Biochemical, physiological, and molecular characterisation of a large collection of aerobic endospore-forming bacteria isolated from Brazilian soils

31 The aerobic endospore -forming bacteria (AEFB) comprise species of Bacillus and 32 related genera, allocated in the phylum Firmicutes. Although Bacillus spp. are among 33 the first bacteria to be characterised, the wide diversity render s appropriate 34 categorisation and generalisations challenging tasks. To determine genetic diversity, 35 analyses at the molecular level are the most accurate. However, gene expression, 36 morphological, biochemical, and physiological aspects must also be considere d. The 37 metabolism of bacteria is adapted to their natural environment or host. Thus, metabolic 38 outlines can be used for identifying AEFB , form the basis of the formal description of 39 bacterial taxa, and are strongly recommended for taxonomic purposes. This work 40 addressed the biochemical and physiological profiles of 312 environmental AEFB —41 designated as SDF (Solo do Distrito Federal )—by performing 30 tests. Out of it, 246 42 were classified by 16S rRNA gene sequences. We summarised the phenotypic test 43 relationships among selected SDF strains using a Pearson correlation -based 44 clustering represented in heatmaps. In practice, biochemical and physiological profiles 45 are often less discriminatory than molecular data and may be unstable because of the 46 loss of traits. Though these test reactions are not universally positive or negative within 47 species, they may define biotypes and be efficient strain markers, enhancing the 48 accuracy of unknown sample identification. It can be also helpful in selecting the best 49 represent the phenotypes of samples. Along with the other phenotypic and genotypic 50 data, the present results will be of great importance for the robust classification of the 51 SDF strains within the scope of the polyphasic approach. 52 53

Bacillales; Bacillaceae; endosporulation; Firmicutes; bacterial 54 identification; bacterial metabolism; phenotyping; taxonomy. 55 56 57 Running head: Soil aerobic endospore-formers phenotypic and molecular profiles 58 59 60 61 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 3

62 Aerobic endospore -forming bacteria (AEFB) enc ompass species from genus 63 Bacillus and related genera and produce dormant and highly resistant cells called 64 spores (Fritze 2004; Logan and Halket 2011; Setlow 2014; Driks and Eichenberger 65 2016). Spores can germinate within seconds when external conditions become 66 favourable (Moir and Cooper 2014). Strains of AEFB are widely distributed in nature, 67 and soil is recognised as the main reservoir (Fritz e 2004; Logan et al. 2009; Mandic -68 Mulec and Prosse r 2011; De Vos 2011). AEFB harbour species of significant 69 importance in health, environment, and biotechnology (Fritze 2004; Logan et al. 2009; 70 Ehling-Schulz and Messelhäusser 2013; Alina et al. 2015). 71 72 AEFB exhibit high levels of genetic, biochemical, and physiological diversity and 73 appreciable resistance to adverse environmental ( De Vos et al. 2009; Logan et al. 74 2009; Logan and Halket 2011; Galperin 2013; Setlow 2014; Driks and Eichenberger 75 2016). The high heterogeneity in the phenotypic and genotypic characteristics h as 76 been hampering the taxonomy of these species (Ash 1991; Fritze 2004; Logan et al. 77 2009; Galperin 2013). 78 79 The first identification and classification schemes of AEFB were based on the 80 morphology of the colonies, vegetative cells , sporangia, spores, Gram-staining 81 response, besides biochemical, physiological, and chemotaxonomic properties (Logan 82 et al. 2009). Today’s polyphasic taxonomy distinguishes and classifies strains based 83 on these classical phenotypic data, supplemented with genotypic and other phenotypic 84

obtained at the molecular level (Colwell 1970; Fritze 2004; Prakash et al. 2007; 85 Logan et al. 2009; Das et al. 2014). Combining classical and molecular data, notably 86 16S rRNA gene sequencing, has revolutionised our understanding of domain Bacteria 87 (Bochner 2009) and led to a rapid increase in the number of descriptions of novel AEFB 88 taxa, especially at genus and species levels (Fritze 2004; Logan et al. 2009; Maughan 89 and Van der Auwera 2011). 90 91 AEFB are allocated in the phylum Firmicutes, with in the class Bacilli, order 92 Bacillales, where seven families harbour aerobic spore -forming genera: Bacillaceae, 93 Alicyclobacillaceae, Paenibacillaceae, Planococcaceae, Pasteuriaceae, 94 Sporolactobacillaceae, and Thermoactinomycetaceae (De Vos et al. 2009; Logan and 95 Halket 2011; Galperin 2013; Parte 2018). 96 97 Phylogenetic studies based on the 16S rRNA gene sequences suggest clusters 98 of closed related AEFB species, designated groups (Ash et al. 1991; Stackebrandt and 99 Swiderski 2002; Fritze 2004; De Vos et al. 2009; Logan 2009; Alina 2015). The early 100 rRNA groups 1 to 5 of Bacillus species proposed by Ash et al. in 1991 were expanded 101 to house alkaliphilic and alkalitolerant species, while other groups of species, such as 102 those allocated in genera Paenibacillus (group 3, Brevibacillus (group 4), and other 103 distinct taxa have been reclassified (Stackebrandt and Swiderski 2002). 104 105 Within genus Bacillus, members of B. cereus group or sensu lato (sl) and B. 106 subtilis complex are composed of highly related members (>99% similarity), restricting 107 species delimitation when considering only the 16S rRNA gene analyses. Differently 108 from the other Bacillus groups described above that harbour high genome identity, B. 109 megaterium and B. aryabhattai share 99.7% of identi ty in the 16S rRNA gene 110 sequences. Nevertheless, the genomes are less than 70% identical (Shivaji et al. 111 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 4 2009). Therefore, the distinction of these two strains using only this technique is also 112 challenging. 113 114 Since observable features from growth condition s and enzymatic reactions are 115 related to the genome expression, the resulting profiles allow detecting phenotypic 116 patterns for the species evaluated. Thus, investigating these intrinsic metabolic 117 activities are still essential for the identification and classification of new AEFB isolates. 118 These assays are highly recommended in the characterization of AEFB strains (Logan 119 et. al 2009). 120 121 To help understand AEFB diversity and explore their biotechnological potential, 122 we isolated 312 strains from soil samples c ollected at random areas of the Federal 123 District, Midwest region of Brazil (Cavalcante et al. 2019; Orem et al. 2019; Martins et 124 al. 2020). These strains, designated SDF0001 -SDF0312 (Solo do Distrito Federal or 125 SDF) are deposited at the Coleção de Bactérias Aeróbias Formadoras de Endósporos 126 (AEFB Collection —AEFBC), hosted at the University of Brasilia. For taxonomic 127 purposes, the SDF strains are being analysed by a polyphasic strategy. 128 129 In the present work, 3 0 biochemical and physiological tests were performed to 130 investigate substrate s utilisation and transformation , in addition to the growth 131 conditions capabilities of 312 SDF strains. Among them, 246 were classified by 16S 132 rRNA sequences. A Pearson correlation based on a clustering method (Gu et al. 2016) 133 was used to construct heatmaps to summarise the relationships of selected SDF 134 strains to these phenotypic tests. 135 136 137

138 Bacterial strains. The 312 SDF strains evaluated in this study were isolated as 139 described in Cavalcante et al. (2019) and Orem et al. ( 2019). The reference strains 140 used as positive and negative controls for the physiological and biochemical tests 141 (Table 1) are deposited at Coleção de Culturas do Gênero Bacillus e Gêneros 142 Correlatos (CCGB), of the Instituto Oswaldo Cruz (LFB-Fiocruz-RJ, Brazil). 143 Ethics statement. Specific permissions required to collect bacterial strains used in 144 this study were endorsed by the Federal Brazilian Authority (CNPq; Authorization of 145 Access and Sample of Genetic Patrimony nº 010439/2015-3). Sampling did not involve 146 endangered or protected species. 147 Biochemical and physiological assays. Strains were grown in nutrient agar (33 °C, 148 24 h) under atmospheric aerobic conditions. Cells from a single colony were 149 transferred to a tube containing nutrient broth and incubated at 33 °C, under constant 150 stirring (200 rpm), for about 16 h. The 30 biochemical and physiological tests (Table 1) 151 were performed according to Bergey's Manual of Systematic Bacteriology (Smith et al. 152 1952; Gordon et al. 1973; Claus and Berkeley 1986; Oliveira and Rabinovitch 1998; 153 De Vos et al. 2009; Rabinovitch and Oliveira 2015). All tests were performed in 154 duplicate in two independent experiments. 155 Taxonomic assignments of SDF strains. DNA preparation, PCR amplification, 156 sequencing, and sequence analyses were performed as described in Orem et al. 157 (2019). Briefly, the nearly full length of both strands of 16S rRNA genes was amplified 158 using total DNA and primers 27F (5’ AGA GTT TGA TCM TGG CTC AG 3’) and 1492R 159 (5’ GGY TAC CTT GTT ACG ACT T 3’). PCR products were bi-directionally sequenced 160 employing the Sanger method. These sequences were filtered for Q≥20 in Phred 161 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 5 scores and taxonomically assigned using BLAST and Classifier as described in Orem 162 et al. (2019). 163 Heatmaps. The biochemical and physiological assays results were arranged in 164 heatmaps (Gu et al. 2016) to enhance the potential of visually revealing patterns and 165 correlations among them. We took the dichotomous values 0 (for Negative) and 1 (for 166 Positive) as binary variables representing the association among the species’ and its 167 biochemical and physiological assay results. Using Pearson’s correlation, the species 168 were clustered taking similar biochemical and physiological results (Hummel et al. 169 2017). R scripts are available at https://github.com/waldeyr/bafes_figures. 170 171 172

and discussion 173 Due to metabolism importance for identification and classification of AEFB new 174 isolates (Fritze 2002; Logan et al. 2009), we applied 30 biochemical and physiological 175 tests (Table 1) to 312 AEFB strains isolated from Brazilian soils, designated SDF 176 strains (Cavalcante et al. 2019; Orem et al. 2019; Martins et al. 2020). T he profiles 177 obtained from enzymatic reactions and growth conditions are described in Table S1, 178 available in the online Supplementary Material. It is important to state that, all the 312 179 SDF strains studied are aerobic or facultative anaerobic endospore -formers, and 180 Gram-positive or Gram -variable cells (Cavalcante et al. 2019; Orem et al. 2019; 181 Martins et al. 2020). The latter characteristics are common to taxa found in the order 182 Bacillales (Fritze 2004; De Vos et al. 2009; Logan et al. 2009; Galperin 2013), where 183 these environmental AEFB strains are allocated. 184 185 Of these 312 SDF strains, the taxonomic assignment s of 246 w ere addressed 186 using the standard tool of taxonomists for bacteria l identification, classification, and 187 phylogenetic relatedness, the 16S rRNA gene sequences (Tringe and Hugenholtz 188 2008; De Vos et al. 2009; Hakorvita et al. 2016), as described in Orem et al. (2019). 189 The lowest and highest inter -species pairwise 16S rRNA gene sequence similarities 190 spanned from 90 % to 100 % (Table S1) . Considering the similarity thresholds for 191 genera 96%, and ≥97% for species (Stackebrandt and Goebel 1994), the classification 192 obtained segregated 238 SDF strains into 6 genera, being 4 part of family Bacillaceae 193 and 2 of Paenibacillaceae (Fig. 1A). Among the SDF strains described in the present 194 work, Bacillus spp., belonging to the family Bacillaceae, are the most prevalent (207 195 strains; 84.14%), followed by species of genera Paenibacillus (14; 5.69%; family 196 Paenibacillaceae), Lysinibacillus (7; 2.84%; Bacillaceae), Brevibacillus (6; 2.43%; 197 Paenibacillaceae), Terribacillus (1; 0.40%; Bacillaceae), and Rummeliibacillus (1; 198 0.40%; Bacillaceae). These findings are not surprising since the selective procedure 199 we used to isolate SDF strains intended to favour non -fastidious AEFB species, 200 excluding strict anaerobic endospore-forming and Gram-negative cells (Cavalcante et 201 al. 2019; Orem et al. 2019; Martins et al. 2020). 202 203 Included in the 224 SDF strains classified at the species level (Table S1) , 204 members of B. pumilus subgroup were predominant (83 strains; 37.05%), followed by 205 B. cereus group species (48; 21.42%), B. megaterium group (35; 15.62%), other 206 members of B. subtilis complex (1 2; 5.35%); B. simplex (7; 3.12%); B. clausii (3; 207 1.33%); B. subterraneus (2; 0.89%); besides 1 ( 0.44%) of each: B. australimaris; B. 208 arbutinovorans; B. circulans; B. kochii; B. luciferensis; B. oleronius; B. siamensis, and 209 B. senegalensis. Outside genus Bacillus, other species belonging to family Bacillaceae 210 were Lysinibacillus sphaericus (3; 1.33%); L. xylanilyticus (2; 0.89%); L. fusiformis (2; 211 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 6 0.89%), and Terribacillus goriensis (1; 0.44%). Paenibacillus spp. (12 strains; 5.35%), 212 and Brevibacillus spp. ( 5 strains; 2.23%) allocated in the family Paenibacillaceae 213 complete the list of SDF strains classified at the species level (see below). The diversity 214 of the SDF strains is represented in Fig. 1B. 215 216 It is mentionable that member s of B. cereus sl and B. subtilis subgroups are 217 composed of very related members (>99% similarity), restricting species delimitation 218 when considering only the 16S rRNA gene analyses. Conversely, B. megaterium and 219 B. aryabhattai share 99.7% of identity in the 16S rRNA gene sequences, even though 220 the genomes are less than 70% identical (Shivaji et al. 2009). Therefore, the distinction 221 of these two species using only this technique is also challenging. 222 223 Thus, our taxonomic assignments based on 16S rRNA gene sequences are a 224 preliminary inference of genera or species. Accordingly, when 16S rRNA gene profiling 225 placed these strains within these AEFB taxa, a sample analysed can belong to two or 226 even more species alternatives within the same affiliation cluster. In these instances, 227 this approach can find groups of bacteria, never theless cannot assign it accurately to 228 a species according to its low discrimination ability. Since 10 SDF strains exhibited 229 similarity rates spanning 90 -95% (Table S1), the 16S rRNA gene -sequencing tool 230 failed to classify these environmental strains even at the genus level. While this genetic 231 marker was insufficient to set up genus or species, low gene-sequence similarity might 232 suggest that novel species could have been isolated (Tindal et al. 2010). Nevertheless, 233 the description of the new taxa is beyond the scope of this article. 234 235 Bacillus is the genus type of order Bacillales, and Bacillus spp. have been isolated 236 from a wide range of environments (Tamames et al. 2010; Mandic-Mulec and Prosser 237 2011; Alina et al. 2015; Orem et al. 2019; Cavalcante 2019; Salgado et al. 2020). Soils, 238 along with freshwaters, are one of the least restrictive for these species. It is worthy to 239 note that certain species found in soils are inactive in these environments. It could be 240 the case for some SDF strains isolated from Brazilian soils. The method of isolation 241 based on heat shock allowed the dormant spores to germinate and grow in vitro. 242 243 B. cereus and B. anthracis are human pathogens causing food-borne illness and 244 anthrax, respectively (Arnesen et al. 2008; Ehling -Schulz and Messelhäusser 2013). 245 On the other hand, the metabolic breadth of Bacillus spp. has been explored by the 246 industry for producing a vast range of antibiotics; plant growth promotion m olecules; 247 hydrolyses; toxins against plants, fungus, insect, and nematode, in addition to other 248 bioproducts (de Maagd et al. 2003; Berkeley et al. 2008; Logan et al. 2009; Galperin 249 2013; Alina 2015). Therefore, despite the danger of few members, most speci es are 250 beneficial. 251 252 Genus Bacillus stays the largest AEFB taxon, accommodating 614 species, as 253 registered at the List of Prokaryotic Names with Standing in Nomenclature (LPSN: 254 https://www.bacterio.net/Bacillus.html; accessed on 01 February 2022). Taxonomy 255 within genus Bacillus is hampered by high heterogeneity at phenotypic and genotypic 256 levels (Ash 1991; Fritze 2004; Logan and De Vos 2009; Logan et al. 2009). Further, 257 these divergencies restrict the distinction between Bacillus spp. and those allocated in 258 other genera inside Bacillaceae. 259 260 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 7 Typically, Bacillus spp. are considered aerobic, although at least 20 species are 261 facultatively anaerobic (Logan and De Vos 2009). Furthermore, nitrate reduction is 262 frequently observed in this genus. Members of Bacillus can be rods or cocci, motile or 263 non-motile, organotrophic or lithotrophic (Fritze 2004; Logan and De Vos 2009; Logan 264 et al. 2009). Cell size, varying from 0.4 to 1.8 μm in diameter and from 0.9 to 10.0 μm 265 in length, can also be used to differentiate Bacillus spp. (Logan and De Vos 2009). 266 Phylogenetically, most recognised species are arranged into subclusters or rRNA 267 groups. 268 269 Due to the significant relevance in economy and health issues, the B. cereus 270 group and B. subtilis complex have been received considerable attention (Fritze 2004; 271 Maughan and Van der Auwera 2011). The B. cereus group hosts B. cereus sensu 272 stricto (or ss or B. cereus ), B. anthracis , B. thuringiensis , B. mycoides , B. 273 pseudomycoides, B. weihenstephanensis , B. toyonensis , and B. cytotoxicus (Fritze 274 2004; Maughan and Van der Auwera 2011; Ehling -Schulz and Messelhäusser 2013). 275 Organisms placed in this group belong to 16S rRNA/DNA group 1. Cells are typically 276 wider than 1 μm, Gram-positive, and endospores are oval to cylindrical paracentral or 277 subterminally localised in unswelling sporangia. 278 279 Traditionally, these bacteria have been differentiated based on phenotypic 280 characteristics, especially pathogenic potential. Nonetheless, this group is a highly 281 homogeneous subdivision inside genus Bacillus (Helgason et al. 2000; Chen and Tsen 282 2002). Furthermore, they are hardly distinguishable with standard biochemical and 283 chemotaxonomic methods or phylogenetically relevant target genes (Bavykin et al. 284 2004; Arnesen et al. 2008). However, specific biochemical and physiological 285 characteristics of the B. cereus sl are advantageous to differentiate these taxa from 286 the other aerobic endospore-forming species. 287 288 Out of 224 SDF strains classified at the species level (Tabl e S1), 48 ( 21.42%) 289 were members of the B. cereus group. Using a Pearson correlation -based clustering 290

(Gu et al., 2016), we constructed a heatmap ( Fig. 2) to summarise the 291 relationships of these 48 environmental strains to the 30 biochemical and physiological 292 tests performed (Table 1). Each column shows the metabolic pattern (bottom side) of 293 individual SDF strain (rows at the right side), classified based on 16S rRNA sequences. 294 The green and the red colours represent positive and negative responses, 295 respectively. Fig. 2 shows an assembling of these essays based on the prevalence of 296 the positive responses. It is doable to distinguish which strains respond similarly to the 297 tests when they are in the same clade. For example, th ose strains in distant clades 298 respond differently. It is also possible to discern which SDF strains respond similarly 299 to each test , as well as discriminate them by correlating rows and columns. The 300 clusters from the upper-side dendrogram (Fig. 2) stands for the similarity of the 30 tests 301 responses. The left-most contains 17 columns, while the right-most contains 13, where 302 the majority of these SDF strains responded positively and negatively, respectively. 303 304 Although many AEFB may not respond positively to the c atalase test, most 305 species rod-shaped, either Gram-positive or Gram-positive only in the initial stages of 306 growth, are catalase -positive, especially members of the genus Bacillus (Logan and 307 De Vos , 2009a). However, in most cases, respiratory metabolism occ urs at low 0 2 308 levels. Here, all the 48 SDF members of the B. cereus group responded positively to 309 this enzyme linked to respiration in the presence of atmospheric 0 2 (Fig. 2). This 310 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 8 positivity seems to be a characteristic of this group of sporulating procar yotes. As 311 assessed in this work, it is worth noting that B. cereus ss can grow under certain 312 anaerobiosis conditions (Logan and De Vos, 2009a). 313 314 The cytochrome C oxidase is especially useful to discriminate Gram -negative 315 pathogens Vibrio spp. (oxidase positive) from the oxidase -negative enteric bacteria 316 (Vila et al. 1992). This enzyme catalyses the oxidation of cytochrome C while reducing 317 oxygen to form water. The oxidation test in vitro employs colourless artificial acceptors 318 like dimethyl or tetramethyl p -phenylenediamine resulting in purple colour when 319 positive. This essay also distinguishes Neisseria and Moraxella (both oxidase positive) 320 from Acinetobacter spp. (oxidase negative) (Henriksen 1976; Powell and Marcon 321 2012). 322 323 From the 48 SDF str ains allocated in the B. cereus group, 25 (52.08%) were 324 oxidase-positive. Logan and De Vos (2009) point out this variability for this genus and 325 related genera, demonstrating apparent inactivity of this enzyme, or even that the 326 traditional method failed to detect the oxidase activity in almost half of these samples. 327 328 Anaerobiosis assays, performed in tubes containing aldehyde -reduced agar 329 medium inoculated with a needle, revealed growth a few centimetres below the 330 interface of the culture medium with atmosph eric air to 24 (50%) of the SDF strains 331 belonging to the B. cereus sl. This effect present in -depth denotes anaerobic growth, 332 a property conserved among AEFB. 333 334 Some Bacillus species do not appear to utilise carbohydrates whatsoever (Logan 335 and De Vos 2009). Yet the acid production profiles from monosaccharides and 336 disaccharides are of great value in the characterisation and identification of these 337 species. Most SDF strains allocated into B. cereus group used D-glucose, L-arabinose, 338 D-xylose, and other fermentable carbohydrates as sole sources of carbon and energy 339 (Table S1; Fig. 2). Probably they have the genetic information to conduct the pathway 340 of Embden-Meyerhof-Parnas, coupled with the Krebs cycle, verified by acid production 341 (Logan and De Vos 2009a). 342 343 Regarding glucose consumption, five SDF strains, one classified as B. 344 thuringiensis (SDF0225), and four as B.cereus ss (SDF0124; SDF0229; SDF0237, and 345 SDF248), responded negatively to the use of this monosaccharide, which is rare 346 among rods AEFB, as they usually assimilate and degrade D -glucose. Though the 347 formation of acid from D-mannitol is frequently negative for members of the B. cereus 348 sl and positive for strains of other groups (Fritze 2002), three SDF strains classified as 349 B. cereus ss (SDF0219; SDF0124, and SDF0022) and B. anthracis SDF0199 were 350 able to ferment this sugar ( Fig. 2). Interestingly, these strains were gathered in the 351 uppermost and downmost rows of the strains' list (right side). Indeed, clustering heat 352 maps can group samples based on the similarity of their phenotypic patterns, thus 353 identifying atypical responses (Zhao et al. 2014). 354 355 The Voges-Proskauer test presented some species such as two B. thuringiensis 356 strains (SDF0161 and SDF0178); three B. anthracis (SDF181; SDF0186, and 357 SDF0199), besides seven B. cereus ss (SDF0155; SDF0159; SDF0182; SDF0184; 358 SDF0239; SDF0270, and SDF0272) responding negatively to the acetyl -359 methylcarbinol production assay, which allows us to suspect that these strains may not 360 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 9 produce enzymes that decarboxylate lactic acid from the glycolytic pathway, or do not 361 have an enzyme capable of bonding two molecules originating from the production of 362 acetate ions. 363 364 Oliveira and Rabinovitch (1998) established a standardised protocol for detection 365 of gelatin hydrolysis by Lysinibacillus sphaericus—former B. sphaericus (Seldin et al. 366 1984; Ash et al. 1994) —showing that 93.3% of strains belonging to this species 367 hydrolyses this incomplete protein after four days of incubation. Here bulk 48 SDF 368 strains accommodated in the B. cereus group could use gelatin. Providing the relatively 369 high number of strains submitted to this type of biochemical test, we considered a very 370 valid verification. 371 372 The development in the presence of lysozyme is another characteristic of the B. 373 cereus group and hardly occur in the other species of other groups (Fritze 2002). 374 However, B. cereus SDF0124 and B. thuringiensis SDF085 did not grow in this 375 condition, indicating that the cell wall of these two strains can be hydrolysed by this 376 enzyme. 377 378 The production of haemolysin, as well as cell morphology in a few strains of B. 379 cereus sl, are also phenotypes with relevance for taxonomic studies (Fritze 2002; 380 Fritze 2004; De Vos and Logan 2009; Logan et al. 2009). B. cereus ss, in general, 381 mobile, is heavily haemolytic, but does not produce rhizoid growth pattern, a 382 characteristic that can be used to differentiate from colonies of B. mycoides strains 383 (Fritze 2002). Most B. anthracis strains are neither mobile nor haemolytic (Fritze 2004; 384 Maughan and Van der Auwera 2011). However, non-mobile B. cereus strains, as well 385 as hemolytic B. anthracis, may hinder the differentiation between these two species. 386 In addition, the latter sp ecies can be differentiated by parasporal crystal formation 387 typically described to B. thuringiensis (Fritze 2002). 388 389 Out of 48 strains, SDF allocated in the B. cereus group, three samples classified 390 as B. cereus (SDF0159, SDF0237, and SDF0270); one B. anthracis SDF0181, and 391 three B. thuringiensis (SDF0161; SDF0085, and SDF0030) presented no haemolysin 392 activity. It is of great significance to mention that B. thuringiensis SDF0030 produces a 393 typical parasporal crystal (Cavalcante et al. 2014), a classical feature distinguishing B. 394 thuringiensis strains from B. cereus ss. (Dagmar 2014). Conversely, three strains 395 classified as B. anthracis (SDF0199, SDF089, and SDF0186) were positive for 396 haemolysin activity. The main phenotypical properties that are frequent ly used to 397 distinguish B. cereus, B. thuringiensis, and B. anthracis are related to the presence or 398 absence of large plasmids, where the replicons are localized (Maughan and Van der 399 Auwera 2011). Future investigation on the extrachromosomal profiles of the se SDF 400 strains will help to understand the evolutionary relatedness of these species. 401 402 B. subtilis ss, the genus Bacillus type-species, is prominent in microbial history, 403 and play a distinct role as a model for Gram-positive bacteria and in the understanding 404 of stress-resistance of bacterial spores (Fritze 2004; Maughan and Van der Auwera 405 2011; Galperin 2013; Driks and Eichenberger 2016). Besides being recognised as a 406 model, this species, along with other highly related accommodated in the B. subtilis 407 complex, is extensively employed in industry and agriculture (Fan et al. 2017). 408 409 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 10 B. subtilis strains are aerobics, although some strict anaerobic growth may be 410 observed in complex media with glucose or (less effectively) nitrate (Logan and De 411 Vos 2009a). This organism is catalase -positive, oxidase variable, and can reduce 412 nitrate to nitrite. The motile rod-cells of 0.7 –0.8 x 2.0 –3.0 μm are Gram -positive and 413 can be frequently observed singly, in pairs, and, occasionally, in chains. This species 414 forms ellipsoidal to cylindrical endospores at the central, paracentral, or subterminal 415 position in unswollen sporangia. 416 417 Although optimal growth ranges from 28 -30 °C, B. subtilis can tolerate 418 temperatures from 5 –20 °C and 45 –55 °C (Logan and De Vos 2009a). Growth can 419 occur from pH 5.5 to 8.5, with no limits recorded. The vegetative cells have a significant 420 role in the early steps of organic matter decomposition. Growth in minimal medium 421 containing glucose and ammonium salt—as sole sources of carbon— and nitrogen is 422 also observed. Most strains can use citrate, as the sole carbon source, and growth 423 occurs in th e presence of up to 7% NaCl, and certain tolerate 10% NaCl. B. subtilis 424 can hydrolyse casein, esculin, gelatin, and starch but not phenylalanine and urea. 425 Extracellular dextran and levan are produced from sucrose. Voges –Proskauer test is 426 positive, and the production of acid without gas can be detected from glucose, besides 427 additional carbohydrates. 428 429 As a taxonomic unit above the species level, the B. subtilis species complex can 430 be split into four clades (Fan et al. 2017). These recognizable monophyletic groups 431 comprise clade I, consisting of three subspecies of B. subtilis (subtilis, spizenii, and 432 inaquosorum), besides B. tequilensis, B. vallismortis, B. mojavensis, and B. 433 atrophaeus; clade II containing species B. amyloliquefaciens , B. siamensis , and a 434 conspecific complex embracing B. methylotrophicus , B. velezensis, and B. 435 amyloliquefaciens subsp. plantarum; clade III encompassing B. licheniformis , B. 436 sonorensis, and related species, and clade IV made of B. pumilus and B. safensis, B. 437 xiamenensis, and a conspecific group involving the type strains of B. altitudinis , B. 438 stratosphericus, and B. aerophilus. Like strains from the B. cereus group, these taxa 439 are placed in 16S rRNA/DNA group 1 and are phylogenetic and physiologically 440 remarkably similar (Fritze 2004). Strains from this complex are usually mesophiles and 441 neutrophiles, but often tolerant to high pH values (Fritze 2004). 442 443 Employing 16S rRNA gene sequences, from the 224 SDF strains classified at the 444 species level 95 (42.41%) were allocated in the B. subtilis complex (Table S1). Among 445 them, the B. pumilus subgroup represented 83 ( 37.05%), most of it or 61 ( 27.23%) 446 classified as B. pumilus; 16 ( 7.14%) as B. safensis, and 6 (2.67%) as B. altitudinis. 447 Seven strains belonged to B. amyloliquefaciens subgroup or 3.12%, being 4 (1.78%) 448 B. amyloliquefaciens strains and 3 ( 1.33%) B. velezensis. The remaining 4 ( 1.78%) 449 SDF strains were accommodated in the B. subtilis subgroup, being 3 (1.33%) B. subtilis 450 ss and 1 (0.44%) B. tequilensis. 451 452 The relationships of these 95 strains to the 30 biochemical and physiological tests 453 described in Table 1 were also analysed. The res ulting heat map (Gu et al. 2016) 454 shown in Fig. 3 revealed two clusters (upper -side dendrogram) encompassing 13 455 columns at the left-most cluster, while the right -most contained 17, where these SDF 456 strains responded positively and negatively, respectively. 457 458 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 11 Species belonging to the so -called B. pumilus subgroup are almost identical in 459 the 16S rRNA gene sequences, sharing above 99.5% similarity (Alina et al. 2015). B. 460 pumilus ss is aerobic, catalase-positive, and enabled to reduce nitrate (Logan and De 461 Vos 2009a). Gram-positive or Gram-variable small rods (0.6–0.7 by 2.0–3.0 μm) cells 462 can be observed as singly or in pairs and are motile. Cylindrical to ellipsoidal 463 endospores can be central, paracentral, and subterminally localised in unswollen 464 sporangia. Thoug h optimal growth occurs at pH 6.0 and 9.5, some strains can 465 reproduce at pH 4.5. This species can tolerate up to 10% NaCl, hydrolyse casein, 466 esculin, and gelatin but cannot break down starch. Phenylalanine is not deaminated, 467 and citrate is utilised as the sole carbon source, but propionate is not. Acid without gas 468 is produced from glucose and many other carbohydrates, and Voges –Proskauer test 469 is positive. 470 471 In general, the SDF strains belonging to this group corroborates the traits 472 described in Bergeys' Firmicutes (Logan and De Vos 2009a). Furthermore, according 473 to Logan and Forsyth, unpublished observations cited in this manual, B. pumilus 474 strains isolated from Antarctic soils and penguin rookeries present phenotypic 475 peculiarities, such as producing a diffusible yellow pigment. 476 477 Outside Bacillaceae, 17/224 (7.58%) SDF strains were allocated in two genera 478 of the family Paenibacillaceae. Paenibacillus spp. accounted for 12 (5.35%) strains 479 being 7 (3.12%) of P. alvei and 1 (0.44%) of each: P. chibensis; P. ginsengagri; P. 480 lautus; P. susongensis, and P. terrígena (Table S1). Five (2.23%) strains of the genus 481 Brevibacillus (quoted here as Br.): Br. laterosporus (4 or 1.78%), and 1 (0.44%) of Br. 482 agrii completed the SDF strains allocated into the family Paenibacillaceae (Table S1). 483 The mutual connection between these 1 8 strains and the 30 biochemical and 484 physiological tests (Table 1) is represented in Fig. 4. The two clusters (upper -side 485 dendrogram) distinguishable by this heat map (Gu et al. 2016) comprehend 11 (left -486 most) and 19 (right-most) columns, embracing most of these SDF strains responding 487 positively and negatively, respectively. 488 489 The genus Paenibacillus was created to reallocate species previou sly 490 accommodated in the RNA group 3 of genus Bacillus (Priest 2009). The family 491 Paenibacillaceae was subsequently proposed to house genus Paenibacillus and close 492 relatives' genera (Ash et al. 1993; Shida et al. 1997). This family encloses two 493 monophyletic clusters, the first consisting of genera Paenibacillus, Brevibacillus, 494 Cohnella, and Thermobacillus, the second of genera Aneurinibacillus, Ammoniphilus, 495 and Oxalophagus (De Vos et al. 2009). The type-genus is Paenibacillus. 496 497 Members of this family may be strictly aerobic, microaerophilic, facultative 498 aerobic, or obligate anaerobic, being catalase -positive or -negative (De Vos et al. 499 2009). Cells are straight to curved rods of 0.5 –1.0 x 2–6 μm, Gram-positive but may 500 stain Gram-negative or variable. Oval or ellipsoidal endospores are frequently formed 501 inside a swelling sporangium. Peritrichous flagella may be observed, but some species 502 are nonmotile. Although they can utilise oxalic acid as the sole carbon and energy 503 source, these cells are organoheterotrophs and grow in complex media, using 504 carbohydrates and amino acids. They can be mesophilic or thermophilic, neutrophilic 505 or alkaliphilic and have been isolated from soil, roots, faeces, blood, and other 506 substrates. 507 508 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 12 After Bacillus, the genus Paenibacillus accommodates the second largest 509 number of AEFB species known (3 42), as registered at the LPSN 510 (https://www.bacterio.net/; accessed on 01 February 2022). Paenibacillus harbours 511 species aerobic or facultative rod -shaped cells (Priest 2009; Galperin 2013; Parte 512 2018) and bears a typical Gram -positive cell -wall structure (Shida et al. 1996). 513 Nevertheless, even young cells react weakly or even negatively to Gra m staining. It 514 should be noted that the 12 SDF strains classified as Paenibacillus spp. in this work 515 stained weakly, or yet, Gram-negative (not shown). 516 517 Brevibacillus species are aerobic, though some strains are microaerophilic and 518 facultatively anaerobic (Logan and De Vos , 2009b). Most species are catalase -519 positive. Oxidase reaction and nitrate reduction can differ among strains. Rod-shaped 520 cells are 0.7–1.0 μm x 3.0–6.0 μm, occur singly, in pairs, in chains, are motile through 521 peritrichous flagella, and a re Gram -positive or Gram -variable. The ellipsoidal 522 endospores swell the sporangia. 523 524 This genus includes a high diversity of thermophilic, psychrophilic, acidophilic, 525 alkalophilic, and halophilic strains that use a variety of carbon sources for either 526 heterotrophic or autotrophic growth (Panda et al. 2014). Carbohydrates may be 527 assimilated, but acid is produced weakly, if at all, by most species. Some amino acids 528 and organic acids may be used as carbon and energy sources (Logan and De Vos 529 2009b). Casein, gelatin, and starch hydrolysis vary among species. Optimum growth 530 occurs at pH 7.0 and can be inhibited by 5% NaCl. The type -species is Br. brevis 531 (Shida et al. 1996), former Bacillus brevis. 532 533 Brevibacillus spp. are used as a factory for the expression of biotechnologically -534 important enzymes (e.g., alpha -amylase, sphingomyelinase, xylanase, CGTase, and 535 chitosanase), as well as heterologous proteins including cytokines (EGF, IL -2, NGF, 536 IFN-c, TNF-a, and GM-CSF), antigens, and adjuvants (Mizukami et al. 2010). Besides, 537 Brevibacillus spp. are considered a valuable tool for structural and functional biology 538 studies (Panda et al. 2014). 539 540 Br. brevis, Br. choshinensis, and Br. laterosporus have attracted considerable 541 interest owing to the production or transformation of valuable compounds and the 542 biocontrol proprieties ( De Vos et al. 2009b). The broad entomopathogenic activity 543 includes species from orders Coleoptera, Lepidoptera, and Diptera and from phyla 544 Nematoda and Mollusca (Ruiu et al. 2013). 545 546 The recent improvements in the tools have been helping in uncovering the vast 547 physiological and genetic diversity within the AEFB, resulting in more appropriate 548 taxonomic arrangements (Fritze 2004; Maughan and Van der Auwera 2011; G alperin 549 2013). As a result, many new descriptions of genera and species, and reclassifications 550 have occurred. 551 552 Molecular methods, especially 16S rRNA gene sequencing, have become the 553 prevailing technique in procaryotic identification, but significant restrictions in our ability 554 to identify environmental bacteria to the genus and species levels remain (Fritze 2004; 555 Maughan and Van der Auwera 2011; Galperin 2013). 556 557 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 13 Here, the performance of the 16S rRNA sequence analysis was excellent. This 558 tool resolved 238 (96.74%) out of 246 SDF strains at the genus level, unrevealing four 559 and two genera within Bacillaceae and Paenibacillaceae, respectively. Among the 246 560 samples, 22 4 SDF samples (91.05%) were classified at the species level. As 561 mentioned above, using this technique, closely related strains such as those belonging 562 to the B. cereus group, B. subtilis complex, and other AEFB taxa cannot be resolved 563 at the species level. Still, our classifica tions are suitable since they clearly show the 564 genera and restrict the identity of part of these SDF strains to one or a few species in 565 the genera described. The positions of the SDF strains in this initial clustering and 566 identification of closely related species may be more accurately determined by 567 incorporating additional data obtained at both genotypic and phenotypic analyses. 568 569 Furthermore, our SDF strain classifications revealed well -known AEFB species, 570 together with others that are scarcely described i n the literature. Identifying multiple 571 species and strains from different genera may help resolve the order Bacillales at the 572 family, genus, and species levels. 573 574

575 In the present study, 30 biochemical and physiological tests provided profiles of 576 all the 312 SDF strains deposited at AEFBC. From the genetic point of view, a large 577 number of samples such as those originating from the environment, as the SDF strains' 578 collection, will hardly display 100% equal answers for all tests, as seen in taxonomic 579 studies of strains isolated from non-clinical substrates (Logan and De Vos 2009; Logan 580 and Halket 2011). In such cases, there are always taxonomically diverging strains. The 581 ubiquitous species B. pumilus , isolated from Antarctic soils and penguin rookeries, 582 corroborate this statement as a phenotypic distinction from other lineages can be 583 observed (Logan and Forsyth, unpublished observations, apud Logan and De Vos, 584 2009a). The divergent samples need to hav e a separate and improved taxonomic 585 study. 586 587 Biochemical and physiological profiles are util e for identifying these 588 microorganisms. These essays are also part of the minimum standards proposed by 589 Logan et al. (2009) for characterising new species of these t axa. However, the value 590 of these tests to accurately identify large numbers of environmental species is limited 591 (Fritze 2004). Therefore, phenotypic similarities cannot be taken with certainty to 592 indicate close evolutionary relatedness. 593 594 However, along with the other phenotypic and genotypic data (Cavalcante et al. 595 2019; Orem et al. 2019; Martins et al. 2020), including complete genome sequences 596 in progress, the profiles described in Table S1 will be significant for robust 597 identification, consequently, clas sification and differentiation of these environmental 598 strains. The biochemical and physiological profiles can also help optimise the culture 599 conditions for further characterisation and the production of bioactive metabolites by 600 the SDF strains. 601 602 Hence, the classification of AEFB at the species levels is not straightforward. And 603 to classify and differentiate closely related SDF strains, these essays should be 604 coupled to other classical and molecular methods involving phenotypic and genotypic 605 types (Cavalcante et al. 2019; Orem et al. 2019; Martins et al. 2020) in a polyphasic 606 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 14 approach (Colwell 1970; Fritze 2004; Prakash et al. 2007; Logan et al. 2009; Das et 607 al. 2014). 608 609 This strategy will facilitate the establishment of accurate classification of the SDF 610 strains. It will also allow responsible exploitation of the extraordinary AEFB 611 biotechnological potential, the reliable use as insect control agents, and the handling 612 of animal pathogens. 613 614 615 Funding 616 The authors have no funding to report. 617 618 619 Competing interests 620 The authors have declared that no competing interests exist. 621 622 623 Acknowledge 624 We thank University of Brasilia, and the Brazilian research funding agencies 625 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and 626 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We are in 627 debt with Arthur S. Araujo, and Liliam de Oliveira F. Marceneiro for excellent technical 628 assistance. 629 630 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 15

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BMC Microbiology 10(1): 1-14. 920 http://www.biomedcentral.com/1471-2180/10/85 921 922 Tindall BJ, Rosselló -Móra R, Busse HJ, Ludwig W, Kämpfer P (2010) Notes on the 923 characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 924 60(1):249-266. https://doi.org/10.1099/ijs.0.016949-0 925 926 Tringe SG, Hugenholtz P (2008) A renaissance for the pioneering 16S rRNA gene. 927 Curr Opin Microbiol 11(5):442-446. https://doi.org/10.1016/j.mib.2008.09.011 928 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 21 929 Vila J, Abdalla S, Gonzalez J, Garcia C, Bombi JA, Jimenez de Anta MT (1992) A one-930 minute oxidase test to detect Vibrio strains isolated from cultures on thiosulphate -931 citrate-bile salts -sucrose (TCBS) medium. 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BioMed Research International 2014:1-6. 948 https://doi.org/10.1155/2014/986048 949 950 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Manuscript #39153_Resubmissão2022 Neotropical Biology and Conservatio 22 Tables and figures captions 951 952 Table 1. Biochemical and physiological tests used in this work and the respective 953 controls 954 955 956 Table S1. Molecular, biochemical, and physiological profiles of SDF strains belonging 957 to the AEFBC 958 959 960 Figure 1. Overall repartition of SDF strains according 16S rRNA gene sequencing 961 classification. (A) Distribution of 238 SDF strains among six genera belonging to 962 families Bacillaceae (Bacillus, Lysinibacillus, Terribacillus, and Rummeliibacillus) and 963 Paenibacillaceae (Paenibacillus and Brevibacillus). (B) Species assignments of 224 964 SDF strains. 965 966 967 Figure 2. Correlation between SDF strains belonging to B. cereus group and growth 968 conditions or enzymes activities. A Person correlation -based clustering method was 969 employed to construct a heat map associating 48 SDF strains allocated in B. cereus 970 group (right) and 30 phenotypical features (bottom) that contribute to AEFB 971 identification and classification. The top dendrogram clustered the SDF strains into two 972 parts based on the prevalence of positive responses (blue) to 30 growth conditions and 973 enzyme reactions described at the bottom of the graphic. Negative responses are 974 shown in red. 975 976 977 Figure 3. Correlation between SDF strains belonging to B. subtilis complex and growth 978 conditions or enzymes activities. A Per son correlation-based clustering method was 979 employed to construct a heat map associating 95 SDF strains allocated in B. subtilis 980 complex (right) and 30 phenotypical features (bottom) that contribute to AEFB 981 identification and classification. The top dendrogram clustered the SDF strains into two 982 parts based on the prevalence of positive responses ( green) to 30 growth conditions 983 and enzyme reactions described at the bottom of the graphic. Negative responses are 984 shown in red. 985 986 987 Figure 4. Correlation between SDF strains belonging to family Paenibacillaceae and 988 growth conditions or enzymes activities. A Person correlation-based clustering method 989 was employed to construct a heat map associating 18 SDF strains allocated in B. 990 subtilis complex (right) and 30 phenotypical features (bottom) that contribute to AEFB 991 identification and classification. The top dendrogram clustered the SDF strains into two 992 parts based on the prevalence of positive responses (orange) to 30 growth conditions 993 and enzyme reactions described at the bottom of the graphic. Negative responses are 994 shown in red. 995 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Table 1. Biochemical and physiological profiles analysed in this work and the respective controls *not determined. CCGB: Coleção de Culturas do Gênero Bacillus e Gêneros Correlatos. CCGB is an integrant of the World Federation for Culture Collec6ons WFCC (#574). Test Control Positive Negative Growth condition Citrate utilization Bacillus cereus CCGB406 Paenibacillus macerans CCGB126 Propionate utilization Bacillus licheniformis CCGB407 Bacillus subtilis CCGB1249 7% NaCl Bacillus amyloliquefaciens CCGB452 Paenibacillus macerans CCGB126 10% NaCl Bacillus amyloliquefaciens CCGB452 Paenibacillus macerans CCGB126 0.001% lysozyme Bacillus cereus CCGB406 Bacillus pumilus CCGB124 45 °C Geobacillus stearothermophilus CCGB412 ND* 65 °C Geobacillus stearothermophilus CCGB412 Bacillus thuringiensis CCGB1163 pH 5.7 Bacillus cereus CCGB406 Paenibacillus alvei CCGB414 Anaerobiosis Bacillus cereus CCG406 Bacillus megaterium CCGB408 Enzyme Catalase Bacillus cereus CCGB406 ND* Oxidase Lysinibacillus sphaericus CCGB745 Bacillus cereus CCGB406 Hemolysin Bacillus thuringiensis CCGB1163 Lysinibacillus sphaericus CCGB745 Nitrate reductatase Bacillus cereus CCGB406 Bacillus megaterium CCGB408 Hydrolysis Casein Bacillus megaterium CCGB408 Paenibacillus macerans CCGB126 Gelatin Bacillus cereus CCGB406 Geobacillus stearothermophilus CCGB412 Esculin Bacillus subtilis CCGB1249 Lysinibacillus fusiformis CCGB743 Starch Bacillus cereus CCGB406 Lysinibacillus sphaericus CCGB745 Amino acid decomposition Phenylalanine degradation Bacillus megaterium CCGB408 Bacillus cereus CCGB406 Tyrosine degradation Bacillus cereus CCGB406 L. sphaericus CCGB745 Arginine dihydrolase Bacillus licheniformis CCGB407 Bacillus megaterium CCGB408 Lysine decarboxylase Bacillus thuringiensis CCGB1163 Bacillus megaterium CCGB408 Ornithine decarboxylase Bacillus thuringiensis CCGB1163 Bacillus megaterium CCGB408 Indole production Paenibacillus alvei CCGB414 Bacillus cereus CCGB406 Production of acid from D-Glucose Bacillus megaterium CCGB408 Lysinibacillus fusiformis CCGB743 L-Arabinose Bacillus megaterium CCGB408 Brevibacillus brevis CCGB052 Lactose Bacillus megaterium CCGB408 Lysinibacillus fusiformis CCGB743 Mannitol Bacillus megaterium CCGB408 Lysinibacillus fusiformis CCGB743 Sucrose Bacillus amyloliquefaciens CCGB452 Lysinibacillus sphaericus CCGB745 D-Xylose Bacillus megaterium CCGB408 Brevibacillus brevis CCGB052 Voges-Proskauer test Bacillus cereus CCGB406 Bacillus megaterium CCGB408 Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 Bacillus: 208 (87.39%) Brevibacillus: 7 (2.94%) Lysinibacillus: 7 (2.94%) Paenibacillus: 14 (5.88%) Rummeliibacillus: 1 (0.42%) Terribacillus: 1 (0.42%) Genera level (238 SDF strains) A Bacillus toyonensis: 1 (0.45%)Bacillus tequilensis: 1 (0.45%)Bacillus arbutinivorans: 1 (0.45%)Bacillus australimaris: 1 (0.45%)Bacillus circulans: 1 (0.45%) Bacillus kochii: 1 (0.45%) Bacillus luciferensis: 1 (0.45%) Bacillus oleronius: 1 (0.45%) Bacillus senegalensis: 1 (0.45%) Bacillus siamensis: 1 (0.45%) Bacillus subterraneus: 2 (0.89%) Bacillus velezensis: 3 (1.34%) Bacillus clausii: 3 (1.34%) Bacillus anthracis: 4 (1.79%)Bacillus amyloliquefaciens: 4 (1.79%)Bacillus subtilis: 4 (1.79%) Bacillus altitudinis: 6 (2.68%)Bacillus simplex: 7 (3.12%) Bacillus aryabhattai: 9 (4.02%) Bacillus thuringiensis: 12 (5.36%) Bacillus safensis: 16 (7.14%) Bacillus megaterium: 26 (11.61%) Bacillus cereus: 31 (13.84%) Bacillus pumilus: 61 (27.23%) Brevibacillus agrii: 1 (0.45%) Brevibacillus brevis: 1 (0.45%) Brevibacillus laterosporus: 4 (1.79%) Lysinibacillus fusiformis: 2 (0.89%) Lysinibacillus xylanilyticus: 2 (0.89%) Lysinibacillus sphaericus: 3 (1.34%) Paenibacillus chibensis: 1 (0.45%) Paenibacillus ginsengagri: 1 (0.45%) Paenibacillus lautus: 1 (0.45%) Paenibacillus susongensis: 1 (0.45%)Paenibacillus terrigena: 1 (0.45%) Paenibacillus alvei: 7 (3.12%)Terribacillus goriensis: 1 (0.45%) Species level (224 SDF strains) B Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 SDF strains Bacillus cereus (98%) (SDF0219) Bacillus cereus (99%) (SDF0124) Bacillus anthracis (97%) (SDF0199) Bacillus cereus (100%) (SDF0119) Bacillus thuringiensis (98%) (SDF0235) Bacillus cereus (99%) (SDF0061) Bacillus thuringiensis (100%) (SDF0288) Bacillus cereus (98%) (SDF0006) Bacillus toyonensis (98%) (SDF0053) Bacillus cereus (98%) (SDF0250) Bacillus anthracis (100%) (SDF0089) Bacillus cereus (97%) (SDF0206) Bacillus cereus (99%) (SDF0299) Bacillus cereus (99%) (SDF0300) Bacillus cereus (99%) (SDF0304) Bacillus anthracis (99%) (SDF0186) Bacillus cereus (99%) (SDF0305) Bacillus cereus (99%) (SDF0200) Bacillus cereus (100%) (SDF0295) Bacillus cereus (99%) (SDF0184) Bacillus cereus (98%) (SDF0239) Bacillus thuringiensis (98%) (SDF0178) Bacillus thuringiensis (99%) (SDF0276) Bacillus cereus (97%) (SDF0272) Bacillus cereus (97%) (SDF0159) Bacillus thuringiensis (100%) (SDF0161) Bacillus thuringiensis (100%) (SDF0294) Bacillus cereus (99%) (SDF0286) Bacillus cereus (100%) (SDF0283) Bacillus cereus (99%) (SDF0310) Bacillus thuringiensis (100%) (SDF0225) Bacillus cereus (100%) (SDF0155) Bacillus cereus (98%) (SDF0194) Bacillus cereus (100%) (SDF0248) Bacillus thuringiensis (98%) (SDF0301) Bacillus thuringiensis (97%) (SDF0303) Bacillus cereus (98%) (SDF0229) Bacillus cereus (97%) (SDF0278) Bacillus thuringiensis (98%) (SDF0100) Bacillus thuringiensis (98%) (SDF0085) Bacillus cereus (98%) (SDF0232) Bacillus cereus (98%) (SDF0032) Bacillus cereus (97%) (SDF0237) Bacillus cereus (98%) (SDF0182) Bacillus anthracis (100%) (SDF0181) Bacillus cereus (97%) (SDF0270) Bacillus cereus (100%) (SDF0022) Bacillus thuringiensis (99%) (SDF0030) CatalaseGelatinpH 5.7 Lysozyme Casein D−Glucose 45 °C Hemolysis Propionate utilization Starch Nitrate Reduction Tyrosine DegradationArginine Dihydrolase Voges−Proskauer SucroseOxidase Anaerobiosis NaCl 7%NaCl 10% LactoseD−Xylose Phenylalanine Citrate utilization L−Arabinose Mannitol Lysine Decarboxylase Esculin Ornithine Decarboxylase 65 °C Indole Production Response to biochemical and physiological tests Positive Negative Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 SDF strains Bacillus pumilus (100%) (SDF0123) Bacillus pumilus (100%) (SDF0034) Bacillus pumilus (100%) (SDF0130) Bacillus pumilus (99%) (SDF0115) Bacillus pumilus (100%) (SDF0135) Bacillus safensis (100%) (SDF0122) Bacillus pumilus (99%) (SDF0141) Bacillus subtilis (97%) (SDF0003) Bacillus pumilus (99%) (SDF0132) Bacillus pumilus (99%) (SDF0145) Bacillus pumilus (100%) (SDF0125) Bacillus pumilus (99%) (SDF0129) Bacillus pumilus (99%) (SDF0149) Bacillus pumilus (99%) (SDF0131) Bacillus pumilus (99%) (SDF0137) Bacillus safensis (100%) (SDF0134) Bacillus pumilus (97%) (SDF0140) Bacillus pumilus (98%) (SDF0202) Bacillus pumilus (100%) (SDF0105) Bacillus safensis (100%) (SDF0106) Bacillus pumilus (100%) (SDF0060) Bacillus pumilus (97%) (SDF0026) Bacillus pumilus (99%) (SDF0046) Bacillus pumilus (100%) (SDF0052) Bacillus safensis (98%) (SDF0103) Bacillus pumilus (99%) (SDF0044) Bacillus pumilus (100%) (SDF0035) Bacillus pumilus (99%) (SDF0004) Bacillus safensis (99%) (SDF0013) Bacillus pumilus (100%) (SDF0120) Bacillus pumilus (99%) (SDF0136) Bacillus safensis (98%) (SDF0098) Bacillus pumilus (99%) (SDF0104) Bacillus altitudinis (97%) (SDF0204) Bacillus pumilus (99%) (SDF0207) Bacillus amyloliquefaciens (100%) (SDF0110) Bacillus pumilus (98%) (SDF0171) Bacillus safensis (100%) (SDF0281) Bacillus pumilus (99%) (SDF0292) Bacillus pumilus (99%) (SDF0087) Bacillus pumilus (99%) (SDF0148) Bacillus safensis (100%) (SDF0093) Bacillus safensis (100%) (SDF0027) Bacillus pumilus (99%) (SDF0070) Bacillus pumilus (98%) (SDF0071) Bacillus pumilus (100%) (SDF0121) Bacillus pumilus (100%) (SDF0126) Bacillus pumilus (98%) (SDF0092) Bacillus pumilus (100%) (SDF0113) Bacillus safensis (98%) (SDF0091) Bacillus safensis (98%) (SDF0043) Bacillus pumilus (98%) (SDF0146) Bacillus safensis (98%) (SDF0147) Bacillus pumilus (99%) (SDF0177) Bacillus pumilus (97%) (SDF0273) Bacillus pumilus (99%) (SDF0152) Bacillus safensis (99%) (SDF0151) Bacillus pumilus (99%) (SDF0001) Bacillus pumilus (98%) (SDF0079) Bacillus pumilus (99%) (SDF0154) Bacillus pumilus (100%) (SDF0011) Bacillus altitudinis (99%) (SDF0153) Bacillus pumilus (98%) (SDF0114) Bacillus altitudinis (99%) (SDF0117) Bacillus pumilus (100%) (SDF0112) Bacillus pumilus (99%) (SDF0090) Bacillus safensis (99%) (SDF0002) Bacillus safensis (98%) (SDF0084) Bacillus pumilus (99%) (SDF0208) Bacillus pumilus (97%) (SDF0201) Bacillus altitudinis (97%) (SDF0205) Bacillus safensis (98%) (SDF0196) Bacillus pumilus (100%) (SDF0198) Bacillus pumilus (100%) (SDF0190) Bacillus pumilus (98%) (SDF0173) Bacillus amyloliquefaciens (98%) (SDF0302) Bacillus subtilis (99%) (SDF0285) Bacillus velezensis (99%) (SDF0280) Bacillus pumilus (99%) (SDF0142) Bacillus amyloliquefaciens (98%) (SDF0269) Bacillus velezensis (99%) (SDF0287) Bacillus tequilensis (99%) (SDF0282) Bacillus altitudinis (99%) (SDF0036) Bacillus amyloliquefaciens (99%) (SDF0279) Bacillus velezensis (100%) (SDF0150) Bacillus subtilis (97%) (SDF0025) Bacillus altitudinis (99%) (SDF0017) Bacillus pumilus (99%) (SDF0179) Bacillus pumilus (100%) (SDF0156) Bacillus pumilus (100%) (SDF0157) Bacillus pumilus (97%) (SDF0175) Bacillus pumilus (99%) (SDF0187) Bacillus pumilus (99%) (SDF0230) Bacillus pumilus (99%) (SDF0231) Catalase pH 5.745 °C NaCl 7%Hemolysis GelatinSucrose D−Glucose Voges−Proskauer MannitolCasein L−Arabinose NaCl 10% Esculin Citrate utilization Ornithine Decarboxylase Propionate utilization Lysozyme Nitrate Reduction Starch Arginine DihydrolaseLysine Decarboxylase Oxidase 65 °C LactoseD−Xylose Anaerobiosis Indole Production Tyrosine Degradation Phenylalanine Response to biochemical and physiological tests Positive Negative Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638 SDF strains Paenibacillus lautus (98%) (SDF0265) Paenibacillus alvei (99%) (SDF0164) Paenibacillus alvei (97%) (SDF0213) Paenibacillus alvei (99%) (SDF0080) Paenibacillus alvei (100%) (SDF0028) Paenibacillus alvei (99%) (SDF0023) Paenibacillus alvei (99%) (SDF0008) Paenibacillus alvei (99%) (SDF0133) Paenibacillus ginsengagri (100%) (SDF0227) Brevibacillus laterosporus (99%) (SDF0195) Brevibacillus agrii (99%) (SDF0188) Brevibacillus laterosporus (99%) (SDF0197) Brevibacillus laterosporus (100%) (SDF0020) Paenibacillus chibensis (98%) (SDF0259) Brevibacillus laterosporus (99%) (SDF0220) Paenibacillus susongensis (99%) (SDF0162) Paenibacillus terrigena (97%) (SDF0240) CatalaseSucrosepH 5.7 Lysozyme Gelatin45 °C Voges−Proskauer D−Glucose Indole Production Oxidase Anaerobiosis Hemolysis CaseinNaCl 7% Propionate utilizationTyrosine Degradation Mannitol Nitrate Reduction StarchLactoseEsculin L−Arabinose D−Xylose Lysine DecarboxylaseArginine Dihydrolase Citrate utilization Ornithine Decarboxylase Phenylalanine NaCl 10% 65 °C Response to biochemical and physiological tests Positive Negative Author-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638