The objective of this study was to assess the effect of a residual disinfecting agent, hypochlorite ion, on CH₄ production in the initial methanogenic anaerobic digestion of pig slurry. In order to investigate the alleviation effect of organic materials present in pig slurry on the disinfecting agent, the effect of hypochlorite ion on CH₄ production in different organic concentrations was also analyzed.

At the end of 20-d fermentation, hypochlorite ion addition had no effect (p>0.05) on pH, NH₄⁺-N, TN and SCOD concentrations in the anaerobic batch reactor fermented in all three organic concentrations (Table 2). Total solids and VS decreased during the fermentation for inhibition assay in both 0 and 52.3 mg/L OCl⁻ cultural serials. For the Control (0 mg/L OCl⁻) culture series, the VS contents were reduced to 27.5, 24.4, and 33.1% at the end of 20-d fermentation, compared with the initial VS contents for PS-I, PS-II, and PS-III, respectively. Besides, for the 52.3 mg/L hypochlorite ion treated culture series, the VS contents were decreased to 17.2, 19.3, and 27.3% over a 20 d fermentation period for PS-I, PS-II, and PS-III, respectively. Despite of the significant VS reduction (p<0.05) in OCl⁻ culture of PS-I with VS loading of 9.9 g/L, none of the reductions in TCOD and SCOD were significant (p>0.05). Additionally, inhibition by OCl⁻ was significant at the lowest organic loading (PS-I) as presented by the significant differences (p<0.05) in VS degradability (VS reduction of 17.2% with OCl⁻, VS reduction of 27.5% without OCl⁻). For the two higher organic concentrations PS-II and PS-III, the differences in VS degradability between cultures of with and without OCl⁻ were (p>0.05) much smaller than the difference of VS degradability between of cultures with and without OCl⁻ in PS-I.

CH₄ production in the Control cultures without OCl⁻ increased exponentially from day zero to six, and continued to increase steadily at a lower rate from day eight to the end of fermentation (d 20). In the assay cultures with OCl⁻, the slow, but steady increase in CH₄ production was observed in the fermentation period from d 0 to 11. Thereafter, the CH₄ yield increased more rapidly than that of fermentation period of d 0 to 6 (Figure 1). During anaerobic batch fermentation, CH₄ content increased steeply and reached 70.0% within 4 d for the culture series without OCl⁻. In contrast, CH₄ content of culture series with OCl⁻ was below 60.0% during 6 d after fermentation set up, gradually increased above 60.0% from 14th d of fermentation (Figure 2). The total CH₄ yields were 0.21, 0.15, and 0.13 L/g-VS_added in the organic concentrations PS-I, PS-II and PS-III without OCl⁻ anaerobic culture series and these values were higher than 0.17 (p = 0.31), 0.13 (p = 0.04), and 0.10 (p = 0.06) L/g-VS_added, respectively, of assay culture series with OCl⁻, (Table 2). These results meant that CH₄ yields (L/g-VS_added) from culture series having higher organic concentration, with or without OCl⁻, showed lower CH₄ yield. These results were attained in the short fermentation period because CH₄ production was completed in the reactor with low organic concentration during the fermentation time of 20 d, while CH₄ production was ongoing in the reactor with higher organic concentration.

Figure 3 shows cumulative CH₄ production and the curve fitted by the modified Gompertz equation in OCl⁻ inhibition assay cultures of pig slurry having different organic concentrations. The maximum specific CH₄ production rate (R_m), CH₄ production potential (P), and lag phase time (λ) that were estimated by the modified Gompertz equation are shown in Table 3. In PS-I with the lowest organic concentration, PS-I without OCl⁻ gave a P, R_m, and λ that were 140.0 ml, 18.2 ml/d, and 2.2 d, respectively. While the PS-I with OCl- gave a P, R_m, and λ that were 438.5 ml, 8.1 ml/d, and 25.6 d, respectively. This result implies that the presence of OCl⁻ caused a CH₄ production rate that was decreased more than two times by the inhibition effect of OCl⁻ in the initial fermentation stage. In this result, the CH₄ production potential (438.5 ml) of PS-I with OCl⁻ was higher than that (140.8 ml) of PS-I without OCl⁻, this was caused by the overestimation of P of PS-I with OCl⁻ due to use of the assay data from an initial short fermentation time (20 d). In PS-III with the highest organic concentration, PS-III without OCl⁻ had a P, R_m, and λ that were 377.1 ml, 40.6 ml/d, and 2.6 d. While P, R_m, and λ of the PS-III with OCl⁻ were 456.0 ml, 15.1 ml/d, and 11.4 d, respectively. Comparing the R_m (8.1 ml/d) and λ (25.6 d) of PS-I with OCl⁻, the maximum specific CH₄ production rate increased to 15.1 ml/d, and the lag phase time was reduced to 11.4 d. These results imply that the higher organic concentration might alleviate the inhibition effect of OCl⁻ by the lessening of lag phase time and increasing the CH₄ production rate in the initial stage of fermentation.

Recently regulations and directives on hygiene and sanitary conditions require a high safety level in livestock food products. To meet these requirements, hazard analysis and a critical control points (HACCP) system were introduced to the breeding step at pig farms in Korea. Increasingly high concentrated disinfecting agents were used in order to combat the recent occurrence of various animal diseases. The OCl⁻ ingredient was occasionally used in high concentrations to over 10 times the recommended usage. Our results showed a noticeable inhibitory effect the residual hypochlorite ion on methanogenesis in the initial phase of the 20 d fermentation period. Higher organic loadings in the anaerobic reactor decreased the OCl⁻ inhibitory effect. Our results are inconsistent with those reported by Lambert and Johnston (2001) where hypochlorite ion effectiveness was significantly reduced by organic material through chemical reactions and spatial reaction barriers. Pig slurry has a high amount of colloidal or particulate material as well as soluble organic matter. Those organic materials may compete with bacteria to react with OCl⁻. The sieving process to remove macro-particles for a uniform initial culture might have diluted the soluble and particulate organic material as well as reducing the anaerobic microorganisms’ population, causing changes in anaerobic microbial activity in our anaerobic fermentation experiment. It is possible that organic materials that bound with microbes in the anaerobic reactors may form the spatial barrier, protecting microbes from the OCl⁻. Additionally, the effectiveness of disinfectant is also affected by the type of microbes and their growth stages (Maillard, 2002; Kitis, 2004). These effects could have contributed to the differences reported from our study and the one by Lambert and Johnston (2001).

The effectiveness of disinfectants also depends on environmental conditions. Beside microbe population and type, other major factors such as interfering substances, aqueous conditions (pH, temperature), contact time and influence the effectiveness of the disinfectant (Bessems, 1998; Chmielewski and Frank, 2003). Therefore in order to assess the various inhibitory mechanisms of hypochlorite ion on anaerobic fermentation, studies on the operational conditions of the anaerobic reactor, species of anaerobic bacteria, and characteristics of feedstock are needed.

There are limited inhibitory studies on anaerobic microorganisms with most reported on the effect of quaternary ammonium compounds (also a disinfectant) on the methanogenic process (Battersby and Wilosn, 1989; Garcia et al., 1999). Tezel et al. (2006) reported that a prolonged fermentation time was necessary for the methanogens to overcome the initial inhibitory the effect of quaternary ammonium compounds. It seems that the inhibitory effect is reduced by the adsorption of quaternary ammonium compounds on organic material since quaternary ammonium compounds are not biodegraded under anaerobic conditions (Tezel et al., 2006). Our OCl⁻ results are consistent with those of Tezel et al. (2006) with quaternary ammonium compounds since CH₄ production was initially depressed and a longer time was needed (11th and 14th d) to reached the maximum CH₄ concentration during our OCl⁻ inhibitory fermentation study. Our results suggest that CH₄ production cultured under 52.3 mg/L OCl⁻ was stabilized after 11 to 14 d in the batch fermentation. Although our results were obtained from the anaerobic batch culture, a commercial anaerobic digester continuously operated under a high organic concentration might have a higher buffering capacity against the variable inhibitory effects by hypochlorite ion.

During the 20-d anaerobic batch culture assay under mesophilic (38°C) conditions, the residual hypochlorite ion at concentration of 52.3 mg/L in pig slurry showed tan inhibitory effect on CH₄ yield and biodegradation of biomass. The inhibitory effect by residual hypochlorite ion could be modified by the chemical reactions with various organic materials during the anaerobic fermentation of pig slurry. CH₄ production was preferentially stabilized in the inhibitory assay culture containing a high organic material content. Our results suggested that a prolonged fermentation time was necessary for the methanogens to overcome the initial inhibitory effect. Moreover, an anaerobic reactor operated in high organic loadings is more beneficial in mitigating the hypochlorite ion inhibitory impact.