Denitrification Biokinetics: Towards Optimization for Industrial Applications

Abstract

Denitrification is a microbial process that converts nitrate (NO₃ ^-) to N₂ and can play an important role in industrial applications such as souring control and microbially enhanced oil recovery (MEOR). The effectiveness of using NO₃ ^- in souring control depends on the partial reduction of NO₃ ^- to nitrite (NO₂ ^-) and/or N₂O while in MEOR complete reduction of NO₃ ^- to N₂ is desired. Thauera has been reported as a dominant taxon in such applications, but the impact of NO₃ ^- and NO₂ ^- concentrations, and pH on the kinetics of denitrification by this bacterium is not known. With the goal of better understanding the effects of such parameters on applications such as souring and MEOR, three strains of Thauera (K172, NS1 and TK001) were used to study denitrification kinetics when using acetate as an electron donor. At low initial NO₃ ^- concentrations (∼1 mmol L^-1) and at pH 7.5, complete NO₃ ^- reduction by all strains was indicated by non-detectable NO₃ ^- concentrations and near-complete recovery (> 97%) of the initial NO₃-N as N₂ after 14 days of incubation. The relative rate of denitrification by NS1 was low, 0.071 mmol L^-1 d^-1, compared to that of K172 (0.431 mmol L^-1 d^-1) and TK001 (0.429 mmol L^-1 d^-1). Transient accumulation of up to 0.74 mmol L^-1 NO₂ ^- was observed in cultures of NS1 only. Increased initial NO₃ ^- concentrations resulted in the accumulation of elevated concentrations of NO₂ ^- and N₂O, particularly in incubations with K172 and NS1. Strain TK001 had the most extensive NO₃ ^- reduction under high initial NO₃ ^- concentrations, but still had only ∼78% of the initial NO₃-N recovered as N₂ after 90 days of incubation. As denitrification proceeded, increased pH substantially reduced denitrification rates when values exceeded ∼ 9. The rate and extent of NO₃ ^- reduction were also affected by NO₂ ^- accumulation, particularly in incubations with K172, where up to more than a 2-fold rate decrease was observed. The decrease in rate was associated with decreased transcript abundances of denitrification genes (nirS and nosZ) required to produce enzymes for reduction of NO₂ ^- and N₂O. Conversely, high pH also contributed to the delayed expression of these gene transcripts rather than their abundances in strains NS1 and TK001. Increased NO₂ ^- concentrations, N₂O levels and high pH appeared to cause higher stress on NS1 than on K172 and TK001 for N₂ production. Collectively, these results indicate that increased pH can alter the kinetics of denitrification by Thauera strains used in this study, suggesting that liming could be a way to achieve partial denitrification to promote NO₂ ^- and N₂O production (e.g., for souring control) while pH buffering would be desirable for achieving complete denitrification to N₂ (e.g., for gas-mediated MEOR).

Keywords: MEOR; NO2– accumulation; NO3– concentration; Thauera; denitrification; denitrification gene transcripts; pH; souring.

FIGURE 1

Time series of NO₃^–, NO₂^– and measured N₂ during denitrification with three different denitrifying Thauera strains (K172, NS1 and TK001) in batch cultures amended with ∼ 0.9 mmol L^–1 NO₃^– and acetate. Error bars represent the standard errors for three to four microcosm replicates. The dashed line indicates the N₂ concentrations calculated assuming decreases in initial NO₃^–-N concentrations are completely reduced to N₂.

FIGURE 2

Time series of NO₃^–, NO₂^–, and measured N₂ during denitrification with three different denitrifying Thauera strains (K172, NS1 and TK001) in batch cultures amended with two different initial NO₃^– concentrations of ∼ 2 mmol L^–1 (N2; upper graphs) or ∼ 5 mmol L^–1 (N4; lower graphs). Error bars represent the standard errors for three to four replicates. The dashed line indicates the N₂ concentrations calculated assuming decreases in initial NO₃-N concentrations are completely reduced to N₂.

FIGURE 3

(A) Percent NO₂^– reduction (Equation 2), and final concentrations of (B) N₂O and (C) N₂ produced after 30 days of incubation in batch cultures of Thauera strains, shown as a function of varying initial NO₂^– concentrations (∼ 1 to 5 mmol L^–1; x-axis). Error bars represent standard errors for three to four replicates. The time series for these batches are shown in Supplementary Figure 4.

FIGURE 4

(A) Final pH and (B) change in total alkalinity (ΔTA; meq L^–1) expressed as a function of initial NO₃^– concentrations (mmol L^–1) (Table 4) produced through acetate oxidation coupled to denitrification by Thauera strains in batch cultures. Error bars represent standard errors for three to four replicates.

FIGURE 5

Percent N₂O_REMAINING calculated as ([N₂O]/[N₂O + N₂])*100 after 15 days incubation of batch cultures of three different Thauera strains (K172, NS1 and TK001) as a function of varying initial N₂O concentrations (0.05 to ∼ 1.0 mmol L^–1), and varying initial pH values (8.10 to 9.80). Error bars represent standard errors for two replicates.

FIGURE 6

Changes in production of N₂ and expression of denitrification genes (NirS and NosZ) as a function of increasing pH in batch cultures of denitrifying Thauera strains (K172, NS1 and TK001) amended with 1 mmol L^–1 of NO₃^– and acetate. The red star (*) indicates optimum pH-7.5 taken as equivalent to 1 for comparison with high pH conditions. Error bars represent standard errors for three replicates.