Inhibition resilience of microbes in pig effluent lagoons

A recent inhibition test protocol (Astals et al. 2015), which has been optimised for cost and speed, allows pork producers to quantify microbial inhibition in piggery effluent lagoons. This is important, because effective effluent treatment relies on healthy microbial activity in effluent lagoons. The inhibition test measures the KI₅₀ value, which is the concentration of a specific inhibitor at which the activity of exposed microbes are reduced by 50% (Astals et al. 2015). Accordingly, if the inhibitor concentration in flush manure fed to a lagoon is less than the KI₅₀, then the lagoon may be uninhibited. The aim of this study was to determine if inhibition test data also provided information about the relative tolerance of microbes, in a piggery lagoon, to unavoidable periodic increases in inhibitor concentrations that are below the KI₅₀.

Ammonia (NH₃) was selected as the model inhibitor because flush manure is rich in NH₃ and it is a key inhibitor of anaerobic digestion. For experiments, a sludge sample (containing microbes for which inhibition is to be tested) was collected from an unmixed covered lagoon at a commercial breeder piggery in NSW. The volatile solids (VS), background NH₃ nitrogen and native pH of the sludge sample were measured (Astals et al. 2015) at 10 g VS/L, 776 mg N/L and pH 7.2, respectively. Glass vials (160 mL) were loaded with the sludge and different amounts of NH₃ (added as NH₄Cl salt) and with 2 g/L acetate as food source. The vials were sealed and incubated at 37°C. The pH in the vials was 7.08–7.72 depending on the amount of NH₄Cl added. Methane produced by microbes in sludge inside the vials was measured at 1, 2 and 3 days of incubation (Astals et al. 2015). Specific methanogenic activity (SMA) was determined as the slope of a linear line fitted to the methane data over time (expressed in units of chemical oxygen demand or COD equivalents, normalized with respect to the amount of VS in the sludge added to each vial). All the experiments were run in triplicate and the error in SMA was estimated at the 95% confidence level (seven degrees of freedom). The SMAs were plotted against NH₃ (symbols, Fig. 1b) and KI₅₀ was estimated by linear interpolation, corresponding to the NH₃ content at which SMA had been reduced to 50% of the highest measured SMA.

Fig. 1.  (a) Cumulative methane produced by the lagoon sludge versus time at 0.77 g N/L (◇), 2.74 g N/L (■), 3.71 g N/L (▵), 5.61 g N/L (×) ammonia (added plus background). Note: slopes of the linear lines of best-fit are the specific methanogenic activities or SMA. (b) SMAs (symbols, estimated from Fig. 1a) versus NH₃ content. KI₅₀ =3.98 ± 0.67 g N/L. The solid line was derived from data of a different lagoon sludge (adapted from Astals et al. 2015).

As expected, increasing NH₃ decreased measured microbial activity/SMA (symbols, Fig. 1b), likely due to inhibition. The estimated KI₅₀ of 3.98 ± 0.7 gTAN/L (given with error at 95% confidence level) was the threshold concentration for NH₃ inhibition of the particular sludge sample being tested. The background NH₃ (776 mgN/L) was noted to be well below this KI₅₀ and thus indicated that the lagoon was not likely to be inhibited by NH₃. Further, the shape of the inhibition profile (symbols, Fig. 1b) showed a gradual decrease in SMA with increasing NH₃, indicating that the microbes were reasonably tolerant to increases in NH₃, albeit with some decrease in SMA. A stronger threshold-type response was observed for another lagoon sludge (solid line, Fig. 1b, Astals et al. 2015), with decrease in activity being more drastic around the KI₅₀ value. These different shapes of the SMA curves (Fig. 1b) suggested differences in tolerance to NH₃. The results in this paper illustrated how inhibition test data can be used to estimate a threshold inhibitor concentration (KI₅₀) as well as to obtain a measure of microbial tolerance to increases in inhibitor concentration.