Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance

Abstract

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH 3 and HNO 2 ) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low K m s for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.Chemolithoautotrophic nitrifying bacteria, i.e., ammoniaoxidizing bacteria (AOB), catalyzing the first oxidation step of ammonia to nitrite and nitrite-oxidizing bacteria (NOB) completing the oxidation of the intermediate nitrite to nitrate are known to be sensitive to low pHs. Optimum growth occurs under neutral to moderately alkaline conditions (pH 7.5 to 8.0). In liquid pure culture, growth is usually restricted to a lower pH of 5.8 (AOB) or 6.5 (NOB) (62) and activity ceases typically below pH 5.5 (28, 31). The failure of AOB to cope with acidic conditions is thought to be primarily based on the unavailability of a substrate: with decreasing pHs, ammonia, the substrate of AOB (58), is increasingly protonated. Nitrite, the substrate of NOB, undergoes protonation to nitric acid, which disproportionates to nitrate and gaseous nitric oxide at low pHs (6). Furthermore, when present at elevated concentrations under low pHs, free nitric acid negatively affects the growth and activity of nitrifying bacteria (4).Despite these limitations, autotrophic nitrifying bacteria have been isolated from, or nitrifying activity has been demonstrated in, acidic environments, such as soils, activated sludge, and biofilms. Numerous nitrifying isolates have been obtained from soils with pHs around 4 (for a review, see reference 18 and references therein) to even as low as 2.5 (45). However, the majority of such isolates do not show nitrifying activity in acidic mineral medium (18). In contrast, autotrophic nitr...

Analysis of How a Biofilm Forms on the Surface of the Aquatic Macrophyte Phragmites australis

“…Microbial biofilms are ubiquitous in aquatic environments and perform various functions such as nutrient recycling (3,9,16), purification of pollutants (5,12,15,36), and preservation of the microbial gene pool (2,7,27). Studies have clarified that the microorganisms living inside biofilms are different from those in the surrounding environment (1,37).…”

Size-dependent calcium carbonate precipitation induced microbiologically in aerobic granules

“…COD concentration and MLVSS was set at around 300 mg/L and 100 mg/L, respectively, for SOUR measurement, which was based on the report by Liu et al [36]. Dissolved oxygen concentration for SOUR was measured with DO-electrode (YSI 5000).…”

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Analysis of Ammonia Monooxygenase and Archaeal 16S rRNA Gene Fragments in Nitrifying Acid-Sulfate Soil Microcosms

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