{"paper_id":"054d9e3f-a39a-49a8-bbcc-796897af9385","body_text":"PREPRINT\nAuthor-formatted, not peer-reviewed document posted on 18/05/2022\nDOI: https://doi.org/10.3897/arphapreprints.e86638\nBiochemical, physiological, and molecular\ncharacterisation of a large collection of aerobic\nendospore-forming bacteria isolated from Brazilian soils\nPaulo Henrique Rosa Martins, Leon Rabinovitch, Juliana Orem,  Waldeyr Silva, Felipe Mesquita, Maria \nMagalhaes, Danilo Cavalcante, Adriana Vivoni, Josiane Brito, Edmar Oliveira, Vera Lima,  Marlene De-\nSouza\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n1 \n \nBiochemical, physiological, and molecular characterisation of a large collection 1 \nof aerobic endospore-forming bacteria isolated from Brazilian soils 2 \n 3 \nPaulo Henrique Rosa Martins1, ph.biomedicina@gmail.com; 4 \nLeon Rabinovitch2, leon@ioc.fiocruz.br; 5 \nJuliana Capela de Orem1, jucorem@gmail.com; 6 \nWaldeyr Mendes C. Silva3, waldeyr.mendes@ifg.edu.br; 7 \nFelipe de Araujo Mesquita1, felipedearaujomesquita@gmail.com; 8 \nMaria Ines Andre de Magalhães1, miamagalhaes@gmail.com; 9 \nDanilo de Andrade Cavalcante1, danilo.ac9@gmail.com; 10 \nAdriana Marcos Vivoni2, avivoni@ioc.fiocruz.br; 11 \nEdmar Justo de Oliveira2, edsea@hotmail.com.br; 12 \nVera Cristina Pessoa de Lima2, vera@ioc.fiocruz.br; 13 \nJosiane Teixeira Brito2, josidebrito@hotmail.com; 14 \nand Marlene Teixeira De-Souza1*, marlts@unb.br. 15 \n 16 \n1Department of Cellular Biology, Institute of Biological Sciences, University of Brasília, 17 \nDF, Brazil.  18 \n2Laboratório de Fisiologia Bacteriana, Instituto Oswaldo Cruz, Fundação Oswaldo 19 \nCruz, Rio de Janeiro, RJ, Brazil 20 \n3Federal Institute of Goias, Formosa, Brazil 21 \n 22 \n 23 \n*Corresponding author 24 \nAddress: Universidade de Brasília; IB/Departamento de Biologia Celular;  25 \nCampus Universitário Darcy Ribeiro; 70.910-900 – Brasília – DF, Brazil.  26 \nTelephone: +55 61 3107-3044 27 \ne-mail: marlts@unb.br 28 \nhttps://orcid.org/0000-0003-1538-2657 29 \n  30 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n2 \n \nAbstract  31 \nThe aerobic endospore -forming bacteria (AEFB) comprise species of Bacillus and 32 \nrelated genera, allocated in the phylum Firmicutes. Although Bacillus spp. are among 33 \nthe first bacteria to be characterised, the wide  diversity render s appropriate 34 \ncategorisation and generalisations challenging tasks. To determine genetic diversity, 35 \nanalyses at the molecular level are the most accurate. However, gene expression, 36 \nmorphological, biochemical, and physiological aspects must also be considere d. The 37 \nmetabolism of bacteria is adapted to their natural environment or host. Thus, metabolic 38 \noutlines can be used for identifying AEFB , form the basis of the formal description of 39 \nbacterial taxa, and  are strongly recommended for taxonomic purposes. This work 40 \naddressed the biochemical and physiological profiles of 312 environmental AEFB —41 \ndesignated as SDF (Solo do Distrito Federal )—by performing 30 tests.  Out of it, 246 42 \nwere classified by 16S rRNA gene sequences. We summarised the phenotypic test 43 \nrelationships among selected SDF strains using a Pearson correlation -based 44 \nclustering represented in heatmaps. In practice, biochemical and physiological profiles 45 \nare often less discriminatory than molecular data and may be unstable because of the 46 \nloss of traits. Though these test reactions are not universally positive or negative within 47 \nspecies, they may define biotypes  and be efficient strain markers, enhancing the 48 \naccuracy of unknown sample identification. It can be also helpful in selecting the best 49 \nrepresent the phenotypes of samples. Along with the other phenotypic and genotypic 50 \ndata, the present results will be of great importance for the robust classification of the 51 \nSDF strains within the scope of the polyphasic approach. 52 \n 53 \nKeywords: Bacillales; Bacillaceae; endosporulation; Firmicutes; bacterial 54 \nidentification; bacterial metabolism; phenotyping; taxonomy. 55 \n 56 \n 57 \nRunning head: Soil aerobic endospore-formers phenotypic and molecular profiles 58 \n 59 \n 60 \n  61 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n3 \n \nIntroduction 62 \nAerobic endospore -forming bacteria (AEFB) enc ompass species from genus 63 \nBacillus and related genera and produce dormant and highly resistant cells called 64 \nspores (Fritze 2004; Logan and Halket 2011; Setlow 2014; Driks and Eichenberger 65 \n2016). Spores can germinate within seconds when external conditions become 66 \nfavourable (Moir and Cooper 2014). Strains of AEFB are widely distributed in nature, 67 \nand soil is recognised as the main reservoir (Fritz e 2004; Logan et al. 2009; Mandic -68 \nMulec and Prosse r 2011; De Vos  2011). AEFB harbour species of significant 69 \nimportance in health, environment, and biotechnology (Fritze 2004; Logan et al. 2009; 70 \nEhling-Schulz and Messelhäusser 2013; Alina et al. 2015). 71 \n 72 \nAEFB exhibit high levels of genetic, biochemical, and physiological diversity and 73 \nappreciable resistance to adverse environmental ( De Vos  et al. 2009; Logan et al. 74 \n2009; Logan and Halket 2011; Galperin 2013; Setlow 2014; Driks and Eichenberger 75 \n2016). The high heterogeneity in the phenotypic and genotypic characteristics h as 76 \nbeen hampering the taxonomy of these species (Ash 1991; Fritze 2004; Logan et al. 77 \n2009; Galperin 2013).  78 \n 79 \nThe first identification and classification schemes of AEFB were based on the 80 \nmorphology of the colonies, vegetative cells , sporangia, spores, Gram-staining 81 \nresponse, besides biochemical, physiological, and chemotaxonomic properties (Logan 82 \net al. 2009). Today’s polyphasic taxonomy distinguishes and classifies strains based 83 \non these classical phenotypic data, supplemented with genotypic and other phenotypic 84 \nresults obtained at the molecular level (Colwell 1970; Fritze 2004; Prakash et al. 2007; 85 \nLogan et al. 2009; Das et al. 2014). Combining classical and molecular data, notably 86 \n16S rRNA gene sequencing, has revolutionised our understanding of domain Bacteria 87 \n(Bochner 2009) and led to a rapid increase in the number of descriptions of novel AEFB 88 \ntaxa, especially at genus and species levels (Fritze 2004; Logan et al. 2009; Maughan 89 \nand Van der Auwera 2011). 90 \n 91 \nAEFB are allocated in the phylum Firmicutes, with in the class  Bacilli, order 92 \nBacillales, where seven families harbour aerobic spore -forming genera: Bacillaceae, 93 \nAlicyclobacillaceae, Paenibacillaceae, Planococcaceae, Pasteuriaceae, 94 \nSporolactobacillaceae, and Thermoactinomycetaceae (De Vos et al. 2009; Logan and 95 \nHalket 2011; Galperin 2013; Parte 2018). 96 \n 97 \nPhylogenetic studies based on the 16S rRNA gene sequences suggest  clusters 98 \nof closed related AEFB species, designated groups (Ash et al. 1991; Stackebrandt and 99 \nSwiderski 2002; Fritze 2004; De Vos et al. 2009; Logan 2009; Alina 2015). The early 100 \nrRNA groups 1 to 5 of Bacillus species proposed by Ash et al. in 1991 were expanded 101 \nto house alkaliphilic and alkalitolerant species, while other groups of species, such as 102 \nthose allocated in genera Paenibacillus (group 3, Brevibacillus (group 4), and other 103 \ndistinct taxa have been reclassified (Stackebrandt and Swiderski 2002). 104 \n 105 \nWithin genus Bacillus, members of B. cereus  group or sensu lato  (sl) and B. 106 \nsubtilis complex are composed of highly related members (>99% similarity), restricting 107 \nspecies delimitation when considering only the 16S rRNA gene analyses. Differently 108 \nfrom the other Bacillus groups described above that harbour high genome identity,  B. 109 \nmegaterium and B. aryabhattai share 99.7% of identi ty in the 16S rRNA gene 110 \nsequences. Nevertheless, the genomes are less than 70% identical (Shivaji et al. 111 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n4 \n \n2009). Therefore, the distinction of these two strains using only this technique is also 112 \nchallenging.  113 \n 114 \nSince observable features from growth condition s and enzymatic reactions are 115 \nrelated to the genome expression, the resulting profiles allow detecting phenotypic 116 \npatterns for the species evaluated. Thus, investigating these intrinsic metabolic 117 \nactivities are still essential for the identification and classification of new AEFB isolates. 118 \nThese assays are highly recommended in the characterization of AEFB strains (Logan 119 \net. al 2009). 120 \n 121 \nTo help understand AEFB diversity and explore their biotechnological potential, 122 \nwe isolated 312 strains from soil samples c ollected at random areas of the Federal 123 \nDistrict, Midwest region of Brazil (Cavalcante et al. 2019; Orem et al. 2019; Martins et 124 \nal. 2020). These strains, designated SDF0001 -SDF0312 (Solo do Distrito Federal or 125 \nSDF) are deposited at the Coleção de Bactérias Aeróbias Formadoras de Endósporos 126 \n(AEFB Collection —AEFBC), hosted at the University of Brasilia. For taxonomic 127 \npurposes, the SDF strains are being analysed by a polyphasic strategy.  128 \n 129 \nIn the present work, 3 0 biochemical and physiological tests were performed to 130 \ninvestigate substrate s utilisation and transformation , in addition to the growth 131 \nconditions capabilities of 312 SDF strains. Among them, 246 were classified by 16S 132 \nrRNA sequences. A Pearson correlation based on a clustering method (Gu et al. 2016) 133 \nwas used to construct heatmaps to summarise the relationships of selected SDF 134 \nstrains to these phenotypic tests.  135 \n 136 \n 137 \nMethods 138 \nBacterial strains. The 312 SDF strains evaluated in this study were isolated as 139 \ndescribed in Cavalcante et al. (2019) and Orem  et al. ( 2019). The reference strains 140 \nused as positive and negative controls for the physiological and biochemical tests 141 \n(Table 1) are deposited at  Coleção de Culturas do Gênero Bacillus e Gêneros 142 \nCorrelatos (CCGB), of the Instituto Oswaldo Cruz (LFB-Fiocruz-RJ, Brazil). 143 \nEthics statement. Specific permissions required to collect bacterial strains used in 144 \nthis study were endorsed by the Federal Brazilian Authority (CNPq; Authorization of 145 \nAccess and Sample of Genetic Patrimony nº 010439/2015-3). Sampling did not involve 146 \nendangered or protected species. 147 \nBiochemical and physiological assays. Strains were grown in nutrient agar (33 °C, 148 \n24 h) under atmospheric aerobic conditions. Cells from a single colony  were 149 \ntransferred to a tube containing nutrient broth and incubated at 33 °C, under constant 150 \nstirring (200 rpm), for about 16 h. The 30 biochemical and physiological tests (Table 1) 151 \nwere performed according to Bergey's Manual of Systematic Bacteriology (Smith et al. 152 \n1952; Gordon et al. 1973; Claus and Berkeley 1986; Oliveira and Rabinovitch 1998;  153 \nDe Vos et al. 2009;  Rabinovitch and Oliveira 2015). All tests were performed in 154 \nduplicate in two independent experiments. 155 \nTaxonomic assignments of SDF strains. DNA preparation, PCR amplification, 156 \nsequencing, and sequence analyses were performed as described in Orem et al. 157 \n(2019). Briefly, the nearly full length of both strands of 16S rRNA genes was amplified 158 \nusing total DNA and primers 27F (5’ AGA GTT TGA TCM TGG CTC AG 3’) and 1492R 159 \n(5’ GGY TAC CTT GTT ACG ACT T 3’). PCR products were bi-directionally sequenced 160 \nemploying the Sanger method. These sequences were filtered for Q≥20 in Phred 161 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n5 \n \nscores and taxonomically assigned using BLAST and Classifier as described in Orem 162 \net al. (2019).  163 \nHeatmaps. The biochemical and physiological assays results were arranged in 164 \nheatmaps (Gu et al. 2016) to enhance the potential of visually revealing patterns and 165 \ncorrelations among them. We took the dichotomous values 0 (for Negative) and 1 (for 166 \nPositive) as binary variables representing the association among the species’ and its 167 \nbiochemical and physiological assay results. Using Pearson’s correlation, the species 168 \nwere clustered taking similar biochemical and physiological results (Hummel et al. 169 \n2017). R scripts are available at https://github.com/waldeyr/bafes_figures. 170 \n 171 \n 172 \nResults and discussion 173 \nDue to metabolism importance for identification and classification  of AEFB new 174 \nisolates (Fritze 2002; Logan et al. 2009), we applied 30 biochemical and physiological 175 \ntests (Table 1) to 312 AEFB strains isolated from Brazilian soils, designated SDF 176 \nstrains (Cavalcante et al. 2019; Orem et al. 2019; Martins et al. 2020). T he profiles 177 \nobtained from enzymatic reactions and growth conditions are described in Table S1, 178 \navailable in the online Supplementary Material. It is important to state that, all the 312 179 \nSDF strains studied are aerobic or facultative anaerobic endospore -formers, and 180 \nGram-positive or Gram -variable cells (Cavalcante et al. 2019; Orem et al. 2019; 181 \nMartins et al. 2020). The latter characteristics are common to taxa found in the order 182 \nBacillales (Fritze 2004; De Vos et al. 2009; Logan et al. 2009; Galperin 2013), where 183 \nthese environmental AEFB strains are allocated.  184 \n 185 \nOf these 312 SDF strains, the taxonomic assignment s of 246 w ere addressed 186 \nusing the standard tool of taxonomists for bacteria l identification, classification, and 187 \nphylogenetic relatedness, the 16S rRNA gene sequences (Tringe and Hugenholtz 188 \n2008; De Vos et al. 2009; Hakorvita et al. 2016), as described in Orem et al. (2019). 189 \nThe lowest and highest inter -species pairwise 16S rRNA gene sequence similarities 190 \nspanned from 90 % to 100 % (Table S1) . Considering the similarity thresholds  for 191 \ngenera 96%, and ≥97% for species (Stackebrandt and Goebel 1994), the classification 192 \nobtained segregated 238 SDF strains into 6 genera, being 4 part of family Bacillaceae 193 \nand 2 of Paenibacillaceae (Fig. 1A). Among the SDF strains described in the present 194 \nwork, Bacillus spp., belonging to the family Bacillaceae, are the most prevalent (207 195 \nstrains; 84.14%), followed by species of genera Paenibacillus (14; 5.69%; family 196 \nPaenibacillaceae), Lysinibacillus (7; 2.84%; Bacillaceae), Brevibacillus (6; 2.43%; 197 \nPaenibacillaceae), Terribacillus (1; 0.40%; Bacillaceae), and Rummeliibacillus (1; 198 \n0.40%; Bacillaceae). These findings are not surprising since the selective procedure 199 \nwe used to isolate SDF strains intended to favour non -fastidious AEFB species, 200 \nexcluding strict anaerobic endospore-forming and Gram-negative cells (Cavalcante et 201 \nal. 2019; Orem et al. 2019; Martins et al. 2020). 202 \n 203 \nIncluded in the 224 SDF strains classified at the species level  (Table S1) , 204 \nmembers of B. pumilus subgroup were predominant (83 strains; 37.05%), followed by 205 \nB. cereus  group species (48; 21.42%), B. megaterium  group (35; 15.62%), other 206 \nmembers of B. subtilis  complex (1 2; 5.35%); B. simplex  (7; 3.12%); B. clausii (3; 207 \n1.33%); B. subterraneus (2; 0.89%); besides 1 ( 0.44%) of each: B. australimaris; B. 208 \narbutinovorans; B. circulans; B. kochii; B. luciferensis; B. oleronius; B. siamensis, and 209 \nB. senegalensis. Outside genus Bacillus, other species belonging to family Bacillaceae 210 \nwere Lysinibacillus sphaericus (3; 1.33%); L. xylanilyticus (2; 0.89%); L. fusiformis (2; 211 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n6 \n \n0.89%), and Terribacillus goriensis (1; 0.44%). Paenibacillus spp. (12 strains; 5.35%), 212 \nand Brevibacillus spp. ( 5 strains; 2.23%) allocated in the family Paenibacillaceae 213 \ncomplete the list of SDF strains classified at the species level (see below). The diversity 214 \nof the SDF strains is represented in Fig. 1B. 215 \n 216 \nIt is mentionable that member s of B. cereus  sl and B. subtilis  subgroups are 217 \ncomposed of very related members (>99% similarity), restricting species delimitation 218 \nwhen considering only the 16S rRNA gene analyses. Conversely, B. megaterium and 219 \nB. aryabhattai share 99.7% of identity in the 16S rRNA gene sequences, even though 220 \nthe genomes are less than 70% identical (Shivaji et al. 2009). Therefore, the distinction 221 \nof these two species using only this technique is also challenging. 222 \n 223 \nThus, our taxonomic assignments based on 16S rRNA gene sequences are a 224 \npreliminary inference of genera or species. Accordingly, when 16S rRNA gene profiling 225 \nplaced these strains within these AEFB taxa, a sample analysed can belong to two or 226 \neven more species alternatives within the same affiliation cluster. In these instances, 227 \nthis approach can find groups of bacteria, never theless cannot assign it accurately to 228 \na species according to its low discrimination ability. Since 10 SDF strains exhibited 229 \nsimilarity rates spanning 90 -95% (Table S1), the 16S rRNA gene -sequencing tool 230 \nfailed to classify these environmental strains even at the genus level. While this genetic 231 \nmarker was insufficient to set up genus or species, low gene-sequence similarity might 232 \nsuggest that novel species could have been isolated (Tindal et al. 2010). Nevertheless, 233 \nthe description of the new taxa is beyond the scope of this article. 234 \n 235 \nBacillus is the genus type of order Bacillales, and Bacillus spp. have been isolated 236 \nfrom a wide range of environments (Tamames et al. 2010; Mandic-Mulec and Prosser 237 \n2011; Alina et al. 2015; Orem et al. 2019; Cavalcante 2019; Salgado et al. 2020). Soils, 238 \nalong with freshwaters, are one of the least restrictive for these species. It is worthy to 239 \nnote that certain species found in soils are inactive in these  environments. It could be 240 \nthe case for some SDF strains isolated from Brazilian soils. The method of isolation 241 \nbased on heat shock allowed the dormant spores to germinate and grow in vitro. 242 \n 243 \nB. cereus and B. anthracis are human pathogens causing food-borne illness and 244 \nanthrax, respectively (Arnesen et al. 2008; Ehling -Schulz and Messelhäusser 2013). 245 \nOn the other hand, the metabolic breadth of Bacillus spp. has been explored by the 246 \nindustry for producing a vast range of antibiotics; plant growth promotion m olecules; 247 \nhydrolyses; toxins against plants, fungus, insect, and nematode, in addition to other 248 \nbioproducts (de Maagd et al. 2003; Berkeley et al. 2008; Logan et al. 2009; Galperin 249 \n2013; Alina 2015). Therefore, despite the danger of few members, most speci es are 250 \nbeneficial.  251 \n 252 \nGenus Bacillus stays the largest AEFB taxon, accommodating 614 species, as 253 \nregistered at the List of Prokaryotic Names with Standing in Nomenclature (LPSN: 254 \nhttps://www.bacterio.net/Bacillus.html; accessed on 01 February 2022). Taxonomy 255 \nwithin genus Bacillus is hampered by high heterogeneity at phenotypic and genotypic 256 \nlevels (Ash 1991; Fritze 2004; Logan and De Vos 2009; Logan et al. 2009). Further, 257 \nthese divergencies restrict the distinction between Bacillus spp. and those allocated in 258 \nother genera inside Bacillaceae. 259 \n 260 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n7 \n \nTypically, Bacillus spp. are considered aerobic, although at least 20 species are 261 \nfacultatively anaerobic (Logan and De Vos  2009). Furthermore, nitrate reduction is 262 \nfrequently observed in this genus. Members of Bacillus can be rods or cocci, motile or 263 \nnon-motile, organotrophic or lithotrophic (Fritze 2004; Logan and De Vos 2009; Logan 264 \net al. 2009). Cell size, varying from 0.4 to 1.8 μm in diameter and from 0.9 to 10.0 μm 265 \nin length, can also be used to differentiate Bacillus spp. (Logan and De Vos 2009). 266 \nPhylogenetically, most recognised species are arranged into subclusters or rRNA 267 \ngroups. 268 \n 269 \nDue to the significant relevance in economy and health issues, the B. cereus 270 \ngroup and B. subtilis complex have been received considerable attention (Fritze 2004; 271 \nMaughan and Van der Auwera 2011). The B. cereus group hosts B. cereus sensu 272 \nstricto (or ss or B. cereus ), B. anthracis , B. thuringiensis , B. mycoides , B. 273 \npseudomycoides, B. weihenstephanensis , B. toyonensis , and B. cytotoxicus  (Fritze 274 \n2004; Maughan and Van der Auwera 2011; Ehling -Schulz and Messelhäusser 2013). 275 \nOrganisms placed in this group belong to 16S rRNA/DNA group 1. Cells are typically 276 \nwider than 1 μm, Gram-positive, and endospores are oval to cylindrical paracentral or 277 \nsubterminally localised in unswelling sporangia. 278 \n 279 \nTraditionally, these bacteria have been differentiated based on phenotypic 280 \ncharacteristics, especially pathogenic potential. Nonetheless, this group is a highly 281 \nhomogeneous subdivision inside genus Bacillus (Helgason et al. 2000; Chen and Tsen 282 \n2002). Furthermore, they are hardly distinguishable with standard biochemical and 283 \nchemotaxonomic methods or phylogenetically relevant target genes (Bavykin et al. 284 \n2004; Arnesen  et al. 2008). However, specific biochemical and physiological 285 \ncharacteristics of the B. cereus sl are advantageous to differentiate these taxa from 286 \nthe other aerobic endospore-forming species.  287 \n 288 \nOut of 224 SDF strains classified at the species level (Tabl e S1), 48 ( 21.42%) 289 \nwere members of the B. cereus group. Using a Pearson correlation -based clustering 290 \nmethod (Gu et al., 2016), we constructed a heatmap ( Fig. 2) to summarise the 291 \nrelationships of these 48 environmental strains to the 30 biochemical and physiological 292 \ntests performed (Table 1). Each column shows the metabolic pattern (bottom side) of 293 \nindividual SDF strain (rows at the right side), classified based on 16S rRNA sequences. 294 \nThe green and the red colours represent positive and negative responses, 295 \nrespectively. Fig. 2 shows an assembling of these essays based on the prevalence of 296 \nthe positive responses. It is doable to distinguish which strains respond similarly to the 297 \ntests when they are in the same clade. For example, th ose strains in distant clades 298 \nrespond differently. It is also possible to discern which SDF strains respond similarly 299 \nto each test , as well as  discriminate them by correlating rows and columns. The 300 \nclusters from the upper-side dendrogram (Fig. 2) stands for the similarity of the 30 tests 301 \nresponses. The left-most contains 17 columns, while the right-most contains 13, where 302 \nthe majority of these SDF strains responded positively and negatively, respectively. 303 \n 304 \nAlthough many AEFB may not respond positively to the c atalase test, most 305 \nspecies rod-shaped, either Gram-positive or Gram-positive only in the initial stages of 306 \ngrowth, are catalase -positive, especially members of the genus Bacillus (Logan and 307 \nDe Vos , 2009a). However, in most cases, respiratory metabolism occ urs at low 0 2 308 \nlevels. Here, all the 48 SDF members of the B. cereus group responded positively to 309 \nthis enzyme linked to respiration in the presence of atmospheric 0 2 (Fig. 2). This 310 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n8 \n \npositivity seems to be a characteristic of this group of sporulating procar yotes. As 311 \nassessed in this work, it is worth noting that B. cereus  ss can grow under certain 312 \nanaerobiosis conditions (Logan and De Vos, 2009a). 313 \n 314 \nThe cytochrome C oxidase is especially useful to discriminate Gram -negative 315 \npathogens Vibrio spp. (oxidase positive) from the oxidase -negative enteric bacteria 316 \n(Vila et al. 1992). This enzyme catalyses the oxidation of cytochrome C while reducing 317 \noxygen to form water. The oxidation test in vitro employs colourless artificial acceptors 318 \nlike dimethyl or tetramethyl p -phenylenediamine resulting in purple colour when 319 \npositive. This essay also distinguishes Neisseria and Moraxella (both oxidase positive) 320 \nfrom Acinetobacter spp. (oxidase negative) (Henriksen 1976; Powell and Marcon 321 \n2012). 322 \n 323 \nFrom the 48 SDF str ains allocated in the B. cereus group, 25 (52.08%) were 324 \noxidase-positive. Logan and De Vos (2009) point out this variability for this genus and 325 \nrelated genera, demonstrating apparent inactivity of this enzyme, or even that the 326 \ntraditional method failed to detect the oxidase activity in almost half of these samples. 327 \n 328 \nAnaerobiosis assays, performed in tubes containing aldehyde -reduced agar 329 \nmedium inoculated with a needle, revealed growth a few centimetres below the 330 \ninterface of the culture medium with atmosph eric air to 24 (50%) of the SDF strains  331 \nbelonging to the B. cereus sl. This effect present in -depth denotes anaerobic growth, 332 \na property conserved among AEFB. 333 \n 334 \nSome Bacillus species do not appear to utilise carbohydrates whatsoever (Logan 335 \nand De Vos 2009). Yet the acid production profiles from monosaccharides and 336 \ndisaccharides are of great value in the characterisation and identification of these 337 \nspecies. Most SDF strains allocated into B. cereus group used D-glucose, L-arabinose, 338 \nD-xylose, and other fermentable carbohydrates as sole sources of carbon and energy 339 \n(Table S1; Fig. 2). Probably they have the genetic information to conduct the pathway 340 \nof Embden-Meyerhof-Parnas, coupled with the Krebs cycle, verified by acid production 341 \n(Logan and De Vos 2009a).  342 \n 343 \nRegarding glucose consumption, five SDF strains, one classified as B. 344 \nthuringiensis (SDF0225), and four as B.cereus ss (SDF0124; SDF0229; SDF0237, and 345 \nSDF248), responded negatively to the use of this monosaccharide, which is rare 346 \namong rods AEFB, as they usually assimilate and degrade D -glucose. Though the 347 \nformation of acid from D-mannitol is frequently negative for members of the B. cereus 348 \nsl and positive for strains of other groups (Fritze 2002), three SDF strains classified as 349 \nB. cereus ss (SDF0219; SDF0124, and SDF0022) and B. anthracis SDF0199 were 350 \nable to ferment this sugar ( Fig. 2). Interestingly, these strains were gathered in the 351 \nuppermost and downmost rows of the strains' list (right side). Indeed, clustering heat 352 \nmaps can group samples based on the similarity of their phenotypic patterns, thus 353 \nidentifying atypical responses (Zhao et al. 2014). 354 \n 355 \nThe Voges-Proskauer test presented some species such as two B. thuringiensis 356 \nstrains (SDF0161 and SDF0178); three B. anthracis (SDF181; SDF0186, and 357 \nSDF0199), besides seven B. cereus ss (SDF0155; SDF0159; SDF0182; SDF0184; 358 \nSDF0239; SDF0270, and SDF0272) responding negatively to the acetyl -359 \nmethylcarbinol production assay, which allows us to suspect that these strains may not 360 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n9 \n \nproduce enzymes that decarboxylate lactic acid from the glycolytic pathway, or do not 361 \nhave an enzyme capable of bonding two molecules originating from the production of 362 \nacetate ions. 363 \n 364 \nOliveira and Rabinovitch (1998) established a standardised protocol for detection 365 \nof gelatin hydrolysis by Lysinibacillus sphaericus—former B. sphaericus (Seldin et al. 366 \n1984; Ash et al. 1994) —showing that 93.3% of strains belonging to this species 367 \nhydrolyses this incomplete protein after four days of incubation. Here bulk 48 SDF 368 \nstrains accommodated in the B. cereus group could use gelatin. Providing the relatively 369 \nhigh number of strains submitted to this type of biochemical test, we considered a very 370 \nvalid verification. 371 \n 372 \nThe development in the presence of lysozyme is another characteristic of the B. 373 \ncereus group and hardly occur in the other species of other groups (Fritze 2002). 374 \nHowever, B. cereus  SDF0124 and B. thuringiensis  SDF085 did not grow in this 375 \ncondition, indicating that the cell wall of these two strains can be hydrolysed by this 376 \nenzyme. 377 \n 378 \nThe production of haemolysin, as well as cell morphology in a few strains of B. 379 \ncereus sl, are also phenotypes with relevance for taxonomic studies (Fritze 2002; 380 \nFritze 2004; De Vos and Logan 2009; Logan et al. 2009). B. cereus ss, in general, 381 \nmobile, is heavily haemolytic, but does not produce rhizoid growth pattern, a 382 \ncharacteristic that can be used to differentiate from colonies of B. mycoides strains 383 \n(Fritze 2002). Most B. anthracis strains are neither mobile nor haemolytic (Fritze 2004; 384 \nMaughan and Van der Auwera 2011). However, non-mobile B. cereus strains, as well 385 \nas hemolytic B. anthracis, may hinder the differentiation between these two species. 386 \nIn addition, the latter sp ecies can be differentiated by parasporal crystal formation 387 \ntypically described to B. thuringiensis (Fritze 2002). 388 \n 389 \nOut of 48 strains, SDF allocated in the B. cereus group, three samples classified 390 \nas B. cereus (SDF0159, SDF0237, and SDF0270); one B. anthracis SDF0181, and 391 \nthree B. thuringiensis (SDF0161; SDF0085, and SDF0030) presented no haemolysin  392 \nactivity. It is of great significance to mention that B. thuringiensis SDF0030 produces a 393 \ntypical parasporal crystal (Cavalcante et al. 2014), a classical feature distinguishing B. 394 \nthuringiensis strains from B. cereus  ss. (Dagmar 2014). Conversely, three strains 395 \nclassified as B. anthracis  (SDF0199, SDF089, and SDF0186) were positive for 396 \nhaemolysin activity. The main phenotypical properties that are frequent ly used to 397 \ndistinguish B. cereus, B. thuringiensis, and B. anthracis are related to the presence or 398 \nabsence of large plasmids, where the replicons are localized (Maughan and Van der 399 \nAuwera 2011). Future investigation on the extrachromosomal profiles of the se SDF 400 \nstrains will help to understand the evolutionary relatedness of these species.  401 \n 402 \nB. subtilis ss, the genus Bacillus type-species, is prominent in microbial history, 403 \nand play a distinct role as a model for Gram-positive bacteria and in the understanding 404 \nof stress-resistance of bacterial spores (Fritze 2004; Maughan and Van der Auwera 405 \n2011; Galperin 2013; Driks and Eichenberger 2016). Besides being recognised as a 406 \nmodel, this species, along with other highly related accommodated in the B. subtilis 407 \ncomplex, is extensively employed in industry and agriculture (Fan et al. 2017). 408 \n 409 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n10 \n \nB. subtilis strains are aerobics, although some strict anaerobic growth may be 410 \nobserved in complex media with glucose or (less effectively) nitrate (Logan and De 411 \nVos 2009a). This organism is catalase -positive, oxidase variable, and can reduce 412 \nnitrate to nitrite. The motile rod-cells of 0.7 –0.8 x 2.0 –3.0 μm are Gram -positive and 413 \ncan be frequently observed singly, in pairs, and, occasionally, in chains. This species 414 \nforms ellipsoidal to cylindrical endospores at the central, paracentral, or subterminal  415 \nposition in unswollen sporangia. 416 \n 417 \nAlthough optimal growth ranges from 28 -30 °C, B. subtilis  can tolerate 418 \ntemperatures from 5 –20 °C and 45 –55 °C (Logan and De Vos 2009a). Growth can 419 \noccur from pH 5.5 to 8.5, with no limits recorded. The vegetative cells have a significant 420 \nrole in the early steps of organic matter decomposition. Growth in minimal medium 421 \ncontaining glucose and ammonium salt—as sole sources of carbon— and nitrogen is 422 \nalso observed. Most strains can use citrate, as the sole carbon source, and growth 423 \noccurs in th e presence of up to 7% NaCl, and certain tolerate 10% NaCl. B. subtilis 424 \ncan hydrolyse casein, esculin, gelatin, and starch but not phenylalanine and urea. 425 \nExtracellular dextran and levan are produced from sucrose. Voges –Proskauer test is 426 \npositive, and the production of acid without gas can be detected from glucose, besides 427 \nadditional carbohydrates. 428 \n 429 \nAs a taxonomic unit above the species level, the B. subtilis species complex can 430 \nbe split into four clades (Fan et al. 2017). These recognizable monophyletic groups 431 \ncomprise clade I, consisting of three subspecies of B. subtilis (subtilis, spizenii, and 432 \ninaquosorum), besides B. tequilensis, B. vallismortis, B. mojavensis, and B. 433 \natrophaeus; clade II containing species B. amyloliquefaciens , B. siamensis , and a 434 \nconspecific complex embracing B. methylotrophicus , B. velezensis, and B. 435 \namyloliquefaciens subsp. plantarum; clade III encompassing B. licheniformis , B. 436 \nsonorensis, and related species, and clade IV made of B. pumilus and B. safensis, B. 437 \nxiamenensis, and a conspecific group involving the type strains of B. altitudinis , B. 438 \nstratosphericus, and B. aerophilus. Like strains from the B. cereus group, these taxa 439 \nare placed in 16S rRNA/DNA group 1 and are phylogenetic and physiologically 440 \nremarkably similar (Fritze 2004). Strains from this complex are usually mesophiles and 441 \nneutrophiles, but often tolerant to high pH values (Fritze 2004).  442 \n 443 \nEmploying 16S rRNA gene sequences, from the 224 SDF strains classified at the 444 \nspecies level 95 (42.41%) were allocated in the B. subtilis complex (Table S1). Among 445 \nthem, the B. pumilus subgroup represented 83 ( 37.05%), most of it or 61 ( 27.23%) 446 \nclassified as B. pumilus; 16 ( 7.14%) as B. safensis, and 6  (2.67%) as B. altitudinis. 447 \nSeven strains belonged to B. amyloliquefaciens subgroup or 3.12%, being 4 (1.78%) 448 \nB. amyloliquefaciens strains and 3 ( 1.33%) B. velezensis. The remaining 4 ( 1.78%) 449 \nSDF strains were accommodated in the B. subtilis subgroup, being 3 (1.33%) B. subtilis 450 \nss and 1 (0.44%) B. tequilensis. 451 \n 452 \nThe relationships of these 95 strains to the 30 biochemical and physiological tests 453 \ndescribed in Table 1 were also analysed. The res ulting heat map (Gu et al. 2016) 454 \nshown in Fig. 3 revealed two clusters (upper -side dendrogram) encompassing 13 455 \ncolumns at the left-most cluster, while the right -most contained 17, where these SDF 456 \nstrains responded positively and negatively, respectively. 457 \n 458 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n11 \n \nSpecies belonging to the so -called B. pumilus subgroup are almost identical in 459 \nthe 16S rRNA gene sequences, sharing above 99.5% similarity (Alina et al. 2015). B. 460 \npumilus ss is aerobic, catalase-positive, and enabled to reduce nitrate (Logan and De 461 \nVos 2009a). Gram-positive or Gram-variable small rods (0.6–0.7 by 2.0–3.0 μm) cells 462 \ncan be observed as singly or in pairs and are motile. Cylindrical to ellipsoidal 463 \nendospores can be central, paracentral, and subterminally localised in unswollen 464 \nsporangia. Thoug h optimal growth occurs at pH 6.0 and 9.5, some strains can 465 \nreproduce at pH 4.5. This species can tolerate up to 10% NaCl, hydrolyse casein, 466 \nesculin, and gelatin but cannot break down starch. Phenylalanine is not deaminated, 467 \nand citrate is utilised as the sole carbon source, but propionate is not. Acid without gas 468 \nis produced from glucose and many other carbohydrates, and Voges –Proskauer test 469 \nis positive. 470 \n 471 \nIn general, the SDF strains belonging to this group corroborates the traits 472 \ndescribed in Bergeys' Firmicutes (Logan and De Vos 2009a). Furthermore, according 473 \nto Logan and Forsyth, unpublished observations cited in this manual, B. pumilus  474 \nstrains isolated from Antarctic soils and penguin rookeries present phenotypic 475 \npeculiarities, such as producing a diffusible yellow pigment. 476 \n 477 \nOutside Bacillaceae, 17/224 (7.58%) SDF strains were allocated in two genera 478 \nof the family Paenibacillaceae. Paenibacillus spp. accounted for 12 (5.35%) strains 479 \nbeing 7 (3.12%) of P. alvei and 1 (0.44%) of each:  P. chibensis; P. ginsengagri; P. 480 \nlautus; P. susongensis, and P. terrígena (Table S1). Five (2.23%) strains of the genus 481 \nBrevibacillus (quoted here as Br.): Br. laterosporus (4 or 1.78%), and 1 (0.44%) of Br. 482 \nagrii completed the SDF strains allocated into the family Paenibacillaceae (Table S1). 483 \nThe mutual connection between these 1 8 strains and the 30 biochemical and 484 \nphysiological tests (Table 1) is represented in Fig. 4. The two clusters (upper -side 485 \ndendrogram) distinguishable by this heat map (Gu et al. 2016) comprehend 11 (left -486 \nmost) and 19 (right-most) columns, embracing most of these SDF strains responding 487 \npositively and negatively, respectively. 488 \n 489 \nThe genus Paenibacillus was created to reallocate species previou sly 490 \naccommodated in the RNA group 3 of genus Bacillus (Priest 2009). The family 491 \nPaenibacillaceae was subsequently proposed to house genus Paenibacillus and close 492 \nrelatives' genera (Ash et al. 1993; Shida  et al. 1997). This family encloses two 493 \nmonophyletic clusters, the first consisting of genera Paenibacillus, Brevibacillus, 494 \nCohnella, and Thermobacillus, the second of genera Aneurinibacillus, Ammoniphilus, 495 \nand Oxalophagus (De Vos et al. 2009). The type-genus is Paenibacillus. 496 \n 497 \nMembers of this family may be strictly aerobic, microaerophilic, facultative 498 \naerobic, or obligate anaerobic, being catalase -positive or -negative (De Vos et al. 499 \n2009). Cells are straight to curved rods of 0.5 –1.0 x 2–6 μm, Gram-positive but may 500 \nstain Gram-negative or variable. Oval or ellipsoidal endospores are frequently formed 501 \ninside a swelling sporangium. Peritrichous flagella may be observed, but some species 502 \nare nonmotile. Although they can utilise oxalic acid as the sole carbon and energy 503 \nsource, these cells are organoheterotrophs and grow in complex media, using 504 \ncarbohydrates and amino acids. They can be mesophilic or thermophilic, neutrophilic 505 \nor alkaliphilic and have been isolated from soil, roots, faeces, blood, and other 506 \nsubstrates. 507 \n 508 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n12 \n \nAfter Bacillus, the genus Paenibacillus accommodates the second largest 509 \nnumber of AEFB species known (3 42), as registered at the LPSN 510 \n(https://www.bacterio.net/; accessed on 01 February  2022). Paenibacillus harbours 511 \nspecies aerobic or facultative rod -shaped cells (Priest 2009; Galperin 2013; Parte 512 \n2018) and bears a typical Gram -positive cell -wall structure (Shida et al. 1996). 513 \nNevertheless, even young cells react weakly or even negatively to Gra m staining. It 514 \nshould be noted that the 12 SDF strains classified as Paenibacillus spp. in this work 515 \nstained weakly, or yet, Gram-negative (not shown). 516 \n 517 \nBrevibacillus species are aerobic, though some strains are microaerophilic and 518 \nfacultatively anaerobic (Logan and De Vos , 2009b). Most species are catalase -519 \npositive. Oxidase reaction and nitrate reduction can differ among strains. Rod-shaped 520 \ncells are 0.7–1.0 μm x 3.0–6.0 μm, occur singly, in pairs, in chains, are motile through 521 \nperitrichous flagella, and a re Gram -positive or Gram -variable. The ellipsoidal 522 \nendospores swell the sporangia. 523 \n 524 \nThis genus includes a high diversity of thermophilic, psychrophilic, acidophilic, 525 \nalkalophilic, and halophilic strains that use a variety of carbon sources for either 526 \nheterotrophic or autotrophic growth (Panda et al. 2014). Carbohydrates may be 527 \nassimilated, but acid is produced weakly, if at all, by most species. Some amino acids 528 \nand organic acids may be used as carbon and energy sources (Logan and De Vos 529 \n2009b). Casein, gelatin, and starch hydrolysis vary among species. Optimum growth 530 \noccurs at pH 7.0 and can be inhibited by 5% NaCl. The type -species is Br. brevis 531 \n(Shida et al. 1996), former Bacillus brevis.  532 \n 533 \nBrevibacillus spp. are used as a factory for the expression of biotechnologically -534 \nimportant enzymes (e.g., alpha -amylase, sphingomyelinase, xylanase, CGTase, and 535 \nchitosanase), as well as heterologous proteins including cytokines (EGF, IL -2, NGF, 536 \nIFN-c, TNF-a, and GM-CSF), antigens, and adjuvants (Mizukami et al. 2010). Besides, 537 \nBrevibacillus spp. are considered a valuable tool for structural and functional biology 538 \nstudies (Panda et al. 2014). 539 \n 540 \nBr. brevis, Br. choshinensis, and Br. laterosporus have attracted considerable 541 \ninterest owing to the production or transformation of valuable compounds and the 542 \nbiocontrol proprieties ( De Vos  et al. 2009b). The broad entomopathogenic activity 543 \nincludes species from orders Coleoptera, Lepidoptera, and Diptera and from phyla 544 \nNematoda and Mollusca (Ruiu et al. 2013). 545 \n 546 \nThe recent improvements in the tools have been helping in uncovering the vast 547 \nphysiological and genetic diversity within the AEFB, resulting in more appropriate 548 \ntaxonomic arrangements (Fritze 2004; Maughan and Van der Auwera 2011; G alperin 549 \n2013). As a result, many new descriptions of genera and species, and reclassifications 550 \nhave occurred. 551 \n 552 \nMolecular methods, especially 16S rRNA gene sequencing, have become the 553 \nprevailing technique in procaryotic identification, but significant restrictions in our ability 554 \nto identify environmental bacteria to the genus and species levels remain (Fritze 2004; 555 \nMaughan and Van der Auwera 2011; Galperin 2013). 556 \n 557 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n13 \n \nHere, the performance of the 16S rRNA sequence analysis was excellent. This 558 \ntool resolved 238 (96.74%) out of 246 SDF strains at the genus level, unrevealing four 559 \nand two genera within Bacillaceae and Paenibacillaceae, respectively. Among the 246 560 \nsamples, 22 4 SDF samples  (91.05%) were classified at the species level. As 561 \nmentioned above, using this technique, closely related strains such as those belonging 562 \nto the B. cereus group, B. subtilis complex, and other AEFB taxa cannot be resolved 563 \nat the species level. Still, our classifica tions are suitable since they clearly show the 564 \ngenera and restrict the identity of part of these SDF strains to one or a few species in 565 \nthe genera described. The positions of the SDF strains in this initial clustering and 566 \nidentification of closely related species may be more accurately determined by 567 \nincorporating additional data obtained at both genotypic and phenotypic analyses. 568 \n 569 \nFurthermore, our SDF strain classifications revealed well -known AEFB species, 570 \ntogether with others that are scarcely described i n the literature. Identifying multiple 571 \nspecies and strains from different genera may help resolve the order Bacillales at the 572 \nfamily, genus, and species levels. 573 \n 574 \nConclusion 575 \nIn the present study, 30 biochemical and physiological tests provided profiles of 576 \nall the 312 SDF strains deposited at AEFBC. From the genetic point of view, a large 577 \nnumber of samples such as those originating from the environment, as the SDF strains' 578 \ncollection, will hardly display 100% equal answers for all tests, as seen in taxonomic 579 \nstudies of strains isolated from non-clinical substrates (Logan and De Vos 2009; Logan 580 \nand Halket 2011). In such cases, there are always taxonomically diverging strains. The 581 \nubiquitous species B. pumilus , isolated from Antarctic soils and penguin rookeries, 582 \ncorroborate this statement as a phenotypic distinction from other lineages can be 583 \nobserved (Logan and Forsyth, unpublished observations, apud Logan and De Vos, 584 \n2009a). The divergent samples need to hav e a separate and improved taxonomic 585 \nstudy. 586 \n 587 \nBiochemical and physiological profiles are util e for identifying these 588 \nmicroorganisms. These essays are also part of the minimum standards proposed by 589 \nLogan et al. (2009) for characterising new species of these t axa. However, the value 590 \nof these tests to accurately identify large numbers of environmental species is limited 591 \n(Fritze 2004). Therefore, phenotypic similarities cannot be taken with certainty to 592 \nindicate close evolutionary relatedness. 593 \n 594 \nHowever, along with the other phenotypic and genotypic data (Cavalcante et al. 595 \n2019; Orem et al. 2019; Martins et al. 2020), including complete genome sequences 596 \nin progress, the profiles described in Table S1 will be significant for robust 597 \nidentification, consequently, clas sification and differentiation of these environmental 598 \nstrains. The biochemical and physiological profiles can also help optimise the culture 599 \nconditions for further characterisation and the production of bioactive metabolites by 600 \nthe SDF strains. 601 \n 602 \nHence, the classification of AEFB at the species levels is not straightforward. And 603 \nto classify and differentiate closely related SDF strains, these essays should be 604 \ncoupled to other classical and molecular methods involving phenotypic and genotypic 605 \ntypes (Cavalcante et al. 2019; Orem et al. 2019; Martins et al. 2020) in a polyphasic 606 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n14 \n \napproach (Colwell 1970; Fritze 2004; Prakash et al. 2007; Logan et al. 2009; Das et 607 \nal. 2014). 608 \n 609 \nThis strategy will facilitate the establishment of accurate classification of the SDF 610 \nstrains. It will also allow responsible exploitation of the extraordinary AEFB 611 \nbiotechnological potential, the reliable use as insect control agents, and the handling 612 \nof animal pathogens. 613 \n 614 \n 615 \nFunding 616 \nThe authors have no funding to report. 617 \n 618 \n 619 \nCompeting interests 620 \nThe authors have declared that no competing interests exist. 621 \n 622 \n 623 \nAcknowledge 624 \nWe thank University of Brasilia, and the Brazilian research funding agencies 625 \nCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and 626 \nConselho Nacional de Desenvolvimento Científico e Tecnológico  (CNPq). We are in 627 \ndebt with Arthur S. Araujo, and Liliam de Oliveira F. Marceneiro for excellent technical 628 \nassistance. 629 \n  630 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. 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DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\n Manuscript #39153_Resubmissão2022\n Neotropical Biology and Conservatio \n22 \n \nTables and figures captions 951 \n 952 \nTable 1. Biochemical and physiological tests used in this work and the respective 953 \ncontrols 954 \n 955 \n 956 \nTable S1. Molecular, biochemical, and physiological profiles of SDF strains belonging 957 \nto the AEFBC 958 \n 959 \n 960 \nFigure 1. Overall repartition of SDF strains according 16S rRNA gene sequencing 961 \nclassification. (A) Distribution of 238 SDF strains among six genera belonging to 962 \nfamilies Bacillaceae (Bacillus, Lysinibacillus, Terribacillus, and Rummeliibacillus) and 963 \nPaenibacillaceae (Paenibacillus and Brevibacillus). (B) Species assignments of 224 964 \nSDF strains.  965 \n 966 \n 967 \nFigure 2. Correlation between SDF strains belonging to B. cereus group and growth 968 \nconditions or enzymes activities. A Person correlation -based clustering method was 969 \nemployed to construct a heat map associating 48 SDF strains allocated in B. cereus 970 \ngroup (right) and 30 phenotypical features (bottom) that contribute to AEFB 971 \nidentification and classification. The top dendrogram clustered the SDF strains into two 972 \nparts based on the prevalence of positive responses (blue) to 30 growth conditions and 973 \nenzyme reactions described at the bottom of the graphic.  Negative responses are 974 \nshown in red. 975 \n 976 \n 977 \nFigure 3. Correlation between SDF strains belonging to B. subtilis complex and growth 978 \nconditions or enzymes activities. A Per son correlation-based clustering method was 979 \nemployed to construct a heat map associating 95 SDF strains allocated in B. subtilis 980 \ncomplex (right) and 30 phenotypical features (bottom) that contribute to AEFB 981 \nidentification and classification. The top dendrogram clustered the SDF strains into two 982 \nparts based on the prevalence of positive responses ( green) to 30 growth conditions 983 \nand enzyme reactions described at the bottom of the graphic. Negative responses are 984 \nshown in red. 985 \n 986 \n 987 \nFigure 4. Correlation between SDF strains belonging to family Paenibacillaceae and 988 \ngrowth conditions or enzymes activities. A Person correlation-based clustering method 989 \nwas employed to construct a heat map associating 18 SDF strains allocated in B. 990 \nsubtilis complex (right) and 30 phenotypical features (bottom) that contribute to AEFB 991 \nidentification and classification. The top dendrogram clustered the SDF strains into two 992 \nparts based on the prevalence of positive responses (orange) to 30 growth conditions 993 \nand enzyme reactions described at the bottom of the graphic. Negative responses are 994 \nshown in red. 995 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\nTable 1. Biochemical and physiological profiles analysed in this work and the respective controls \n*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). \nTest Control \nPositive  Negative \nGrowth condition \nCitrate utilization Bacillus cereus CCGB406 Paenibacillus macerans CCGB126 \nPropionate utilization Bacillus licheniformis CCGB407 Bacillus subtilis CCGB1249 \n7% NaCl Bacillus amyloliquefaciens CCGB452 Paenibacillus macerans CCGB126 \n10% NaCl Bacillus amyloliquefaciens CCGB452 Paenibacillus macerans CCGB126 \n0.001% lysozyme  Bacillus cereus CCGB406 Bacillus pumilus CCGB124 \n45 °C Geobacillus stearothermophilus CCGB412 ND* \n65 °C Geobacillus stearothermophilus CCGB412 Bacillus thuringiensis CCGB1163 \npH 5.7 Bacillus cereus CCGB406 Paenibacillus alvei CCGB414 \nAnaerobiosis Bacillus cereus CCG406 Bacillus megaterium CCGB408 \nEnzyme \nCatalase Bacillus cereus CCGB406 ND* \nOxidase Lysinibacillus sphaericus CCGB745 Bacillus cereus CCGB406 \nHemolysin Bacillus thuringiensis CCGB1163 Lysinibacillus sphaericus CCGB745 \nNitrate reductatase Bacillus cereus CCGB406 Bacillus megaterium CCGB408 \nHydrolysis \nCasein Bacillus megaterium CCGB408 Paenibacillus macerans CCGB126 \nGelatin Bacillus cereus CCGB406 Geobacillus stearothermophilus CCGB412 \nEsculin Bacillus subtilis CCGB1249 Lysinibacillus fusiformis CCGB743 \nStarch Bacillus cereus CCGB406 Lysinibacillus sphaericus CCGB745 \nAmino acid decomposition \nPhenylalanine degradation Bacillus megaterium CCGB408 Bacillus cereus CCGB406 \nTyrosine degradation Bacillus cereus CCGB406 L. sphaericus CCGB745 \nArginine dihydrolase Bacillus licheniformis CCGB407 Bacillus megaterium CCGB408 \nLysine decarboxylase Bacillus thuringiensis CCGB1163 Bacillus megaterium CCGB408 \nOrnithine decarboxylase Bacillus thuringiensis CCGB1163 Bacillus megaterium CCGB408 \nIndole production Paenibacillus alvei CCGB414 Bacillus cereus CCGB406 \nProduction of acid from \nD-Glucose Bacillus megaterium CCGB408 Lysinibacillus fusiformis CCGB743 \nL-Arabinose Bacillus megaterium CCGB408 Brevibacillus brevis CCGB052 \nLactose Bacillus megaterium CCGB408 Lysinibacillus fusiformis CCGB743 \nMannitol Bacillus megaterium CCGB408 Lysinibacillus fusiformis CCGB743 \nSucrose Bacillus amyloliquefaciens CCGB452 Lysinibacillus sphaericus CCGB745 \nD-Xylose Bacillus megaterium CCGB408 Brevibacillus brevis CCGB052 \nVoges-Proskauer test Bacillus cereus CCGB406 Bacillus megaterium CCGB408 \nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\nBacillus: 208 (87.39%)\nBrevibacillus: 7 (2.94%)\nLysinibacillus: 7 (2.94%)\nPaenibacillus: 14 (5.88%)\nRummeliibacillus: 1 (0.42%)\nTerribacillus: 1 (0.42%)\nGenera level (238  SDF strains)\nA\nBacillus toyonensis: 1 (0.45%)Bacillus tequilensis: 1 (0.45%)Bacillus arbutinivorans: 1 (0.45%)Bacillus australimaris: 1 (0.45%)Bacillus circulans: 1 (0.45%)\nBacillus kochii: 1 (0.45%)\nBacillus luciferensis: 1 (0.45%)\nBacillus oleronius: 1 (0.45%)\nBacillus senegalensis: 1 (0.45%)\nBacillus siamensis: 1 (0.45%)\nBacillus subterraneus: 2 (0.89%)\nBacillus velezensis: 3 (1.34%)\nBacillus clausii: 3 (1.34%)\nBacillus anthracis: 4 (1.79%)Bacillus amyloliquefaciens: 4 (1.79%)Bacillus subtilis: 4 (1.79%)\nBacillus altitudinis: 6 (2.68%)Bacillus simplex: 7 (3.12%)\nBacillus aryabhattai: 9 (4.02%)\nBacillus thuringiensis: 12 (5.36%)\nBacillus safensis: 16 (7.14%)\nBacillus megaterium: 26 (11.61%)\nBacillus cereus: 31 (13.84%)\nBacillus pumilus: 61 (27.23%)\nBrevibacillus agrii: 1 (0.45%)\nBrevibacillus brevis: 1 (0.45%)\nBrevibacillus laterosporus: 4 (1.79%)\nLysinibacillus fusiformis: 2 (0.89%)\nLysinibacillus xylanilyticus: 2 (0.89%)\nLysinibacillus sphaericus: 3 (1.34%)\nPaenibacillus chibensis: 1 (0.45%)\nPaenibacillus ginsengagri: 1 (0.45%)\nPaenibacillus lautus: 1 (0.45%)\nPaenibacillus susongensis: 1 (0.45%)Paenibacillus terrigena: 1 (0.45%)\nPaenibacillus alvei: 7 (3.12%)Terribacillus goriensis: 1 (0.45%)\nSpecies level (224 SDF strains)\nB\nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\nSDF strains\nBacillus cereus (98%) (SDF0219)\nBacillus cereus (99%) (SDF0124)\nBacillus anthracis (97%) (SDF0199)\nBacillus cereus (100%) (SDF0119)\nBacillus thuringiensis (98%) (SDF0235)\nBacillus cereus (99%) (SDF0061)\nBacillus thuringiensis (100%) (SDF0288)\nBacillus cereus (98%) (SDF0006)\nBacillus toyonensis (98%) (SDF0053)\nBacillus cereus (98%) (SDF0250)\nBacillus anthracis (100%) (SDF0089)\nBacillus cereus (97%) (SDF0206)\nBacillus cereus (99%) (SDF0299)\nBacillus cereus (99%) (SDF0300)\nBacillus cereus (99%) (SDF0304)\nBacillus anthracis (99%) (SDF0186)\nBacillus cereus (99%) (SDF0305)\nBacillus cereus (99%) (SDF0200)\nBacillus cereus (100%) (SDF0295)\nBacillus cereus (99%) (SDF0184)\nBacillus cereus (98%) (SDF0239)\nBacillus thuringiensis (98%) (SDF0178)\nBacillus thuringiensis (99%) (SDF0276)\nBacillus cereus (97%) (SDF0272)\nBacillus cereus (97%) (SDF0159)\nBacillus thuringiensis (100%) (SDF0161)\nBacillus thuringiensis (100%) (SDF0294)\nBacillus cereus (99%) (SDF0286)\nBacillus cereus (100%) (SDF0283)\nBacillus cereus (99%) (SDF0310)\nBacillus thuringiensis (100%) (SDF0225)\nBacillus cereus (100%) (SDF0155)\nBacillus cereus (98%) (SDF0194)\nBacillus cereus (100%) (SDF0248)\nBacillus thuringiensis (98%) (SDF0301)\nBacillus thuringiensis (97%) (SDF0303)\nBacillus cereus (98%) (SDF0229)\nBacillus cereus (97%) (SDF0278)\nBacillus thuringiensis (98%) (SDF0100)\nBacillus thuringiensis (98%) (SDF0085)\nBacillus cereus (98%) (SDF0232)\nBacillus cereus (98%) (SDF0032)\nBacillus cereus (97%) (SDF0237)\nBacillus cereus (98%) (SDF0182)\nBacillus anthracis (100%) (SDF0181)\nBacillus cereus (97%) (SDF0270)\nBacillus cereus (100%) (SDF0022)\nBacillus thuringiensis (99%) (SDF0030)\nCatalaseGelatinpH 5.7\nLysozyme\nCasein\nD−Glucose\n45 °C\nHemolysis\nPropionate utilization\nStarch\nNitrate Reduction\nTyrosine DegradationArginine Dihydrolase\nVoges−Proskauer\nSucroseOxidase\nAnaerobiosis\nNaCl 7%NaCl 10%\nLactoseD−Xylose\nPhenylalanine\nCitrate utilization\nL−Arabinose\nMannitol\nLysine Decarboxylase\nEsculin\nOrnithine Decarboxylase\n65 °C\nIndole Production\nResponse to biochemical and physiological tests\nPositive Negative\nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\nSDF strains\nBacillus pumilus (100%) (SDF0123)\nBacillus pumilus (100%) (SDF0034)\nBacillus pumilus (100%) (SDF0130)\nBacillus pumilus (99%) (SDF0115)\nBacillus pumilus (100%) (SDF0135)\nBacillus safensis (100%) (SDF0122)\nBacillus pumilus (99%) (SDF0141)\nBacillus subtilis (97%) (SDF0003)\nBacillus pumilus (99%) (SDF0132)\nBacillus pumilus (99%) (SDF0145)\nBacillus pumilus (100%) (SDF0125)\nBacillus pumilus (99%) (SDF0129)\nBacillus pumilus (99%) (SDF0149)\nBacillus pumilus (99%) (SDF0131)\nBacillus pumilus (99%) (SDF0137)\nBacillus safensis (100%) (SDF0134)\nBacillus pumilus (97%) (SDF0140)\nBacillus pumilus (98%) (SDF0202)\nBacillus pumilus (100%) (SDF0105)\nBacillus safensis (100%) (SDF0106)\nBacillus pumilus (100%) (SDF0060)\nBacillus pumilus (97%) (SDF0026)\nBacillus pumilus (99%) (SDF0046)\nBacillus pumilus (100%) (SDF0052)\nBacillus safensis (98%) (SDF0103)\nBacillus pumilus (99%) (SDF0044)\nBacillus pumilus (100%) (SDF0035)\nBacillus pumilus (99%) (SDF0004)\nBacillus safensis (99%) (SDF0013)\nBacillus pumilus (100%) (SDF0120)\nBacillus pumilus (99%) (SDF0136)\nBacillus safensis (98%) (SDF0098)\nBacillus pumilus (99%) (SDF0104)\nBacillus altitudinis (97%) (SDF0204)\nBacillus pumilus (99%) (SDF0207)\nBacillus amyloliquefaciens (100%) (SDF0110)\nBacillus pumilus (98%) (SDF0171)\nBacillus safensis (100%) (SDF0281)\nBacillus pumilus (99%) (SDF0292)\nBacillus pumilus (99%) (SDF0087)\nBacillus pumilus (99%) (SDF0148)\nBacillus safensis (100%) (SDF0093)\nBacillus safensis (100%) (SDF0027)\nBacillus pumilus (99%) (SDF0070)\nBacillus pumilus (98%) (SDF0071)\nBacillus pumilus (100%) (SDF0121)\nBacillus pumilus (100%) (SDF0126)\nBacillus pumilus (98%) (SDF0092)\nBacillus pumilus (100%) (SDF0113)\nBacillus safensis (98%) (SDF0091)\nBacillus safensis (98%) (SDF0043)\nBacillus pumilus (98%) (SDF0146)\nBacillus safensis (98%) (SDF0147)\nBacillus pumilus (99%) (SDF0177)\nBacillus pumilus (97%) (SDF0273)\nBacillus pumilus (99%) (SDF0152)\nBacillus safensis (99%) (SDF0151)\nBacillus pumilus (99%) (SDF0001)\nBacillus pumilus (98%) (SDF0079)\nBacillus pumilus (99%) (SDF0154)\nBacillus pumilus (100%) (SDF0011)\nBacillus altitudinis (99%) (SDF0153)\nBacillus pumilus (98%) (SDF0114)\nBacillus altitudinis (99%) (SDF0117)\nBacillus pumilus (100%) (SDF0112)\nBacillus pumilus (99%) (SDF0090)\nBacillus safensis (99%) (SDF0002)\nBacillus safensis (98%) (SDF0084)\nBacillus pumilus (99%) (SDF0208)\nBacillus pumilus (97%) (SDF0201)\nBacillus altitudinis (97%) (SDF0205)\nBacillus safensis (98%) (SDF0196)\nBacillus pumilus (100%) (SDF0198)\nBacillus pumilus (100%) (SDF0190)\nBacillus pumilus (98%) (SDF0173)\nBacillus amyloliquefaciens (98%) (SDF0302)\nBacillus subtilis (99%) (SDF0285)\nBacillus velezensis (99%) (SDF0280)\nBacillus pumilus (99%) (SDF0142)\nBacillus amyloliquefaciens (98%) (SDF0269)\nBacillus velezensis (99%) (SDF0287)\nBacillus tequilensis (99%) (SDF0282)\nBacillus altitudinis (99%) (SDF0036)\nBacillus amyloliquefaciens (99%) (SDF0279)\nBacillus velezensis (100%) (SDF0150)\nBacillus subtilis (97%) (SDF0025)\nBacillus altitudinis (99%) (SDF0017)\nBacillus pumilus (99%) (SDF0179)\nBacillus pumilus (100%) (SDF0156)\nBacillus pumilus (100%) (SDF0157)\nBacillus pumilus (97%) (SDF0175)\nBacillus pumilus (99%) (SDF0187)\nBacillus pumilus (99%) (SDF0230)\nBacillus pumilus (99%) (SDF0231)\nCatalase\npH 5.745 °C\nNaCl 7%Hemolysis\nGelatinSucrose\nD−Glucose\nVoges−Proskauer\nMannitolCasein\nL−Arabinose\nNaCl 10%\nEsculin\nCitrate utilization\nOrnithine Decarboxylase\nPropionate utilization\nLysozyme\nNitrate Reduction\nStarch\nArginine DihydrolaseLysine Decarboxylase\nOxidase\n65 °C\nLactoseD−Xylose\nAnaerobiosis\nIndole Production\nTyrosine Degradation\nPhenylalanine\nResponse to biochemical and physiological tests\nPositive Negative\nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638\n\nSDF strains\nPaenibacillus lautus (98%) (SDF0265)\nPaenibacillus alvei (99%) (SDF0164)\nPaenibacillus alvei (97%) (SDF0213)\nPaenibacillus alvei (99%) (SDF0080)\nPaenibacillus alvei (100%) (SDF0028)\nPaenibacillus alvei (99%) (SDF0023)\nPaenibacillus alvei (99%) (SDF0008)\nPaenibacillus alvei (99%) (SDF0133)\nPaenibacillus ginsengagri (100%) (SDF0227)\nBrevibacillus laterosporus (99%) (SDF0195)\nBrevibacillus agrii (99%) (SDF0188)\nBrevibacillus laterosporus (99%) (SDF0197)\nBrevibacillus laterosporus (100%) (SDF0020)\nPaenibacillus chibensis (98%) (SDF0259)\nBrevibacillus laterosporus (99%) (SDF0220)\nPaenibacillus susongensis (99%) (SDF0162)\nPaenibacillus terrigena (97%) (SDF0240)\nCatalaseSucrosepH 5.7\nLysozyme\nGelatin45 °C\nVoges−Proskauer\nD−Glucose\nIndole Production\nOxidase\nAnaerobiosis\nHemolysis\nCaseinNaCl 7%\nPropionate utilizationTyrosine Degradation\nMannitol\nNitrate Reduction\nStarchLactoseEsculin\nL−Arabinose\nD−Xylose\nLysine DecarboxylaseArginine Dihydrolase\nCitrate utilization\nOrnithine Decarboxylase\nPhenylalanine\nNaCl 10%\n65 °C\nResponse to biochemical and physiological tests\nPositive Negative\nAuthor-formatted, not peer-reviewed document posted on 18/05/2022. DOI:  https://doi.org/10.3897/arphapreprints.e86638","source_license":"CC-BY-4.0","license_restricted":false}