Nitrification and nitrifiers in acidic soils

Highlights

• AOA generally make a greater contribution than AOB in acidic soils.

• Heterotrophic nitrifiers play an important role in acidic forest soils.

• Isotope labelling techniques have improved the detection of nitrification rates.

• Pure culture studies have advanced our understanding of nitrification processes.

Abstract

Nitrification, as a crucial step in nitrogen cycling and plant nutrition, is a biologically mediated process responsible for enormous losses of nitrogen fertilizer and a contributor to environmental pollution. The recent progress in our understanding of nitrification and nitrifiers, specifically in acidic soils, is discussed and reviewed. At one time it was assumed that rates of nitrification are relatively low in acidic soils. However, more recent studies have demonstrated nitrification down to pH 3.0 and that the rate of nitrification can equal, or even exceed, that found in neutral soils. Studies on acidic forest soils in Europe noted that they have a high potential for nitrate production. Furthermore, using the ¹⁵N isotope-dilution technique it was shown that net nitrification measurements can markedly underestimate gross nitrification in these natural and highly organic systems. Using selective inhibitors it has been demonstrated that heterotrophic nitrifiers can contribute to nitrification. While heterotrophic nitrification can be performed by a wide range of bacteria and fungi, inhibitor studies point to fungi to be mainly responsible. Autotrophic ammonia-oxidizing bacteria (AOB), such as Nitrosomonas and Nitrosospira, have been known for some considerable time but have generally found to be inactive in acidic conditions. The discovery of ammonia monooxygenase in uncultured archaea that were functionally active at low pH pointed to an autotrophic microbial group (ammonia oxidizing archaea, AOA) that might be adapted to low substrate (ammonia) concentrations and responsible for nitrification in the wider range of acidic grassland and cultivated soils. Obligately acidophilic AOA have more recently been cultivated while stable isotope probing has been used to confirm the dominance of AOA over AOB in acidic soils. Detailed molecular studies using both 16S rRNA and amoA (ammonia monooxygenase sub-unit A) gene sequencing are continuing to expand our appreciation of the diversity of both AOB and AOA and how this varies over different pH ranges and in different ecosystems. Similar work is being directed towards nitrite oxidizing bacteria (NOB) but to date we do not fully know the role of pH in controlling NOB activity. Such understanding of nitrification and nitrifiers will help develop new effective nitrification inhibitors and aid the management of nitrogen cycling in acidic soils.

Introduction

Acidic soils (defined as pH < 5.5) occupy 30% of the world's ice-free land and mainly support forest, woodland and grassland, with a minor fraction used for arable crops (Vonuexkull and Mutert, 1995). Nitrification is a crucial step in nitrogen biogeochemical cycling and plant nutrition in soil-plant ecosystems. Nitrification in soil is generally considered to be a two-step process where ammonia is first oxidized to nitrite by ammonia oxidizers, and subsequently to nitrate by nitrite-oxidizing bacteria (NOB). However, the recent discovery of some species of Nitrospira capable of complete ammonia oxidation (comammox) in water systems (Daims et al., 2015, van Kessel et al., 2015) and detection in soil (Pjevac et al., 2017) indicates that this process could also be relevant. Nitrification can lead to nitrate leaching, losses of nitrogen-based fertilizers and the increased emission of the greenhouse gas nitrous oxide (Wrage et al., 2001). In particular, nitrification in acidic soils can lead to further acidification and aluminum toxicity (He et al., 2012). It was previously assumed that nitrification was relatively low in acidic soils since the availability of the substrate ammonia (NH₃) for the ammonia monoxygenase (AMO) enzyme of ammonia oxidizers would be limited and all isolated bacterial ammonia oxidizers did not grow in standard laboratory medium with a pH < 5.5 (De Boer and Kowalchuk, 2001). However, many recent studies have suggested that nitrification can occur at pH values as low as 3.0 (Norton and Stark, 2011) and that rates of nitrification based on mineralized organic nitrogen can be equal or even greater than that typically found in neutral pH soils (Booth et al., 2005).

There are probably three breakthrough discoveries that have promoted scientific interest and discussion on nitrification mechanisms in acidic soils. Firstly, in the 1980s, acidic forest soils in northwestern Europe were found to have a high potential for nitrate production (Vanbreemen and Vandijk, 1988) and subsequently resulted in increased public concern regarding the potentially damaging effects of nitrate leaching and pollution. Secondly, Stark and Hart (1997) used the ¹⁵N isotope-dilution technique in intact soil cores to measure gross nitrification and microbial assimilation in a large number of acidic forest soils. They demonstrated that standard measurements of net nitrification could be significantly lower than gross nitrification rates determined using ¹⁵N-based measurements and that microbial communities play an important role in promoting nitrate loss in acidic soil ecosystems. Thirdly, homologues of bacterial AMO-encoding genes were found in metagenomic fragments of uncultured archaea (Schleper et al., 2005, Venter et al., 2004) followed by the isolation of Nitrosopumilus maritimus (Könneke et al., 2005), confirming the potential for ammonia oxidation by organisms belonging to the (then-described) mesophilic crenarchaeota, and subsequently termed ammonia-oxidizing archaea (AOA). These organisms were found to be functionally important in soils and sediments (Leininger et al., 2006, Schleper and Nicol, 2010), especially in acidic soil ecosystems (Prosser and Nicol, 2008, Yao et al., 2011b). The discovery of obligately acidophilic AOA in these soils indicated that they possess specific adaptations allowing them to grow at low pH (Lehtovirta-Morley et al., 2011, Lehtovirta-Morley et al., 2014).

Since the turn of the century there has been a continual increase in the number of studies examining nitrification and nitrifiers in acidic soils. The majority of studies have focused on the distinct ecological niches of ammonia oxidizing bacteria and archaea, their relative importance to autotrophic nitrification and their environmental drivers (Erguder et al., 2009, Hu et al., 2014, Yao et al., 2013, Zhalnina et al., 2012). Some studies report on the methodology for the measurement of nitrification, the functioning of isolated microorganisms and the mechanisms responsible for nitrification.