# Nitrosotalea devaniterrae gen. nov., sp. nov. and Nitrosotalea sinensis sp. nov., two acidophilic ammonia oxidising archaea isolated from acidic soil, and proposal of the new order Nitrosotaleales ord. nov. within the class Nitrososphaeria of the phylum Nitrososphaerota

Laura E Lehtovirta-Morley, Chaorong Ge, Jenna Ross, Huaiying Yao, Christina Hazard, Cécile Gubry-Rangin, James I Prosser, Graeme W Nicol

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Laura E Lehtovirta-Morley, Chaorong Ge, Jenna Ross, Huaiying Yao, Christina Hazard, et al.. Nitrosotalea devaniterrae gen. nov., sp. nov. and Nitrosotalea sinensis sp. nov., two acidophilic ammonia oxidising archaea isolated from acidic soil, and proposal of the new order Nitrosotaleales ord. nov. within the class Nitrososphaeria of the phylum Nitrososphaerota. International Journal of Systematic and Evolutionary Microbiology, 2024, 74 (9), 10.1099/ijsem.0.006387. hal-04797969

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*Nitrosotalea devaniterrae* gen. nov., sp. nov., and *Nitrosotalea sinensis* sp. nov., two acidophilic ammonia oxidising archaea isolated from acidic soil, and proposal of the new order *Nitrosotaleales* ord. nov. within the class *Nitrososphaeria* of the phylum *Nitrososphaerota*

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Laura E. Lehtovirta-Morley¹, Chaorong Ge², Jenna Ross³, Huaiying Yao²,⁴, Christina

Hazard⁵, Cécile Gubry-Rangin³, James I Prosser³ and Graeme W. Nicol⁵*

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¹School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

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²Wuhan Institute of Technology, 206 Guanggu 1st Road, Wuhan 430205, People's Republic of China

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³Institute of Biological Sciences, University of Aberdeen, Cruickshank Building, St Machar Drive, Aberdeen, Scotland, AB24 3UU, UK

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⁴Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen, 361021, China

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⁵Univ Lyon, CNRS, INSA Lyon, Université Claude Bernard Lyon 1, Ecole Centrale de Lyon, Ampère, UMR5005, 69134 Ecully, France

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*Correspondence author: Graeme W. Nicol, Email: graeme.nicol@cnrs.fr, Telephone: +33 (0) 472 18 60 88

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**Keywords:** *Nitrosotalea*, soil, archaeal ammonia oxidiser, nitrification, ammonia oxidation, acidophile

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**Accession numbers:** NCBI accession numbers for genome sequences are PRJEB10948 (complete) for Nd1T and GCF_900143675 (draft) for Nd2T. NCBI accession numbers for 16S rRNA gene sequences are JN227488 for Nd1T and KJ540205 for Nd2T.

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**Abbreviations:** AOA, ammonia-oxidising archaea; AOB, ammonia-oxidising bacteria; *amoA*, ammonia monooxygenase sub-unit A; ANI, average nucleotide identity; ATU, allylthiourea; DAPI, 4',6-diamidino-2-phenylindole; DCD, dicyandiamide; DOPE-FISH, double labelling of oligonucleotide probes for fluorescence in situ hybridization; GDGT, glycerol dibiphytanyl glycerol tetraethers; GTDB, Genome Taxonomy Database; GTGT, glycerol trialkyl glycerol tetraethers; IPL, intact polar lipids; MeO-AR, methoxy archaeol; MES, 2-(N-morpholino)ethanesulfonic acid.
30 **Abstract**

Two obligately acidophilic, mesophilic and aerobic soil ammonia-oxidising archaea were isolated from a pH 4.5 arable sandy loam (UK) and pH 4.7 acidic sulphate paddy soil (China) and designated strains Nd1T and Nd2T, respectively. Strains share >99% 16S rRNA gene sequence identity and their genomes are <2 Mb in length, sharing 79% average nucleotide identity, 81% average amino acid identity and a G+C mol% ~37%. Strains are chemolithotrophs that fix carbon dioxide and gain energy by oxidising ammonia to nitrite with no evidence of mixotrophic growth. Neither strain is capable of using urea as a source of ammonia. Strains are non-motile in culture although Nd1T does possess genes encoding flagella and therefore may be motile under certain conditions. Cells are small angular rods 0.5 - 1 µm in length and grow in the pH range 4.0 – 6.1 and temperature range 20 – 40°C. Strains Nd1T and Nd2T are distinct in genomic and physiological features and are assigned as the type strains for the species *Nitrosotalea devaniterrae* sp. nov. (type strain, Nd1T=NCIMB 15248T=DSM 110862T) and *Nitrosotalea sinensis* sp. nov. (type strain, Nd2T=NCIMB 15249T=DSM 110863T), respectively, within the genus *Nitrosotalea* gen. nov. The family *Nitrosotaleaceae* fam. nov. and order *Nitrosotaleales* ord. nov. are also proposed officially.

46 **Introduction**

Nitrification, the oxidation of ammonia to nitrite and subsequently to nitrate, is a key process within the terrestrial and aquatic nitrogen cycles and in aerobic wastewater treatment processes.

The first step in this process, ammonia oxidation to nitrite, was considered to be performed exclusively by ammonia-oxidising bacteria (AOB), following their initial cultivation from soil in 1890 (1). Ammonia oxidation provides energy to these chemolithoautotrophs and their presence and activity have been demonstrated in a wide range of environments. This view of ammonia oxidation was overturned by isolation of the first ammonia-oxidising archaeon, *Nitrosopumilus maritimus* SCM1T (2), from a tropical marine aquarium, after earlier evidence
from marine and soil metagenomes (3,4). All currently isolated ammonia-oxidising archaea (AOA) are autotrophic and belong to the phylum *Nitrososphaerota* (5) (formally *Thaumarchaeota* (6) and within the *Thermoproteota* phylum in GTDB taxonomy (7)), within the *Archaea*. Extensive molecular ecology studies have provided evidence for ubiquity of AOA in terrestrial and aquatic environments. They appear to dominate ammonia-oxidising activity in marine ecosystems (8,9) and contribute to ammonia oxidation in soil systems together with AOB and complete ammonia-oxidising *Nitrospira* (10).

Fundamental physiological differences between members of the archaeal and bacterial domains stimulated investigation of potential niche specialisation and differentiation of AOA and AOB (11,12) and physiological diversity within these two groups has led to their co-existence in the majority of soils. All isolated betaproteobacterial AOB grow optimally at near neutral pH although some *Nitrosospira* strains can demonstrate growth down to pH 5.0 (13,14) and an acid-tolerant gammaproteobacterial AOB has recently been cultivated from soil (15). However, there is strong evidence for significant contributions of AOA to ammonia oxidation in acid soils (16,17). Here we describe the isolation and taxonomic placement of two obligately acidophilic AOA belonging to the class *Nitrososphaeria* that provide a convincing explanation for this observation.

## Habitat and isolation

Strains Nd1T and Nd2T were both isolated from acidic soils. Strain Nd1T was enriched from an acidic agricultural soil (loamy sand) sampled from Scotland's Rural College Craibstone Woodlands Field pH plots (latitude/longitude: 57.18 N/2.21 W), with an 8-year crop rotation cycle (18). Soil pH had been maintained at pH 4.5 since 1961 and other soil characteristics are described in Kemp et al. (19) and Paterson et al. (20). Isolation of a pure culture of strain Nd1T from this enrichment is described in Lehtovirta-Morley et al. (21). Strain Nd2T was isolated from an acidic sulphate paddy soil (pH 4.7) sampled in Taishan County, Guangdong Province
(latitude/longitude: 22.11 N/112.81 E) in Southern China that had been subjected to urea fertilisation and double rice-cropping since 2003 (21).

While autotrophic soil ammonia oxidisers can form colonies on solidified medium after several weeks or months (22,23) they are difficult to separate from faster growing heterotrophs.

Ammonia oxidisers are therefore typically enriched by inoculation of liquid mineral salts medium, containing ammonium, with soil and incubated for several weeks, with or without shaking. Enrichment of strain Nd1T was achieved by inoculation of soil (1% w/v) into 100 ml glass bottles containing 50 ml mineral salts medium (18) with 500 µM ammonium chloride and 50 mg l⁻¹ streptomycin, adjusted to pH 4.5 with HCl and incubation at 28°C in the dark without shaking. Ammonia oxidation was assessed by an increase in nitrite concentrations and Nd1T enriched by routinely transferring 2% of mid-exponential culture (~50 µM nitrate produced). After repeated subcultures in liquid medium to reduce the richness and abundance of contaminating heterotrophs, a highly enriched culture was obtained with Nd1T representing approximately 90% of all cells as assessed by using double labelled oligonucleotide probes and fluorescence in situ hybridization (DOPE-FISH) and 4',6-diamidino-2-phenylindole (DAPI) staining (18). Growth of Nd1T was also assessed by quantification of ammonia monooxygenase sub-unit A (amoA) gene abundance (18). A pure culture was obtained by filtering out larger contaminating bacteria (pore size 0.45 µm) but growth was initially only possible following supplementation of medium with 80 mg l⁻¹ casamino acids or with filter-sterilised (pore size 0.22 µm) spent medium from the mixed enrichment culture previously incubated for 2 days.

After a further 10 subcultures, however, autotrophic growth in mineral salts medium containing ammonium was achieved without supplementation with organic carbon (21). Purity of cultures was confirmed by plating on 10% Tryptone Soya Agar plates for growing potential heterotrophic contaminants, DOPE-FISH fluorescence microscopy for bacterial and archaeal cells and a lack of PCR amplification of bacterial 16S rRNA genes.
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A similar approach was used to enrich strain Nd2T, but with incubation at 37°C and replacement of streptomycin with 50 mg l⁻¹ kanamycin. A pure culture of this strain was obtained after several subcultures without the need for filtration or supplementation with organic carbon (21).

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Both species are routinely cultured by 2% (v/v) inoculation of late exponential phase cultures into mineral salts medium containing 500 µM ammonium chloride adjusted to pH 5.3 with the addition of 5 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer. Cultures are incubated at 25°C (strain Nd1T) and 37°C (strain Nd2T).

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## Phylogeny

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The two strains possess a close phylogenetic relationship with 99.2% 16S rRNA identity. These sequences were placed within the well-supported monophyletic Group I.1a-associated cluster (candidatus order *Nitrosotaleales*) and share a maximum of 90% 16S rRNA gene identity with organisms placed within the Group I.1a cluster (order *Nitrosopumilales*) (Fig. 1a). The 16S rRNA gene phylogeny was also congruent with phylogenomic analysis based on concatenated single copy genes (Fig. 1b). The strains shared 95.5% *amoA* gene sequence identity and were placed within the C14 (24) and NT-α-1 (25) *amoA* gene-defined lineages. Despite the high identity of the marker gene sequences, the average nucleotide identity (ANI) between Nd1T, Nd2T and the *Nitrosotalea* strain *Ca*. *Nitrosotalea okcheonensis* (26) all ranged between 79-83% (27), which is considerably below the species threshold of 95% (28). All three strains (including *Ca*. *Nitrosotalea okcheonensis*) have an ANI lower than 70% with representatives of the order *Nitrosopumilales*, which is the minimum level of identity between 11 representatives of the *Nitrosopumilales* (27). We therefore propose that strains Nd1T and Nd2T be assigned to two different species as *Nitrosotalea devaniterrae* sp. nov. (formally described as *Ca*. *Nitrosotalea devanaterra* (18)) and *Nitrosotalea sinensis* sp. nov., respectively. We also propose *Nitrosotalea* as the type genus within the family *Nitrosotaleaceae* fam. nov. (29) and order *Nitrosotaleales* ord. nov. within the class *Nitrososphaeria* (30).
130 **Genome features**

131 The genomes of Nd1T and Nd2T were 1.81 and 1.60 Mb in length, which is comparable to type
132 strains from the order *Nitrosopumilales*, but smaller than soil-derived isolates from the order
133 *Nitrososphaerales* which are >2.5 Mb in size. The G+C mol% content of both genomes was
134 37% and slightly higher than seven type strains of the *Nitrosopumilales* (33-34%) of both
135 marine and soil origin but lower than *Nitrososphaera viennensis* EN76T (52.7%) (Table 1).
136 Initial characterisation of the *N. devaniterrae* genome is described in Lehtovirta-Morley *et al.*
137 (31), with subsequent comparative analysis by Herbold *et al.* (27) of genomes of strains Nd1T
138 and Nd2T, the enrichment culture *Ca. Nitrosotalea okcheonensis* and a *Nitrosotalea*
139 metagenome-assembled genome (SbT1) with 19 other *Nitrososphaerota*. This analysis
140 identified a nitrososphaerotal core genome of 743 gene families, of which 103 formed a
141 *Nitrosotalea* core genome. This core is smaller than that of *Nitrososphaera* but larger than
142 those of other nitrososphaerotal genera. Within this core genome, 46 gene families were
143 homologous to genes in other *Nitrososphaerota* and there was evidence that 8 gene families
144 had been recruited by horizontal gene transfer from non-*Nitrososphaerota*, mainly acidophilic
145 organisms, suggesting their potential roles in acidophily adaptation.

146 **Phenotypic characterisation**

147 Characterisation of the initial enrichment culture of *N. devaniterrae* is described in Lehtovirta-
148 Morley *et al.* (18) and characterisation of pure cultures of both species is described in
149 Lehtovirta-Morley *et al.* (21). Cells of strains Nd1T and Nd2T are morphologically
150 indistinguishable under light or scanning electron microscopy and are rod-shaped (Fig. 2).
151 Cells have a slightly angular appearance, typical of AOA within the order *Nitrosopumilales*
152 (32), but distinct from the cocci or irregular cocci of the order *Nitrososphaerales* (33,34) and
153 *Ca. Nitrosocaldales* (35,36). The cells have electron-dense poles at which ribosomes are
154 concentrated (18) and have apparent intracellular particles and inclusion bodies the function of
which is unknown (Fig. 2c,d). Pure cultures of both species grow on inorganic, mineral salts medium containing ammonium, obtaining cell carbon by CO₂ fixation and energy through oxidation of ammonia to nitrite. Both are obligate acidophiles, with similar pH ranges for growth, similar pH optima and no growth above pH 6.1. Maximum specific growth rates are similar to (strain Nd2T) or slightly lower than (strain Nd1T) those of other *Nitrososphaerota*. Cell yields are similar to those of other *Nitrososphaerota*, but cell-specific activities are lower (21), and Nd1T growth is inhibited at much lower concentrations of nitrite compared to neutrophilic type strains (Table 1), likely due to the higher proportion of nitrite converted to nitrous acid at low pH.

Membrane lipid compositions of strain Nd1T and other AOA indicate that there is a characteristic lipid profile specific to *Nitrososphaerota* that is distinct from that found in other major archaeal phyla *Crenarchaeota* and *Euryarchaeota*. The cell membranes of all *Nitrososphaerota* investigated to date contain a relatively complex pattern of core lipids, with glycerol dibiphytanyl glycerol tetraethers (GDGT), glycerol diphytanyl diethers (archaeols, AR), hydroxylated GDGTs (OH-GDGTs) and glycerol trialkyl glycerol tetraethers (GTGTs), with intact polar lipids (IPL) consisting of monoglycosyl, diglycosyl, phosphohexose and hexose-phosphohexose headgroups (37). GDGTs and GTGTs are found in all three archaeal phyla, but the GDGT lipid crenarchaeol and methoxy archaeol (MeO-AR) are exclusive to *Nitrososphaerota* (37). The relative abundance of MeO-AR in strain Nd1T is highest (20.9%) of all AOA strains investigated (31,37). The composition of core lipids is consistent with 16S rRNA-gene phylogeny, providing both a basis for chemotaxonomy and potential links to phenotype, adaptation and niche specialisation. There are also some correlations between IPL content and habitat, with similarities between IPLs in strain Nd1T, other terrestrial mesophiles and terrestrial thermophiles. These results provide good evidence for phylogenetic relationships between lipid composition and archaeal phyla. However, the importance of lipid
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composition in *Nitrosotalea* species, its correlation with phylogeny and robust links between phenotype and ecological distributions require analysis of more isolates.

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A number of nitrification inhibitors have been developed for physiological studies but also for commercial use to reduce loss of ammonia-based fertilisers in agricultural soils (38). Although originally designed to target AOB, these inhibitors are generally similarly effective against AOA. The sensitivity of strain Nd1T to three such inhibitors, allylthiourea (ATU), dicyandiamide (DCD) and nitrapyrin, has been investigated by Lehtovirta-Morley *et al*. (39) at concentrations that lead to inhibition of AOB. Inhibition of ammonia oxidation by strain Nd1T in liquid batch culture by DCD and nitrapyrin is similar to that typically observed in AOB (38), with complete inhibition at concentrations of 5 mM and 10 µM, respectively. ATU, however, is less effective, with only 85% inhibition at a concentration of 100 µM, which would typically inhibit AOB activity. Nitrapyrin has a similar inhibitory effect on growth as for AOB.

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There is currently no direct evidence of mixotrophic growth of AOA, including *Nitrosotalea*. Early suggestions of mixotrophy arose through stimulation of growth of another soil AOA, *N. viennensis* EN76T, by pyruvate (33) and of *N. maritimus* SCM1T by pyruvate and a-ketoglutarate (32). There is now strong evidence that the α-keto acids pyruvate and oxaloacetate are not assimilated but stimulate growth by detoxifying intracellular H₂O₂ produced during ammonia oxidation (43). Intermediates of the tricarboxylic acid cycle have differing effects on growth of strain Nd1T and strain Nd2T (21) and, in some cases, effects of individual organic acids on specific growth rate and yield differ within the same species. Pyruvate reduces the growth yield of both species and reduces the maximum specific growth rate of strain Nd1T, but not strain Nd2T. In general, strain Nd1T is more sensitive to inhibition

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by organic acids than strain Nd2T. Both species are inhibited by nitrous acid and strain Nd1T is
inhibited at ammonia concentrations >50 mM.

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Ecology

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The major ecological significance of the *Nitrosotalea* genus is its contribution to explaining
the paradoxical occurrence of high rates of ammonia oxidation and nitrification in acidic soils,
which comprise approximately 30% of soil globally. There is evidence of adaptation of some
AOB phylogenetic lineages to acidic conditions (44), the growth range or activity of cultivated
neutrophilic AOB can extend down to pH 5.0 such as for *Nitrosospira* sp. AF (13) and
*Nitrosospira lacus* (14) and AOB can been isolated from acid soils (45). However, with
exception of the gammaproteobacterial genera *Candidatus* Nitrosoglobus (15,46) and
*Candidatus* Nitrosacidococcus (47), no cultivated AOB grow optimally on ammonium at pH
values below 7 or are obligately acidophilic. This was believed to be through increasing
ionisation of NH₃ (the proposed substrate for ammonia monooxygenase) to NH₄⁺ with
decreasing pH, thus reducing available substrate concentration. Consistent with the substrate
limitation at low pH, strains Nd1T and Nd2T have remarkably low half-saturation constants and
high affinities for ammonia (K<sub>m(app)</sub> = ~0.6–2.8 nM NH₃) (48). Molecular data provided
evidence that AOA were more abundant and active than AOB in acidic soils (16,17) and the
potential existence of acidophilic AOA. This was confirmed by cultivation of the acidophile
strain Nd1T (18), subsequent isolation and enrichment of other acidophilic *Nitrosotalea* strains
(21,26) and demonstration of autotrophic growth of *Nitrosotalea* phylotypes in acid soil
(17,18). This therefore provides the most parsimonious explanation for this paradox, with
consequences for mitigation of fertiliser loss and nitrous oxide emissions in acid soils (49).

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Description of *Nitrosotalea* gen. nov.
Nitrosotalea (Ni.tro.so.ta'le.a. N.L. masc. adj. nitrosus, nitrous; L. fem. n. talea, slender rod; N.L. fem. n. Nitrosotalea, a slender rod producing nitrite). Aerobic. Chemolithoautotrophic growth by ammonia oxidation to nitrite. Organic carbon obtained by CO₂ fixation. Mesophilic, with optimal temperatures between 25°C and 37°C. Acidophilic, growing in the pH range 4.2 – 6.1. Free living in terrestrial ecosystems, particularly acidic soils. Nitrosotalea strains possess >99% 16S rRNA gene identity with each other and 89-90% 16S rRNA gene identity within representatives of the order Nitrosopumilales. G+C content is low, as is typical for many AOA, ranging from 37.1-37.4 mol%. Currently comprises two isolates in pure culture, the type species Nitrosotalea devaniterrae, Nitrosotalea sinensis and an enrichment culture, Ca. Nitrosotalea okcheonensis CS (27).

## Description of *Nitrosotalea devaniterrae* sp. nov.

*Nitrosotalea devaniterrae* sp. nov. (de.va.ni.ter'rae. L. fem. n. Devana the Roman name for Aberdeen; L. fem. n. terra soil; N.L. gen. n. devaniterrae of soil from Aberdeen, describing its habitat of isolation).

Displays the following properties in addition to those described for the genus. Rod-shaped cells (0.33 µm × 0.89 µm). Maximum specific growth rate 0.011 h⁻¹, cell yield 6.45 x 10⁴ cells (µM NH₄⁺)⁻¹ and specific cell activity 0.072 fmol NO₂⁻ cell⁻¹ h⁻¹ (equivalent to approx. 7 µmol (NH₄⁺)mg protein⁻¹ h⁻¹). Growth in the pH range 4.2 – 5.6, optimum pH 5.2 and temperature range 20°C - 30°C, optimum 25°C. Yield increased by oxaloacetate and decreased by pyruvate and citrate. Specific growth rate reduced by pyruvate, citrate, α-ketoglutarate, succinate, fumarate, and malate. Growth inhibited by 0.91 – 3.5 µM HNO₂.

The genome is a single circular 1.81-Mb chromosome with 2,103 coding sequences and a G+C content of 37.1 mol% and an ANI of ≤83% with other cultivated representatives of the
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*Nitrosotalea*. NCBI accession numbers for complete genome and 16S rRNA gene sequences are PRJEB10948 and JN227488, respectively.

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The type strain is *Nitrosotalea devaniterrae* Nd1T (=NCIMB 15248T =DSM 110862T), which was isolated from an acidic (pH 4.5) agricultural soil located near Aberdeen (Scotland, UK).

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**Description of *Nitrosotalea sinensis* sp. nov.**

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*Nitrosotalea sinensis* sp. nov. (si.nen'sis. N.L. fem. adj. sinensis, from China, describing its habitat of isolation).

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Displays the following properties in addition to those described for the genus. Rod-shaped cells
0.22 µm × 0.76 µm. Maximum specific growth rate 0.025 h⁻¹, cell yield 7 x 10⁴ cells (µM
NH₄⁺and specific cell activity 0.065 fmol NO₂⁻ cell⁻¹ h⁻¹ (equivalent to approx. 6.3 µmol NH₄⁺
mg protein⁻¹ h⁻¹). Growth in the pH range 4.0 – 6.1, optimum pH 5.2, and temperature range
20°C – 42°C, optimum 35°C. Yield simulated by oxaloacetate and inhibited by pyruvate and
fumarate. Growth inhibited by 1.61 - 5.7 µM HNO₂.

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The genome is a 1.60-Mb chromosome with 1,888 coding sequences and a G+C content of 37.4 mol% and an ANI of ≤79% with other cultivated representatives of the *Nitrosotalea*.

NCBI accession numbers for draft genome and 16S rRNA gene sequences are GCF_900143675 and KJ540205, respectively.

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The type strain is *Nitrosotalea sinensis* Nd2T (=NCIMB 15249T =DSM 110863T), which was isolated from an acidic sulphate paddy soil (pH 4.7) sampled in Taishan County, Guangdong Province in Southern China.

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271 **Description of *Nitrosotaleaceae* fam. nov.**

272 *Nitrosotaleaceae* (Ni.tro.so.ta.le.a.ce'ae. N.L. fem. n. *Nitrosotalea*, type genus of the family; -aceae, ending to denote a family; N.L. fem. pl. n. *Nitrosotaleaceae*, the family of the genus *Nitrosotalea*).

275 Mesophilic, rod-shaped cells. Obligately acidophilic and obligately aerobic,
276 Chemolithoautotrophic, fixing carbon dioxide and oxidising ammonia to nitrite. Phylogeny of
277 both *amoA* and 16S rRNA genes provides support for a distinct branch within the phylum
278 *Nitrososphaerota*. Currently comprises a single genus, *Nitrosotalea*. Further details of the
279 family are provided in the description of the order *Nitrosotaleales*.

280 **Description of *Nitrosotaleales* ord. nov.**

281 *Nitrosotaleales* (Ni.tro.so.ta.le.a'les. N.L. fem. n. *Nitrosotalea*, type genus of the order; -ales, 282 ending to denote an order; N.L. fem. pl. n. *Nitrosotaleales*, the order of the genus *Nitrosotalea*). 283 Small rods, 0.5 - 1 µm in length with a slight angular appearance and electron-dense poles. 284 Obligate aerobes and obligate acidophiles, growing in the pH range 4.0 – 6.1 and the 285 temperature range 20 – 40°C. Chemolithotrophs that fix carbon dioxide and gain energy by 286 oxidising ammonia to nitrite. The order *Nitrosotaleales* refers to the Group I.1a-associated 287 *Nitrososphaerota*, which is phylogenetically distinct from the orders *Nitrosopumilales* (which 288 refers to Group I.1a and is represented by *Nitrosopumilus maritimus* SCM1T) (32), 289 *Nitrososphaerales* (which refers to Group I.1b and is represented by *Nitrososphaera viennensis* 290 EN76T) (30) and ‘*Candidatus* Nitrosocaldales’ (50) (which refers to the ThAOA group and is 291 represented by ‘*Candidatus* Nitrosocaldus yellowstonensis’ HL72) (51) within the class 292 *Nitrososphaeria*. Found in soil, particularly acidic soil. This order contains the family 293 *Nitrosotaleaceae*. The type genus is *Nitrosotalea*.

294 **Conflicts of interest**
295 The authors declare that there are no conflicts of interest.

296 **Funding information**

297 This work was financially supported by a NERC postgraduate studentship (LLM), NERC

298 Advanced Fellowship NE/D010195/1 (GWN), NERC standard research grant NE/1027835/1

299 (GWN, JIP), Royal Society International Exchange grant IE111001 (JIP), National Science

300 Foundation of China Grant 81101283 (HY, CG), Royal Society University Research

301 Fellowship UF150571 (CGR), Royal Society Dorothy Hodgkin Research Fellowship

302 DH150187 (LLM) and an AXA Research Fund Chair (GWN).

303

304
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484
485 **Figure legends**

486 **Figure 1.** Phylogenetic relationship of strains Nd1T and Nd2T with other candidate or validated
487 strains of the class *Nitrososphaeria*. Maximum likelihood phylogenies were generated using
488 PhyML (52) with automatic model selection. (a) Phylogenetic analysis of 16S rRNA genes
489 using 1,423 unambiguously aligned positions using the general time reversible (GTR)
490 substitution model with estimated gamma and invariant distribution of sites. The scale bar
491 represents an estimated 0.05 changes per nucleotide position and circles at nodes describe
492 bootstrap support (1,000 replicates). (b) Phylogenetic analysis of concatenated protein
493 sequences (7,951 unambiguously aligned amino acid positions) inferred from 45 single copy
494 genes generated using GToTree (53) and determined using the LG substitution model with
495 freerate variation across sites. The scale bar represents an estimated 0.1 changes per nucleotide
496 position and circles at nodes describe bootstrap support (100 replicates).

497 **Figure 2.** Scanning electron micrographs (a, b) and transmission and electron micrographs (c, d) of strains Nd1T (a, c) and Nd2T (b, d). Scale bars are 1 µm (a, b) and 0.5 µm (c, d) and arrows indicate subcellular particles. (From (21), with permission.)
498
499
<figure><img src="image_2.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_3.png" /></figure>
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>

D

<figure><img src="image_7.png" /></figure>
<table><caption>**Table 1.** Characteristics of ND1T and ND2T compared to validated strains within the class *Nitrososphaeria*. Data for *Nitrosopumilus maritimus* SCM1T, *Nitrosopumilus cobalaminigenes* HCA1T, *Nitrosopumilus oxyclinae* HCE1T and *Nitrosopumilus ureiphilus* PS0T were previously reported by Qin et al. [32], *Nitrosopumilus piranensis* DC3T and *Nitrosopumilus adriaticus* NF5T by Bayer et al. [54], *Nitrosopumilus zosterae* NM25T by Nakagawa et al. [55], *Nitrosarchaeum koreense* MY1T by Jung et al. [42] and *Nitrososphaera viennensis* EN76T by Stieglmeier et al. [30]. ND, no data; -, negative; +, positive.</caption><thead><tr><th>Characteristic</th><th>Nd1T</th><th>Nd2T</th><th>D3CT</th><th>HCA1T</th><th>HCE1T</th><th>NF5T</th><th>NM25T</th><th>PS0T</th><th>SCM1T</th><th>MY1T</th><th>EN76T</th></tr></thead><tbody><tr><td>Validated genus</td><td>Nitrosotalea</td><td>Nitrosotalea</td><td>Nitrosopumilus</td><td>Nitrosopumilus</td><td>Nitrosopumilus</td><td>Nitrosopumilus</td><td>Nitrosopumilus</td><td>Nitrosopumilus</td><td>Nitrosopumilus</td><td>Nitrosoarchaeum</td><td>Nitrososphaera</td></tr><tr><td>Habitat source</td><td>Soil (pH 4.5)</td><td>Soil (pH 4.7)</td><td>Seawater</td><td>Seawater</td><td>Seawater</td><td>Seawater</td><td>Sediment</td><td>Sediment</td><td>Marine aquarium</td><td>Soil (pH 5.6)</td><td>Soil (pH 8.0)</td></tr><tr><td>Growth temperature (°C)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Range</td><td>20-30</td><td>20-42</td><td>15-37</td><td>10-30</td><td>04-30</td><td>15-34</td><td>20-37</td><td>10-30</td><td>16-35</td><td>15-30</td><td>28-47</td></tr><tr><td>Optimum</td><td>25</td><td>35</td><td>32</td><td>25</td><td>25</td><td>30-32</td><td>30</td><td>26</td><td>32</td><td>25</td><td>42</td></tr><tr><td>Growth pH</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Range</td><td>4-5.5</td><td>4-6.1</td><td>6.8-8.0</td><td>6.8-8.1</td><td>6.4-7.8</td><td>6.8-8.0</td><td>6.1-7.7</td><td>5.9-8.1</td><td>6.8-8.1</td><td>6.0-8.0</td><td>6.0-8.0</td></tr><tr><td>Optimum</td><td>5.2</td><td>5.2</td><td>7.1-7.3</td><td>7.3</td><td>7.3</td><td>7.1</td><td>7.1</td><td>6.8</td><td>7.3</td><td>7</td><td>7.5</td></tr><tr><td>Max. ammonia oxidation (fmol cell<sup>-1</sup> day<sup>-1</sup>)</td><td>1.7</td><td>1.5</td><td>10.9</td><td>6</td><td>5.8</td><td>9.6</td><td>ND</td><td>2.9</td><td>12.7</td><td>12.7</td><td>ND</td></tr><tr><td>Min. generation time (h)</td><td>57</td><td>28</td><td>27</td><td>30</td><td>33</td><td>34</td><td>28</td><td>28</td><td>45</td><td>19</td><td>28</td></tr><tr><td>Ammonium tolerance (mM)</td><td>&gt;10 mM</td><td>ND</td><td>30</td><td>10</td><td>1</td><td>25</td><td>ND</td><td>20</td><td>12.7</td><td>10</td><td>15</td></tr><tr><td>Nitrite tolerance (mM)</td><td>&lt;0.1</td><td>ND</td><td>10</td><td>ND</td><td>ND</td><td>15</td><td>ND</td><td>ND</td><td>2</td><td>5</td><td>10</td></tr><tr><td>Use of urea</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Genome size (Mb)</td><td>1.81</td><td>1.60</td><td>1.71</td><td>1.56</td><td>1.59</td><td>1.8</td><td>1.76</td><td>2.17</td><td>1.65</td><td>1.61</td><td>2.52</td></tr><tr><td>DNA G+C content (mol%)</td><td>37.1</td><td>37.4</td><td>33.8</td><td>33.0</td><td>33.1</td><td>33.4</td><td>33.8</td><td>33.4</td><td>34.2</td><td>32.7</td><td>52.7</td></tr></tbody></table>