Assessment of Native Cadmium-Resistant Bacteria in Cacao (Theobroma Cacao L.) - Cultivated Soils 2 3 Henry A

bioRxiv preprint doi: https://doi.org/10.1101/2021.08.06.455168; this version posted August 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Assessment of native cadmium-resistant bacteria in cacao (Theobroma cacao L.) - cultivated soils 2 3 Henry A. Cordoba-Novoa1 §, Jeimmy Cáceres-Zambrano2, §, Esperanza Torres-Rojas2* 4 5 1Faculty of Agricultural and Environmental Sciences, McGill University, Montreal, QC, Canada. 2Faculty of 6 Agricultural Sciences, Universidad Nacional de Colombia, Bogotá D.C., Colombia. §These authors 7 contributed equally. 8 9 *Correspondence: 10 [email protected] 11 +57 3165000 ext. 19072 12 13 Abstract 14 Traces of cadmium (Cd) have been reported in some chocolate products due to soils with Cd and the high 15 ability of cacao plants to extract, transport, and accumulate it in their tissues. An agronomic strategy to 16 minimize the uptake of Cd by plants is the use of cadmium-resistant bacteria (Cd-RB). However, knowledge 17 about Cd-RB associated with cacao soils is scarce. This study was aimed to isolate and characterize Cd-RB 18 associated with cacao-cultivated soils in Colombia that may be used in the bioremediation of Cd-polluted 19 soils. Diversity of culturable Cd-RB, qualitative functional analysis related to nitrogen, phosphorous, carbon, 20 and Cd were performed. Thirty different Cd-RB morphotypes were isolated from soils with medium (NC, Y1, 21 Y2) and high (Y3) Cd concentrations using culture media with 6 mg Kg-1 Cd. Cd-RB were identified based on 22 morphological and molecular analyses. The most abundant morphotypes (90%) were gram-negative belong to 23 Phylum Proteobacteria and almost half of them showed the capacity to fix nitrogen, solubilize phosphates and 24 degrade cellulose. Unique morphotypes were isolated from Y3 soils where Burkholderia and Pseudomonas 25 were the dominant genera indicating their capacity to resist high Cd concentrations. P. putida GB78, P. 26 aeruginosa NB2, and Burkholderia sp. NB10 were the only morphotypes that grew on 18 up to 90 (GB78) 27 and 140 mg Kg-1 Cd (NB2-NB10); however, GB78 showed the highest Cd bioaccumulation (5.92 mg g-1). 28 This study provides novel information about culturable Cd-RB soil diversity with the potential to develop 29 biotechnology-based strategies. 30 31 Key Words: Culturable Bacterial Diversity, cellulose, phosphorous, nitrogen, soil remediation, 32 bioaccumulation. 33 34 Introduction 35 36 Cadmium (Cd) is the most prevalent toxic agent to microorganisms, plants, and human beings. It could be 37 present in the environment naturally as a product of volcanic eruptions, weathering of bedrock, and burning of 38 vegetation (Cullen and Maldonado 2013); or as a result of anthropogenic activities such as mining,

39 manufacturing of plastics, paint pigments, batteries, and others (Kirkham 2006). Cd enrichment in agricultural 40 soil is due to soil evolution (Gramlich et al. 2018) and rocks or mineral composition. Cd can be solubilized

41 from different types of minerals such as Greenockite (CdS), Otavite (CdCO3), Cadmoselite (CdSe), 42 Monteponite (CdO), and Metacinnabar Cd ([Hg, Cd]S) (Cullen and Maldonado 2013). The physical-chemical 43 properties (e.g. texture, pH, organic matter composition, effective cation exchange capacity, total metal 44 content), microbial community and climate conditions play also an important role (Amari et al. 2017). 45 Additionally, agricultural practices such as the application of phosphate-based fertilizers that could reach Cd 46 contents up to 130 mg Kg-1 (Siripornadulsil and Siripornadulsil 2013), crop irrigation with untreated 47 wastewater, and contaminated raw organic material recycling for the same crop (Maddela et al. 2020) may 48 increase Cd concentration and its mobility in soils. 49 The presence of Cd in plants is related to both the ability of the plant to extract, transport, and accumulate Cd 50 in its tissues; and the availability of Cd in soil (Taki 2013). Cd has been detected in vegetables such as lettuce, 51 swiss chard, spinach, fresh fruits, and some cereals (Quezada-Hinojosa et al. 2015). For cacao, which is 52 considered a traditional exportable crop with a global production of 4.7 million tons (in 2018/19), where 68% 53 comes from developing countries (ICCO, 2018; Maddela et al. 2020), Cd has also been reported (Mounicou et 54 al. 2003; Chavez et al. 2015; Gramlich et al. 2016, 2018; Arévalo-gardini et al. 2017). The European 55 Commission has regulated the maximum limits of permissible Cd for chocolate and other derived products 56 depending on the percentage of raw cacao present in the final product (Commission regulation (EU) No. 57 488/2014 2014) where the levels range from 0.1 – 0.8 mg Kg-1 Cd dry matter (Meter et al. 2019). 58 Concentrations of Cd in cacao-based chocolate and raw cacao beans above these critical levels have been 59 reported in some South and Centro American countries (Chavez et al. 2015; Bertoldi et al. 2016). This 60 situation represents a major concern because of the impact on food chain that compromises human health 61 (Clemens et al. 2013), food safety, and the cacao economy (Maddela et al. 2020). 62 63 It has been reported different agronomic strategies to reduce Cd uptake by plants that include the management 64 of pH, organic matter content, mineral nutrition, and replacement of contaminated fertilizers (Rai et al. 2019). 65 The reduction of Cd levels can also be achieved through the application of biological amendments. The use of 66 microorganisms with the ability to adsorb, bioaccumulate and biotransform Cd is an attractive approach to 67 carry out bioremediation processes of contaminated soils (Ashraf et al. 2017). Soil bacteria have several 68 resistance mechanisms to overcome toxic levels of Cd such as biosorption, intracellular sequestration, 69 extracellular binding, complexation, and removal by efflux systems. A reduction of Cd uptake by plants using 70 Cd resistant bacteria (Cd-RB) and Cd resistant plant growth-promoting rhizobacteria (PGPR) has been 71 reported in wheat and maize (Ahmad et al. 2014), rice (Treesubsuntorn et al. 2017), mustard (Sinha and 72 Mukherjee 2008), tomato (Madhaiyan et al. 2007), pea (Safronova et al. 2006), and soybean (Guo and Chi 73 2014). Among Cd-RB with the potential to reduce the availability of this heavy metal at the interchangeable 74 soil phase, Pseudomonas aeruginosa (Chakraborty and Das 2014), Pseudomonas putida (Li et al. 2014), 75 Serratia spp. (Sarma et al. 2016), Burkholderia spp. (Jin et al. 2013), Halomonas spp. (Asksonthong et al. 76 2016) and Enterobacter spp. (Chen et al. 2016) have been commonly reported. 2

77 78 In Colombia, where the spatial distribution and concentration of Cd in cacao-cultivated soils has been mainly 79 associated with a geogenic origin (Rodríguez Albarrcín et al. 2019; Bravo and Benavides-Erazo 2020), Cd- 80 RB belonging to genera Enterobacter sp., Burkholderia sp., Pseudomonas sp., and Methylobacterium sp. 81 from cacao-cultivated soils with Cd have been isolated (Bravo et al. 2018). However, the knowledge of the 82 taxonomic diversity, community structure, and functional activity of Cd-RB associated with cacao crop are 83 still scarce. The purpose of this study was to isolate and characterize culturable Cd-RB associated with cacao- 84 cultivated soils with Cd, to contribute to the knowledge of this bacterial community that may be used in 85 integral soil management programs. 86 87 Materials and methods

88 Study area and sampling 89 90 Soil samples were obtained from two municipalities of Cundinamarca, Colombia: Nilo (NC) 4°18´25´Ń - 91 74°37´12Ó, and Yacopí (Y) 5°27´35´Ń - 74°20´18Ó (Rodríguez Albarrcín et al. 2019). These regions have 92 an average temperature of 26.5 °C and 21°C with annual precipitation of 1292 and 1500 mm, respectively. 93 Three cacao-producing farms were sampled in Y and one in NC (Fig 1). In each sampling site, three 94 producing trees were randomly selected, and four soil sub-samples (0.5 Kg each) were collected at 0.3 m deep 95 with 0.5 m from the trunk base. Sub-samples were mixed to get a composite sample per farm. These samples 96 were used for the determination of physical and chemical properties, and taxonomic classification 97 (Supplemental Table 1; Table 1). 98 99 Soil cadmium determination 100 101 Composite samples were used to quantify the total Cd and potentially available Cd. Total Cd was determined

102 by digestion of 1 g of soil sample with 7 mL of HNO3 65% (v/v) and 1 mL of HCl 37% (v/v). Samples and 103 acids were mixed using a single reaction chamber UltraWAVE (ECR; Milestone, Shelton, CT), the 104 temperature rose from 20 °C to 220 °C in 20 min and was maintained for 10 min for a total digestion time of 105 30 min. After digestion, 10 mL of deionized water was added, and samples were filtered using 0.2 µm x 47 106 mm sterile Swinnex filters (Merck-Millipore, Darmstadt, Germany). Potentially available Cd was 107 quantitatively determined with DTPA extraction solution [0.005 M diethylenetriaminepentaacetic acid

108 (DTPA), 0.01 M calcium chloride dihydrate (CaCl2 2H2O) and 0.1 M triethanolamine (TEA)] in a 1:2 109 proportion (w/v). 110 111 Determinations were performed using an Agilent 700 series Inductively Coupled Plasma Optical Emission 112 Spectroscopy (ICP-OES) spectrometer (Agilent Technologies, Inc., CA, USA) with five milliliters of the 113 digested sample and a running time of 90 s per sample. The ICP Expert II software was used to analyze raw

114 ICP data. A standard curve was created using CdCl2 aliquots (Sigma-Aldrich Corp., 99% purity w/w, CA) in 115 eight concentrations ranging from 0 to 500 mg L-1; Supplemental Fig. 1). Additionally, aliquots of the 116 reference material WEPAL-ISE-997 (Sandy soil with 0.400 mg Kg-1 of Cd2+, obtained from the Wageningen 117 evaluating program for analytical laboratories, Wageningen Agricultural University, the Netherlands) were 118 included (Bravo et al. 2018). 119 120 In Colombia, neither reference values for soil Cd are available for agricultural production. In non-polluted 121 soils, Cd concentrations range from 0.01 to 1.1 mg Kg-1 with an average of 0.41 mg Kg-1 (Kabata-Pendias and 122 Pendias 2011). According to this, in this study Cd levels in the four locations were categorized as medium 123 (>1.1 to 5.0 mg Kg-1) and high (>5.0 mg Kg-1; Table 1). 124 125 126 Culturable Cd-RB isolation, morphological characterization, and molecular identification 127 128 Cd-RB were isolated by plating out serial dilutions of soil as described previously (Avellaneda-Torres et al. 129 2020) with slight modifications. 50 g of sifted soil (0.2 mm) were suspended in 450 mL of NaCl 0.85%, 130 mixed for 10 min and, 1 mL of the dilution was transferred to a new assay tube with 9 mL of NaCl and mixed 131 with vortex for 1 min. The process was made up to 10-7 dilution and 100 µl of the dilutions 10-3, 10-5 and, 10-7 -1 132 were plated out on Mergeay agar (Mergeay 1995) pH 7 with 6 mg Kg of Cd from CdCl2 and incubating at 133 37 °C for 3 days. Following incubation, the colony-forming unit (CFU) was calculated per gram of dry soil as 134 an indirect measure to determine total abundance by location (Bressan et al. 2015). Morphotypes with -1 135 differential morphological characteristics were subcultured in Mergeay agar with 6 mg Kg of CdCl2 and 136 were preserved in glycerol stocks at - 70 °C. 137 138 Micro and macro morphological characterization were performed to each isolated assessing color using the 139 Pantone scale, shape, elevation, surface appearance, and colony consistency. Also, Gram-stain and endospore 140 stain with malachite green dye in contrast with safranin were performed. Molecular identification was made 141 by extracting DNA according to Chen y Kuo (Chen and Kuo 1993) and partial amplification of the 16S rRNA 142 gene with the universal primers 27F and 1492R as previously described (Avellaneda-Torres et al. 2020). All 143 sequences were submitted to the GenBank (NCBI). 144 145 Analysis of structural diversity and phylogeny of culturable Cd-RB 146 147 Cd-RB structural diversity was assessed by (i) richness and abundance; richness was considered as the 148 number of present genera per sample and the abundance as the number of isolated morphotypes for each 149 location (NC, Y1, Y2, Y3) (Lyngwi et al. 2013; dos Passos et al. 2014) and (2) phylogenetic analysis of the 150 16 rRNA genes from isolates. The phylogenetic tree was inferred using the Muscle multiple alignment 151 method and Neighbor-joining algorithm (Saitou and Nei 1987). The evolutionary distances were computed 4

152 using the p-distance method and nodes are supported in the bootstrap method using 1000 pseudoreplicates 153 (Felsenstein 1985). Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). 154 155 Functional analysis of isolated Cd-BR morphotypes associated with C, P, and N 156 157 The cellulolytic potential of isolated morphotypes was assessed in LB agar with carboxymethyl cellulose 158 CMC, as the unique carbon source, and staining with Congo red (Avellaneda-Torres et al. 2014). Phosphate 159 solubilization activity was assessed in SMRS agar (Paul and Sundara 1971), and the solubilization factor (SF) 160 was calculated using bromocresol purple. Hydrolysis and solubilization factors establish the ratio of 161 degradation or solubilization diameter and colony growth diameter (Paul and Rao 1971). Finally, atmospheric 162 nitrogen fixation activity was observed on Rennie agar (Rennie 1981) with bromothymol blue as an indicator, 163 through growth and turning color of this medium that lacks N. Three replicates per morphotype were used for 164 each functional group.

165 Effect of Cd on bacterial growth, Cd bioaccumulation, and Minimum Inhibitory Concentration 166 167 Morphotypes previously isolated on Mergeay agar with 6 mg Kg-1 of Cd were inoculated in LB agar with 12 168 and 18 mg Kg-1 of Cd. Bacterial growth curves were analyzed for bacteria resistant to 18 mg Kg-1 of Cd. LB 169 broth with (18 mg Kg-1) and without Cd was inoculated with 0.1% (v/v) of bacterial cultures of 18 h (Ivanova 170 et al. 2002; Cristani et al. 2012). Bacteria were incubated at 37 °C with shaking at 200 rpm. With three 171 replicates for each morphotype, optic density at 600 nm (OD600) was determined every 2 h during 36 h using 172 a NanoDropTM ONE spectrophotometer (ThermoFisher Scientific, USA). 173 174 Cd bioaccumulation test was made twice at different moments. The bacteria were inoculated into LB broth 175 containing 18 mg Kg-1 of Cd and incubated at 37 °C with shaking (200 rpm) with three replicates per bacteria. 176 The bacterial cells were harvested at 18 h after inoculation by centrifugation at 5000 rpm for 15 min and then 177 washed with sterile deionized water to remove free heavy metal ions. The supernatants were sterilized by 178 filtration with a 0.22 µm membrane filter (MILLIEX®GP Millipore Express®) and cell pellets were dried at 179 100 °C until constant weight. Media without adding Cd or inoculating bacteria were used as controls. The 180 concentration of Cd in supernatants was measured by Atomic Absorption Spectrophotometry (AAS; Perkin 181 Elmer AAnalyst 300, USA) and the metal bioaccumulation of bacteria was calculated as follows (Micheletti 182 et al. 2008): 183 ̽͝ Ǝ ͚̽ ͥƔƬ ư͐ ͡ 184

185 where q (mg g-1) is the Cd bioaccumulation of bacterial cells, Ci is the initial concentration of heavy metal 186 used (mg L-1), Cf is the final concentration of heavy metal (mg L-1), m is the dried weight of cell pellet (g) and 187 V is the volume of the liquid medium (0.05 L in the experiments). 188 189 The bacteria resistant to 18 mg Kg-1 of Cd were grown in 15 mL conical bottom tubes with 7 mL of LB broth 190 supplemented with 18, 24, 30, 40 up to 200 mg Kg-1 of Cd in intervals of 10 mg Kg-1 of Cd. 20 µl of bacterial 191 culture of 18 h were inoculated and incubated at 37 °C with constant agitation at 200 rpm for 18 h. Following 192 incubation, turbidity was observed. The minimum Cd concentration at which bacterial isolate did not show 193 growth was considered as its Minimum inhibitory concentration (MIC). 194 Data analysis 195 196 Data from hydrolysis and solubilization factors and Cd bioaccumulation were analyzed using a completely 197 randomized design (CRD) with a one-way analysis of variance (ANOVA) in SAS v9.4 statistical software 198 (SAS Institute Inc., USA). Shapiro-Wilk normality test and Bartlett homogeneity of variance tests were made. 199 Media were compared using Tukey’s test (p<0.05). 200 Results 201 Culturable of Cd-RB isolation, morphological characterization, and molecular identification 202 203 A total of 30 Cd-RB morphotypes were isolated from the different soil samples in culture media with 6 mg 204 kg-1 Cd. One isolate of each morphotype was morphologically characterized and identified. The number of 205 CFU of Cd-RB was less than 106 CFU g-1 dry soil in all locations without significant differences 206 (Supplemental Fig. 2). The micro and macroscopic characterization are shown in Supplemental Table 2, and 207 the Gram analysis showed that 90% of the isolated (n=27) corresponded to Gram-negative bacteria and the 208 remaining 10% (n=3) to Gram-positive (Table 2). Based on the BLAST search results of the 16S rRNA gene, 209 it was identified eight genera: Burkholderia, Pseudomonas, Enterobacter, Serratia, Bacillus, Halomonas, 210 Herbaspirillum, and Rhodococcus (Supplemental Table 3). 211 212 Analysis of structural diversity and phylogeny of culturable Cd-RB 213 214 It also was found a greater abundance, unique morphotypes, and dominance of isolated Cd-RB belonging to 215 the genera Burkholderia and Pseudomonas in soil with the highest natural concentration of Cd (location Y3; 216 Fig. 2). Additionally, Y1 and Y2 shown the greatest similarity according to the isolated morphotypes (Fig. 2). 217 218 The phylogenetic analysis showed that 90% (n=27) of the isolated bacteria belong to the phylum 219 Proteobacteria, 6.7% to Firmicutes (n=2), and 3.3% to Actinobacteria (n=1) (Fig. 3; Supplemental Table 3). 220 In the phylogenetic tree, Rhodococcus sp. GB17, Bacillus flexus YB22, and Bacillus sp. GB18 were used as 221 an outgroup due to their relationship with the other isolated morphotypes. Within the phylum proteobacteria, 222 two classes stand out, Gamma and Beta Proteobacteria. Gammaproteobacteria were represented by the genera 6

223 Serratia (6 morphotypes), Enterobacter (4 morphotypes), Pseudomonas (7 morphotypes), and Halomonas (1 224 morphotype) belonging to the families Enterobacteriaceae (Enterobacteriales), Pseudomonadaceae 225 (Pseudomonadales), and Halomonadaceae (Oceanospirillales), respectively. Beta Proteobacteria had the 226 genera Burkholderia, which presented the highest number of morphotypes found (8), and Herbaspirillum (1); 227 belonging to the family Burkholderiaceae and Oxalobacteraceae, respectively, and both to order 228 Burkholderiales. The genera Bacillus (2 morphotypes) and Rhodococcus (1) were the least represented, 229 belonging to the Bacillaceae (Bacillales) and Nocardiaceae (Actinomycetales) class, respectively. 230 231 Functional analysis of isolated morphotypes associated with C, P, and N 232 233 Some isolated bacteria presented functional activities related to C, P, and N in addition to their Cd resistance. 234 The 46.7% (n=14) of the isolated morphotypes showed the ability to degrade cellulose, highlighting strains 235 such as Enterobacter sp. YB11, Pseudomonas sp. GB86, Herbaspirillun sp. GB90 and Burkholderia sp. 236 GB68 with hydrolysis factors above 2.5 (Table 2). Regarding P, a higher percentage of morphotypes were 237 able to solubilize phosphates (53%, n=16) compared with other functional groups analyzed. Bacteria from 238 genera Enterobacter, Burkholderia, and Serratia showed high solubilization factors ranging from 1.56 to 4.39 239 (Table 2). Finally, atmospheric nitrogen fixation ability was observed in half of the isolated morphotypes. 240 241 Effect of Cd on bacterial growth, Cd bioaccumulation, and Minimum Inhibitory Concentration 242 243 Among the 30 isolated morphotypes only Pseudomonas sp. GB73, Burkholderia sp. NB10, P. aeruginosa 244 NB2, P. putida GB78 grew on media with 18 mg Kg-1 Cd, and the last three were considered for further 245 analysis. The effect of Cd on the growth curve varied among the assessed strains. In presence of 18 mg Kg-1,

246 P. aeruginosa NB2 showed an average reduction of more than one unit of OD600 during the log phase from 14 247 h after inoculation compared to control without Cd (Fig. 4A). In contrast, P. putida GB78 did not show any

248 difference in OD600 with and without Cd during all growth phases (Fig. 4B). Burkholderia sp. NB10 had a

249 slight reduction of OD600 with Cd from 2 to 20 h post-inoculation, however, from 20 h the OD600 of bacteria 250 grown with Cd reached similar magnitudes to culture without Cd. 251 252 Regarding Cd bioaccumulation (q), P. putida GB78 accumulated 5.92 mg g-1, which was significantly higher 253 than P. aeruginosa NB2 and Burkholderia sp. NB10 accumulation (around 1 mg g-1; Fig. 5). Finally, it was 254 found that Burkholderia sp. NB10 and P. aeruginosa NB2 showed a MIC of 140 mg Kg-1, which is 1.5 times 255 higher than the MIC recorded for P. putida GB78 (90 mg Kg-1). 256 257 Discussion 258 Culturable of Cd-RB isolation, morphological characterization, and molecular identification 259

260 The Log CFU g-1 of dry soil is an indirect measure of the abundance of microorganisms present in soil and for 261 agricultural soils, values between 108 – 1010 cells per gram have been reported (Marteinsson et al. 2015). In 262 this study, the bacterial abundances for all studied locations were considered low (below 106), which could be 263 attributed to the presence of Cd as a selection factor in the isolation culture media with 6 mg Kg-1 Cd (Torsvik 264 et al. 2002; Itävaara et al. 2016). Although it has been found that heavy metals affect adversely the 265 composition of the microbial community (Etesami 2018), in this study, it was selected cacao-cultivated soil 266 and culture media with a high concentration of Cd to isolated bacteria, due to these conditions would allow a 267 better selection of morphotypes with an enhanced capacity to survive under Cd stress condition in vitro. 268 269 The morphological characterization showed that 90% of the isolated morphotypes corresponded to Gram- 270 negative bacteria. Gram-negative and Gram-positive bacteria respond differently to the presence of heavy 271 metals (Bruins et al. 2000; Limcharoensuk et al. 2015). Bruins et al. (Bruins et al. 2000) assert that some 272 Gram-negative bacteria (genus Pseudomonas) can tolerate 5 to 30 times more Cd than Gram-positive bacteria 273 (Staphylococcus aureus, S. faecium, Bacillus subtilis). In this regard, Wu et al. (Wu et al. 2009) reported that 274 Azotobacter chroococum, a Gram-negative bacteria, had a greater capacity for binding to Cd than Bacillus 275 megaterium, a Gram-positive one. This differential response can be explained due to the composition of the 276 cell wall and the resistance mechanisms used. 277 278 Gram-positive bacteria have a thick layer of peptidoglycan (20-80 nm) in which they fix acids, proteins, and 279 polysaccharides. This layer forms a barrier to avoid the entry of heavy metals into the cell, prevailing passive 280 resistance mechanisms such as adsorption (Bruins et al. 2000; Mounaouer et al. 2014). In contrast, Gram- 281 negative bacteria have thinner walls (5-10 nm) that allow the influx and efflux of heavy elements and force 282 the bacteria to develop active mechanisms or flow systems and to have a higher protein synthesis in presence 283 of Cd (Bruins et al. 2000). Nevertheless, both types of bacteria can present passive and active resistance 284 mechanisms to Cd and other heavy metals at the same time (Sharma and Archana 2016; Etesami 2018). This 285 indicates that resistance depends also on the other factors such as type of strain, type, and concentration of the 286 heavy metals, physicochemical properties of soil, and environmental conditions (Sharma and Archana 2016). 287 In this regard, Lima et al. (Lima e Silva et al. 2012) observed a greater presence of Gram-positive Cr resistant 288 bacteria, while Hg and Ag resistant bacteria were mostly Gram-negative. This demonstrates that the pollutant 289 also determines the type of bacteria that survive in contaminated environments. 290 291 Burkholderia, Pseudomonas, Enterobacter, and Serratia were the most frequent isolated genera. These genera 292 have been reported in the management of cultivated soils. Strains of Burkholderia spp. have the potential to 293 promote plant growth, biocontrol pathogens, and biodegrade toxic molecules (Castanheira et al. 2016). 294 Enterobacter spp. can improve bioremediation processes of soils contaminated with heavy metals (Qiu et al. 295 2014; Marteinsson et al. 2015) and within the genus Pseudomonas, growth-promoting strains have also been 296 reported with the ability to mitigate stress in plants caused by the presence of heavy metals such as Cd and Pb, 297 and metals such as Zn that are toxic at high levels (Lin et al. 2016; Rojjanateeranaj et al. 2017). Serratia sp. 8

298 has been reported as a bacteria that can confer tolerance to heavy metal stress in cereals (Ahmad et al. 2014). 299 Additionally, genera found in less frequency such as Halomonas and Herbaspirillum, have been involved in 300 bioremediation processes of Cr and Sr in symbiosis with algae, and in free life (Achal et al. 2012; Murugavelh 301 and Mohanty 2012; Gupta and Diwan 2017), with the potential to fix nitrogen, and chelate heavy metals such 302 As, Pb, and Cu (Govarthanan et al. 2014; Li et al. 2018). The bacteria isolated in this study, constitute a 303 potential resource of native microorganisms to manage Cd in cacao cultivated soils. However, further studies 304 to characterize and evaluate the performance of bacteria in the field and their interaction with plants, are 305 needed. 306 307 Analysis of structural diversity and phylogeny of culturable Cd-RB 308 309 The structure of microbial communities can be affected by the presence of heavy elements. In this study, 310 results showed that there was not a relationship between the richness of isolated morphotypes and the level of 311 Cd present in the soil. However, the presence of unique morphotypes and the dominance of Burkholderia and 312 Pseudomonas genera (Fig. 2), show that the presence of Cd in soils may alter the uniformity of the 313 community of Cd-RB present in the studied soil. It has been reported that more sensitive microorganisms to 314 heavy metals are suppressed by the toxic element and replaced by more resistant with adaptive characteristics, 315 occupying their ecological niche (Azarbad et al. 2015). The presence of unique morphotypes isolated from Y3 316 soils agrees with the results reported by Tipayno et al. (Tipayno et al. 2018) who studied sites with different 317 levels of Cd and other heavy metals (As and Pb) and found that the site with the highest concentration of Cd 318 also presented the highest number of unique morphotypes. 319 320 Most of the isolated morphotypes (90%) in this study corresponded to the Proteobacteria phylum. This 321 phylum represents the largest and most diverse group of prokaryotes and contemplates the vast majority of 322 Gram-negative bacteria studied to date (Spain et al. 2009). Also, it is usually reported as the most frequent in 323 studies related to heavy metals, so strains belonging to several genera have been isolated, characterized, and 324 reported by their resistance (Xiao et al. 2019). Additionally, Chen et al. (Chen et al. 2018) highlight that the 325 Proteobacteria phylum together with Bacteroidetes and Firmicutes harbor the largest set of heavy metal 326 resistance genes. 327 328 Functional analysis of isolated Cd-RB morphotypes associated with C, P, and N 329 330 Cellulolytic potential, free nitrogen fixation, and phosphate solubilization were determined in all isolated 331 morphotypes using selective media to promote an integrated crop cacao management. The use of these 332 bacteria could enhance nutrient cycling, decrease the use of chemical fertilizers, and increase the 333 environmental sustainability of the crop. In agroforestry production systems such as cacao, some interactions 334 and processes favor nutrient cycling, as a result of the decomposition of organic matter from crop residues 335 and pruning such as litter in the crop, stems, among others (Aikpokpodion 2010). The decomposition of 9

336 cellulose increases the levels of organic matter, and carbon can be used as a source of energy for edaphic 337 processes and plant physiology (Gupta et al. 2012). Organic matter is a fraction to which heavy metals such as 338 Cd can bind and be adsorbed by chelates or other substances. Therefore, when levels of organic matter 339 increase in soil, the bioavailability of Cd for plants can be reduced (Gadd 2000). 340 341 The solubilization of phosphates and the promotion of plant growth have been reported for the genus 342 Enterobacter isolated from soil (Beltrán 2014). The main cellular mechanism through which bacteria 343 solubilize phosphates is the production of organic acids such as gluconic acid, 2-ketogluconic acid, lactic 344 acid, isovaleric acid, isobutyric acid, and acetic acid (Beltrán 2014). These molecules have a negative charge 345 and therefore can form complexes with metal cations in solution and be involved in the chelation of Cd (Yang 346 et al. 2018). The bacteria involved in the atmospheric nitrogen fixation (ANF-B) have also played an 347 important role in the remediation of Cd polluted soils (Chen et al. 2020). Ivishina et al. (Ivshina et al. 2014) 348 showed that ANF-B can secrete extracellular polymeric substances (EPS) and various organic acids and 349 amino acids that may alter the Cd availability in the soil by adsorbing and trapping metals due to the presence 350 of many anionic functional groups. 351 352 The management of Cd-polluted agricultural soils is a complex issue where the Cd-RB could have an 353 important function not only to decrease the Cd availability but also to improve soil fertility. The presence of 354 activity associated with the cycling of C, P, and N in Cd-RB is an advantage within holistic crop soil 355 management strategies. In this study, 11 from 30 isolated morphotypes showed the ability to degrade 356 cellulose, solubilize phosphate and, fix atmospheric nitrogen, simultaneously. It would be valuable additional 357 analyses that quantify the activity of involved enzymes and determine the effect of bacteria on plant growth 358 and physiology. 359 360 Effect of Cd on bacterial growth, Cd bioaccumulation, and Minimum Inhibitory Concentration 361 362 The effect of Cd on bacterial growth varies depending on bacterial strain. Heavy metals affect bacterial 363 growth by causing multiple metabolic alterations such as DNA damage, decreasing protein synthesis, 364 disruption of respiration and cell division, and amino acid biosynthesis (Chen et al. 2016). These effects 365 explain the growth decrease of P. aeruginosa NB2 at 18 mg Kg-1 of Cd along with the growth phases (Fig. 366 4A). In Burkholderia sp. NB10, a reduction of the growth was also detected in the lag and log phases (Fig. 367 4C); however, the cells were able to adapt and resume growth at the stationary phase. It can be explained by 368 the repair of cadmium-mediated cellular damage and adjustment of the cell physiology to limit the 369 distribution of toxic ions in the cell (Mohamed and Abo-Amer, 2006). 370 371 Despite Cd effects, bacteria have different Cd resistance strategies dependent and independent on intracellular 372 accumulation (Ashraf et al. 2017; Etesami 2018). Mechanisms of resistance include metal efflux systems, 373 transportation, precipitation, transformation, and sequestration by metallothionein proteins and thiol 10

374 compounds such as glutathione (Nies 2003). The results showed that resistance mechanisms of P. putida 375 GB78 are efficient enough to resist 18 mg Kg-1 of Cd without affecting its growth. These results can also be 376 explained based on the difference between the efficiency of resistance mechanisms of strains. Chen et al. 377 (Chen et al. 2014) suggest that bacteria can activate resistance mechanisms such as metal efflux bombs at 378 particular moments which results in a depletion of intracellular Cd content. 379 380 The evaluation of Cd bioaccumulation (q) is a measure of the potential of microorganisms to be used in 381 bioremediation processes and management of toxic concentrations of heavy metal in agricultural soils 382 (Cánovas et al. 2003). A higher q detected in P. putida GB78 suggests that this strain favors intracellular 383 accumulation processes (Fig. 5). In contrast, Burkholderia sp. NB10 and P. aeruginosa NB2 showed a lower 384 q, which may be attributed to greater efficiency in extracellular Cd outflow mechanisms. 385 386 The MIC is an indication of the resistance capacity that a microorganism has to grow in the presence of Cd 387 and highlights its potential for applications at different heavy metal concentrations in soils (Xu et al. 2012). 388 Results showed a higher resistance in Burkholderia sp. NB10 and P. aeruginosa NB2 (140 mg Kg-1) 389 compared to P. putida GB78 (90 mg Kg-1). Different Pseudomonas and Burkholderia species have been 390 widely reported for their resistance to Cd, other heavy metals such as Cu and Pb, and high concentrations of 391 Zn (Naik and Dubey 2011; Jin et al. 2013). It has been reported MICs from 100 to 190 mg Kg-1 for P. putida 392 (Lee et al. 2001; Leedjärv et al. 2008), up 900 mg Kg-1 for P. aeruginosa (Ghaima et al. 2017), and 200 mg 393 Kg-1 for Burkholderia sp. (Jin et al. 2013). These values show the wide variation that exists in the level of 394 resistance in bacteria. Overall, the bacterial growth and the MIC results suggest that 18 mg Kg-1 of Cd is not a 395 limiting concentration for P. putida GB78. However, it can resist up to 90 mg Kg-1 of Cd by favoring 396 intracellular bioaccumulation. On the contrary, Burkholderia sp. NB10 and P. aeruginosa NB2, with a MIC 397 of 140 mg Kg-1 of Cd, showed growth rate reductions that may be an acclimatization strategy to maintain 398 viability for a longer time and activate resistance mechanisms such as efflux systems (lower q). 399 400 Conclusions 401 Cacao-cultivated soils harbor a diversity of bacteria that exhibit resistance to Cd and functional activity 402 associated with the cycling of C, P, and N. The structural diversity of culturable Cd-RB bacterial community 403 is affected by the presence of Cd in soil samples where high levels of Cd enhance the selection of unique and 404 dominant morphotypes such as Burkholderia spp. and Pseudomonas spp. These genera can resist up to 90 to 405 140 Kg-1 of Cd showing their potential to use in soil bioremediation. In this study, multifunctional Cd-RB 406 morphotypes were identified with the potential to develop integrative soil management of Cd-polluted soils. 407 408 409 Supplementary Information 410 The online version contains supplementary material available at 411 11

412 Acknowledgements The authors thank the cacao farmers for letting them to collect samples. The authors 413 thank the funding organization and the Universidad Nacional de Colombia for supporting the research group. 414 415 Authors’ contributions ETR, HCN, and JCZ conceived the project and designed the experiments. ETR, 416 HCN and JCZ collected the samples. HCN and JCZ performed the experiments and analyzed the data. ETR 417 supervised the research and analyzed the data. All authors wrote, and read and approved the final manuscript. 418 419 Funding This research was supported by the Cundinamarca-Colombia government (Contract No. 2 420 Framework Derivative Agreement of Agro-industrial Technological Corridor (No. 395 - 2012), and 421 Universidad Nacional de Colombia internal grant for research projects. This work was conducted under 422 Ministerio de Ambiente, Vivienda y Desarrollo Territorial (MAVDT) collection permit 0255, March 14th, 423 2014. 424 425 Data availability The datasets used and/or analyzed during the current study are available from the 426 corresponding author on reasonable request. The 16S rRNA sequencing are available at NCBI according to 427 GenBank accession numbers. 428 429 Declarations 430 431 Ethics approval and consent to participate This work was conducted under Ministerio de Ambiente, 432 Vivienda y Desarrollo Territorial (MAVDT) collection permit 0255, March 14th, 2014. Not applicable for 433 animal or human data or tissue. 434 435 Consent for publication: Not applicable. 436 437 Competing interest: The authors declare that they have no competing interests. 438 439 References 440 Achal V, Pan X, Zhang D (2012) Bioremediation of strontium (Sr) contaminated aquifer quartz sand based on 441 carbonate precipitation induced by Sr resistant Halomonas sp. Chemosphere 89:764–768. 442 https://doi.org/http://dx.doi.org/10.1016/j.chemosphere.2012.06.064 443 Ahmad I, Akhtar MJ, Zahir ZA, et al (2014) Cadmium-tolerant bacteria induce metal stress tolerance in 444 cereals. Environ Sci Pollut Res 21:11054–11065. https://doi.org/10.1007/s11356-014-3010-9 445 Aikpokpodion PE (2010) Nutrients dynamics in cocoa Soils, leaf and beans in Ondo State, Nigeria. J Agric 446 Sci 1:1–9. https://doi.org/10.1080/09766898.2010.11884647 447 Amari T, Ghnaya T, Abdelly C (2017) Nickel, cadmium and lead phytotoxicity and potential of halophytic 448 plants in heavy metal extraction. South African J Bot 111:99–110. 449 https://doi.org/10.1016/j.sajb.2017.03.011 450 Arévalo-gardini E, Arévalo-hernández CO, Baligar VC, He ZL (2017) Heavy metal accumulation in leaves 451 and beans of cacao (Theobroma cacao L.) in major cacao growing regions in Peru. Sci Total Environ

452 605–606:792–800. https://doi.org/10.1016/j.scitotenv.2017.06.122 453 Ashraf MA, Hussain I, Rasheed R, et al (2017) Advances in microbe-assisted reclamation of heavy metal 454 contaminated soils over the last decade: A review. J Environ Manage 198:132–143. 455 https://doi.org/10.1016/j.jenvman.2017.04.060 456 Asksonthong R, Siripongvutikorn S, Usawakesmanee W (2016) Evaluation of harmful heavy metal (Hg, Pb 457 and Cd) reduction using Halomonas elongata and Tetragenococcus halophilus for protein hydrolysate 458 product. Funct Foods Heal Dis 6:195–205. https://doi.org/10.31989/ffhd.v6i4.240 459 Avellaneda-Torres LM, Guevara CP, Torres E (2014) Assessment of cellulolytic microorganisms in soils of 460 Nevados Park, Colombia. Brazilian J Microbiol 45:1211–1220. https://doi.org/10.1590/s1517- 461 83822014000400011 462 Avellaneda-Torres LM, Sicard TL, Castro EG, Rojas ET (2020) Potato cultivation and livestock effects on 463 microorganism functional groups in soils from the neotropical high andean Páramo. Rev Bras Cienc do 464 Solo 44: e0190122. https://doi.org/10.36783/18069657rbcs20190122 465 Azarbad H, Niklińska M, Laskowski R, et al (2015) Microbial community composition and functions are 466 resilient to metal pollution along two forest soil gradients. FEMS Microbiol Ecol 91:1–11. 467 https://doi.org/10.1093/femsec/fiu003 468 Beltrán E (2014) Phosphate solubilization as a microbial strategy for promoting plant growth. Corpoica Cienc 469 Tecnol Agropecu 15:101–113. https://doi.org/https://doi.org/10.21930/rcta.vol15_num1_art:401 470 Bertoldi D, Barbero A, Camin F, et al (2016) Multielemental fingerprinting and geographic traceability of 471 Theobroma cacao beans and cocoa products. Food Control 65:46–53. 472 https://doi.org/10.1016/j.foodcont.2016.01.013 473 Bravo D, Benavides-Erazo J (2020) The use of a two-dimensional electrical resistivity tomography (2D-ERT) 474 as a technique for cadmium determination in Cacao crop soils. Appl Sci 10:. 475 https://doi.org/10.3390/APP10124149 476 Bravo D, Pardo-Díaz S, Benavides-Erazo J, et al (2018) Cadmium and cadmium-tolerant soil bacteria in 477 cacao crops from northeastern Colombia. J Appl Microbiol 124:1175–1194. 478 https://doi.org/10.1111/jam.13698 479 Bressan M, Trinsoutrot Gattin I, Desaire S, et al (2015) A rapid flow cytometry method to assess bacterial 480 abundance in agricultural soil. Appl Soil Ecol 88:60–68. https://doi.org/10.1016/j.apsoil.2014.12.007 481 Bruins MR, Kapil S, Oehme FW (2000) Microbial resistance to metals in the environment. Ecotoxicol 482 Environ Saf 45:198–207. https://doi.org/10.1006/eesa.1999.1860 483 Cánovas D, Durán C, Rodríguez N, et al (2003) Testing the limits of biological tolerance to arsenic in a 484 fungus isolated from the River Tinto. Environ Microbiol 5:133–138. https://doi.org/10.1046/j.1462- 485 2920.2003.00386.x 486 Castanheira N, Dourado AC, Kruz S, et al (2016) Plant growth-promoting Burkholderia species isolated from 487 annual ryegrass in Portuguese soils. J Appl Microbiol 120:724–739. https://doi.org/10.1111/jam.13025 488 Chakraborty J, Das S (2014) Characterization and cadmium-resistant gene expression of biofilm-forming 489 marine bacterium Pseudomonas aeruginosa JP-11. Environ Sci Pollut Res 21:14188–14201. 490 https://doi.org/10.1007/s11356-014-3308-7 491 Chavez E, He ZL, Stoffella PJ, et al (2015) Concentration of cadmium in cacao beans and its relationship with 492 soil cadmium in southern Ecuador. Sci Total Environ 533:205–214. 493 https://doi.org/10.1016/j.scitotenv.2015.06.106 494 Chen M, Li Y, Zhang L, et al (2014) Analysis of Gene Expression Provides Insights into the Mechanism of 495 Cadmium Tolerance in Acidithiobacillus ferrooxidans. Curr Microbiol. https://doi.org/10.1007/s00284- 496 014-0710-9 497 Chen W, Kuo T (1993) A simple and rapid method for the preparation of gram-negative bacterial genomic 498 DNA. Nucleic Acids Res 21:2260–2260. https://doi.org/10.1093/nar/21.9.2260 499 Chen Y, Chao Y, Li Y, et al (2016) Survival strategies of the plant-associated bacterium Enterobacter sp.

500 strain EG16 under cadmium stress. Appl Environ Microbiol 82:1734–1744. 501 https://doi.org/10.1128/AEM.03689-15 502 Chen Y, Chen FF, Xie M Di, et al (2020) The impact of stabilizing amendments on the microbial community 503 and metabolism in cadmium-contaminated paddy soils. Chem Eng J 395:125132. 504 https://doi.org/10.1016/j.cej.2020.125132 505 Chen Y, Jiang Y, Huang H, et al (2018) Long-term and high-concentration heavy-metal contamination 506 strongly influences the microbiome and functional genes in Yellow River sediments. Sci Total Environ 507 637–638:1400–1412. https://doi.org/10.1016/j.scitotenv.2018.05.109 508 Clemens S, Aarts MGM, Thomine S, Verbruggen N (2013) Plant science: The key to preventing slow 509 cadmium poisoning. Trends Plant Sci 18:92–99. https://doi.org/10.1016/j.tplants.2012.08.003 510 Commision regulation (EU) No. 488/2014 . (2014) Statement on maximum levels of cadmium in foodstuffs, 511 Amending Regulation (EC) No. 1881/2006. Off J Eur Union 138:75–79. 512 https://doi.org/10.2903/j.efsa.2011.1975 513 Cristani M, Naccari C, Nostro A, et al (2012) Possible use of Serratia marcescens in toxic metal biosorption 514 (removal). Environ Sci Pollut Res 19:161–168. https://doi.org/10.1007/s11356-011-0539-8 515 Cullen J, Maldonado T (2013) Biogeochemistry of Cadmium and Its Release to the Environment. In: Sigel A, 516 Sigel H, Sigel R (eds) Cadmium: From Toxicity to Essentiality. Volume II. Springer, New York, pp 31– 517 62 518 dos Passos J, da Costa PB, Costa MD, et al (2014) Cultivable bacteria isolated from apple trees cultivated 519 under different crop systems: diversity and antagonistic activity against Colletotrichum gloeosporioides. 520 Genet Mol Biol 37:560–572. https://doi.org/10.1590/S1415-47572014000400013 521 Etesami H (2018) Bacterial mediated alleviation of heavy metal stress and decreased accumulation of metals 522 in plant tissues: Mechanisms and future prospects. Ecotoxicol Environ Saf 147:175–191. 523 https://doi.org/10.1016/j.ecoenv.2017.08.032 524 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Soc Study Evol 525 Stable 39:783–791. https://doi.org/10.2307/2408678 526 Gadd GM (2000) Bioremedial potential of microbial mechanisms of metal mobilization and immobilization. 527 Curr Opin Biotechnol 11:271–279. https://doi.org/10.1016/S0958-1669(00)00095-1 528 Ghaima KK, Mohamed AI, Meshhdany WY, Abdulhassan AA (2017) Resistance and bioadsorption of 529 Cadmium by Pseudomonas aeruginosa isolated from agricultural soil. Int J Appl Environ Sci 12:1649– 530 1660 531 Govarthanan M, Lee G, Park J, et al (2014) Bioleaching characteristics, influencing factors of Cu 532 solubilization and survival of Herbaspirillum sp. GW103 in Cu contaminated mine soil. Chemosphere 533 109:42–48. https://doi.org/10.1016/j.chemosphere.2014.02.054 534 Gramlich A, Tandy S, Andres C, et al (2016) Cadmium uptake by cocoa trees in agroforestry and 535 monoculture systems under conventional and organic management. Sci Total Environ 580:677–686. 536 https://doi.org/10.1016/j.scitotenv.2016.12.014 537 Gramlich A, Tandy S, Gauggel C, et al (2018) Soil cadmium uptake by cocoa in Honduras. Sci Total Environ 538 612:370–378. https://doi.org/10.1016/j.scitotenv.2017.08.145 539 Guo J, Chi J (2014) Effect of Cd-tolerant plant growth-promoting rhizobium on plant growth and Cd uptake 540 by Lolium multiflorum Lam. and Glycine max (L.) Merr. in Cd-contaminated soil. Plant Soil 375:205– 541 214. https://doi.org/10.1007/s11104-013-1952-1 542 Gupta P, Diwan B (2017) Bacterial Exopolysaccharide mediated heavy metal removal: A Review on 543 biosynthesis, mechanism and remediation strategies. Biotechnol Reports 13:58–71. 544 https://doi.org/10.1016/j.btre.2016.12.006 545 Gupta P, Samant K, Sahu A (2012) Isolation of cellulose-degrading bacteria and determination of their 546 cellulolytic potential. Int J Microbiol 2012: https://doi.org/10.1155/2012/578925 547 International cocoa organization (2018) Quarterly Bulletin of Cocoa Statistics.

548 https://www.icco.org/statistics/. Accessed 27 August 2020 549 Itävaara M, Salavirta H, Marjamaa K, Ruskeeniemi T (2016) Chapter one: geomicrobiology and 550 metagenomics of terrestrial deep subsurface microbiomes. In: Advances in applied microbiology. pp. 551 Volume 94. Academic Press, pp 3–33. https://doi.org/10.1016/bs.aambs.2015.12.001 552 Ivanova EP, Kurilenko V V., Kurilenko A V., et al (2002) Tolerance to cadmium of free-living and associated 553 with marine animals and eelgrass marine gamma-proteobacteria. Curr Microbiol 44:357–362. 554 https://doi.org/10.1007/s00284-001-0017-5 555 Ivshina IB, Kostina L V., Kamenskikh TN, et al (2014) Soil microbiocenosis as an indicator of stability of 556 meadow communities in the environment polluted with heavy metals. Russ J Ecol 45:83–89. 557 https://doi.org/10.1134/S1067413614020039 558 Jin ZM, Sha W, Zhang YF, et al (2013) Isolation of Burkholderia cepacia JB12 from lead- and cadmium- 559 contaminated soil and its potential in promoting phytoremediation with tall fescue and red clover. Can J 560 Microbiol 59:449–455. https://doi.org/10.1139/cjm-2012-0650 561 Kabata-Pendias A, Pendias H (2011) Trace Elements in Soils and Plants, 4th Editio. CRC Press, Boca Raton. 562 Kirkham MB (2006) Cadmium in plants on polluted soils: Effects of soil factors, hyperaccumulation, and 563 amendments. Geoderma 137:19–32. https://doi.org/10.1016/j.geoderma.2006.08.024 564 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for 565 Bigger Datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054 566 Lee S, Glickmann E, Cooksey DA (2001) Chromosomal locus for cadmium resistance in Pseudomonas 567 putida consisting of a cadmium-transporting ATPase and a MerR family response regulator. 568 Microbiology 67:1437–1444. https://doi.org/10.1128/AEM.67.4.1437 569 Leedjärv A, Ivask A, Virta M (2008) Interplay of different transporters in the mediation of divalent heavy 570 metal resistance in Pseudomonas putida KT2440. J Bacteriol 190:2680–2689. 571 https://doi.org/10.1128/JB.01494-07 572 Li X, Xu H, Gao B, et al (2018) Efficient biosorption of Pb (II) from aqueous solutions by a PAH-degrading 573 strain Herbaspirillum chlorophenolicum FA1. J Ind Eng Chem 57:64–71. 574 https://doi.org/10.1016/j.jiec.2017.08.008 575 Li Y, Wu Y, Wang Q, et al (2014) Biosorption of copper, manganese, cadmium, and zinc by Pseudomonas 576 putida isolated from contaminated sediments. Desalin Water Treat 52:7218–7224. 577 https://doi.org/10.1080/19443994.2013.823567 578 Lima e Silva A, Ribeiro de Carvalho M, De Souza S, et al (2012) Heavy metal tolerance (Cr, Ag and Hg) in 579 bacteria isolated from sewage. Brazilian J Microbiol 43(4):1620–1631. https://doi.org/10.1590/S1517- 580 83822012000400047 581 Limcharoensuk T, Sooksawat N, Sumarnrote A, et al (2015) Bioaccumulation and biosorption of Cd2+ and 582 Zn2+ by bacteria isolated from a zinc mine in Thailand. Ecotoxicol Environ Saf 122:322–330. 583 https://doi.org/10.1016/j.ecoenv.2015.08.013 584 Lin X, Mou R, Cao Z, et al (2016) Characterization of cadmium-resistant bacteria and their potential for 585 reducing accumulation of cadmium in rice grains. Sci Total Environ 569–570:97–104. 586 https://doi.org/10.1016/j.scitotenv.2016.06.121 587 Lyngwi NA, Koijam K, Sharma D, Joshi SR (2013) Cultivable bacterial diversity along the altitudinal 588 zonation and vegetation range of tropical Eastern Himalaya. Int J Trop Biol Conserv 61:467–490. 589 https://doi.org/10.15517/RBT.V61I1.11141 590 Maddela NR, Kakarla D, García LC, et al (2020) Cocoa-laden cadmium threatens human health and cacao 591 economy: A critical view. Sci Total Environ 720: https://doi.org/10.1016/j.scitotenv.2020.137645 592 Madhaiyan M, Poonguzhali S, Sa T (2007) Metal tolerating methylotrophic bacteria reduces nickel and 593 cadmium toxicity and promotes plant growth of tomato (Lycopersicon esculentum L.). Chemosphere 594 69:220–228. https://doi.org/10.1016/j.chemosphere.2007.04.017 595 Marteinsson V, Klonowski A, Reynisson E, et al (2015) Microbial colonization in diverse surface soil types in

596 Surtsey and diversity analysis of its subsurface microbiota. Biogeosciences 12:1191–1203. 597 https://doi.org/10.5194/bg-12-1191-2015 598 Mergeay M (1995) Heavy metal resistance in microbial ecosystems. Mol Microb Ecol Man 439–455. 599 https://doi.org/10.1007/978-94-011-0351-0 600 Meter A, Atkinson RJ, Laliberte B (2019) Cadmium in Cacao from Latin America and the Caribbean. A 601 Review of Research and Potential Mitigation Solutions. Rome (Italy): Biodiversity International 73 p 602 Micheletti E, Colica G, Viti C, et al (2008) Selectivity in the heavy metal removal by exopolysaccharide- 603 producing cyanobacteria. J Appl Microbiol 105:88–94. https://doi.org/10.1111/j.1365- 604 2672.2008.03728.x 605 Mounaouer B, Nesrine A, Abdennaceur H (2014) Identification and characterization of heavy metal-resistant 606 bacteria selected from different polluted sources. Desalin Water Treat 52:7037–7052. 607 https://doi.org/10.1080/19443994.2013.823565 608 Mounicou S, Szpunar J, Andrey D, et al (2003) Concentrations and bioavailability of cadmium and lead in 609 cocoa powder and related products. Food Addit Contam 20:343–52. 610 https://doi.org/10.1080/0265203031000077888 611 Murugavelh S, Mohanty K (2012) Bioreduction of hexavalent chromium by free cells and cell free extracts of 612 Halomonas sp. Chem Eng J J 203:415–422. https://doi.org/10.1016/j.cej.2012.07.069 613 Naik MM, Dubey SK (2011) Lead-enhanced siderophore production and alteration in cell morphology in a 614 Pb-resistant Pseudomonas aeruginosa strain 4EA. Curr Microbiol 62:409–414. 615 https://doi.org/10.1007/s00284-010-9722-2 616 Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27:313–339. 617 https://doi.org/10.1016/S0168-6445(03)00048-2 618 Paul NB, Sundara WVB (1971) Phosphate-dissolving bacteria in the rhizosphere of some cultivated legumes. 619 Plant Soil 35:127–132 620 Qiu Z, Tan H, Zhou S, Cao L (2014) Enhanced phytoremediation of toxic metals by inoculating endophytic 621 Enterobacter sp. CBSB1 expressing bifunctional glutathione synthase. J Hazard Mater 267:17–20. 622 https://doi.org/10.1016/j.jhazmat.2013.12.043 623 Quezada-Hinojosa R, Föllmi KB, Gillet F, Matera V (2015) Cadmium accumulation in six common plant 624 species associated with soils containing high geogenic cadmium concentrations at Le Gurnigel, Swiss 625 Jura Mountains. Catena 124:85–96. https://doi.org/10.1016/j.catena.2014.09.007 626 Rai PK, Lee SS, Zhang M, et al (2019) Heavy metals in food crops: Health risks, fate, mechanisms, and 627 management. Environ Int 125:365–385. https://doi.org/10.1016/j.envint.2019.01.067 628 Rennie RJ (1981) A single medium for the isolation of acetylene-reducing (dinitrogen-fixing) bacteria from 629 soils. Can J Microbiol 27:8–14. https://doi.org/10.1139/m81-002 630 Rodríguez Albarrcín HS, Darghan Contreras AE, Henao MC (2019) Spatial regression modeling of soils with 631 high cadmium content in a cocoa producing area of Central Colombia. Geoderma Reg 15: e00214. 632 https://doi.org/10.1016/j.geodrs.2019.e00214 633 Rojjanateeranaj P, Sangthong C, Prapagdee B (2017) Enhanced cadmium phytoremediation of Glycine max L 634 . through bioaugmentation of cadmium-resistant bacteria assisted by biostimulation. Chemosphere 635 185:764–771. https://doi.org/10.1016/j.chemosphere.2017.07.074 636 Safronova VI, Stepanok V V., Engqvist GL, et al (2006) Root-associated bacteria containing 1- 637 aminocyclopropane-1-carboxylate deaminase improve growth and nutrient uptake by pea genotypes 638 cultivated in cadmium supplemented soil. Biol Fertil Soils 42:267–272. https://doi.org/10.1007/s00374- 639 005-0024-y 640 Saitou N, Nei M (1987) The Neighbor-joining Method: A New Method for Reconstructing Phylogenetic 641 Trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 642 Sarma B, Acharya C, Joshi SR (2016) Characterization of metal tolerant Serratia spp. isolates from sediments 643 of uranium ore deposit of domiasiat in Northeast India. Proc Natl Acad Sci India Sect B - Biol Sci

644 86:253–260. https://doi.org/10.1007/s40011-013-0236-0 645 Sharma RK, Archana G (2016) Cadmium minimization in food crops by cadmium resistant plant growth 646 promoting rhizobacteria. Appl Soil Ecol 107:66–78. https://doi.org/10.1016/j.apsoil.2016.05.009 647 Sinha S, Mukherjee SK (2008) Cadmium-induced siderophore production by a high Cd-resistant bacterial 648 strain relieved Cd toxicity in plants through root colonization. Curr Microbiol 56:55–60. 649 https://doi.org/10.1007/s00284-007-9038-z 650 Siripornadulsil S, Siripornadulsil W (2013) Cadmium-tolerant bacteria reduce the uptake of cadmium in rice: 651 Potential for microbial bioremediation. Ecotoxicol Environ Saf 94:94–103. 652 https://doi.org/10.1016/j.ecoenv.2013.05.002 653 Spain AM, Krumholz LR, Elshahed MS (2009) Abundance, composition, diversity and novelty of soil 654 Proteobacteria. ISME J 3:992–1000. https://doi.org/10.1038/ismej.2009.43 655 Taki M (2013) Imaging and Sensing of Cadmium in Cells. In: Sigel A, Sigel H, Sigel R (eds) Cadmium: 656 From Toxicity to Essentiality. Springer, Dordrecht, pp 99–114 657 Tipayno SC, Truu J, Samaddar S, et al (2018) The bacterial community structure and functional profile in the 658 heavy metal contaminated paddy soils, surrounding a nonferrous smelter in South Korea. Ecol Evol 659 8:6157–6168. https://doi.org/10.1002/ece3.4170 660 Torsvik V, Øvreås L, Thingstad TF (2002) Prokaryotic diversity — magnitude, dynamics, and controlling 661 factors. Science (80- ) 296:1064–6. https://doi.org/10.1126/science.1071698 662 Treesubsuntorn C, Dhurakit P, Khaksar G, Thiravetyan P (2017) Effect of microorganisms on reducing 663 cadmium uptake and toxicity in rice (Oryza sativa L.). Environ Sci Pollut Res 1–12. 664 https://doi.org/10.1007/s11356-017-9058-6 665 Wu SC, Peng XL, Cheung KC, et al (2009) Adsorption kinetics of Pb and Cd by two plant growth promoting 666 rhizobacteria. Bioresour Technol 100:4559–4563. https://doi.org/10.1016/j.biortech.2009.04.037 667 Xiao S, Zhang Q, Chen X, et al (2019) Speciation distribution of heavy metals in uranium mining impacted 668 soils and impact on bacterial community revealed by high-throughput sequencing. Front Microbiol 669 10:1–15. https://doi.org/10.3389/fmicb.2019.01867 670 Xu X, Xia L, Huang Q, et al (2012) Biosorption of cadmium by a metal-resistant filamentous fungus isolated 671 from chicken manure compost. Environ Technol (United Kingdom) 33:1661–1670. 672 https://doi.org/10.1080/09593330.2011.641591 673 Yang P, Zhou XF, Wang LL, et al (2018) Effect of phosphate-solubilizing bacteria on the mobility of 674 insoluble cadmium and metabolic analysis. Int J Environ Res Public Health 15:1–12. 675 https://doi.org/10.3390/ijerph15071330 676 677 678 679 680 Fig. 1 Location of sampling sites. Each dot represents a cacao farm where previously soil cadmium levels 681 have been documented 682 683 Fig. 2 Venn diagram of the culturable Cd-RB morphotypes isolated from locations studied. Each gray circle 684 represents the location, referred to with a label. Black circles represent the isolated morphotypes, the different 685 morphotypes belonging to the same genera are represented by the red contour circle. The morphotypes were 686 isolated from cacao cultivated soils with medium (>1.0 to 5.0 mg Kg-1; CN, Y1 and Y2) and high (>5.0 mg 687 Kg-1; Y3) total Cd levels 688

689 Fig. 3 Phylogenetic tree of strains isolated in Mergeay agar with 6 mg kg-1 of Cd from cacao culture soil. The 690 evolutionary history was inferred using the neighbor-joining method. The bootstrap test was inferred with 691 1000 replicates and the analysis was performed with 36 sequences. Positions containing gaps and missing 692 data were eliminated. The evolutionary distances were computed with the p-distance method. Evolutionary 693 analyses were performed in MEGA 7. Sequences indicated with black squares were used as a template 694 695 Fig. 4 Growth of A) P. aeruginosa NB2, B) P. putida GB78 and C) Burkholderia sp. NB10 in the presence 696 and absence of 18 mg Kg-18 of Cd. The bars represent the standard error (n=6) 697 698 Fig. 5 Bioaccumulation of Cd in bacteria at 18 h of growth on media with 18 mg Kg-1 of Cd. The bars 699 represent the typical error (n=3). Means with different letters indicate significant differences by Tukey’s test 700 (p<0.05) 701 702 Table 1 Location, soil taxonomy, and total and potentially available Cd concentrations in sampling sites in 703 Nilo (NC) and Yacopí (Y). Medium (>1.0 to 5.0 mg Kg-1) and high (>5.0 mg Kg-1) total Cd level 704 705 Table 2 Cellulose hydrolysis and phosphate solubilization factors, and free nitrogen-fixing activity of Cd-RB. 706 Letters in the same columns represent significant differences (p<0.05) between strains according to Tukey’s 707 multiple comparison test. + and – indicate the presence or absence of growth/fixation on Rennie agar at 3 days 708 post-inoculation (dpi) 709 710 711

712 713 714 715 716 717 718 719 720 721 722 723 724 725

726 727 728 729 730 731

732 733 734

735 22

736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753

Total Cd Available Cd Location Natural region Soil Taxonomy Cd level (mg Kg-1) (mg Kg-1) Dry Tropical NC Dystric Eutrudepts Medium 1.68 1.28 Forest Y1 Typic Udorthents Medium 1.74 0.20 Tropical Y2 Premontane Typic Udorthents Medium 4.24 2.12 Moist Forest Y3 Typic Hapludands High 27.21 21.18 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784

Cellulose P Growth at N No. ID NCBI ID Loc. Morphotype hydrolysis solubilization fixation factor factor 12 mg Kg-1 18 mg Kg-1

1 NB2 MN719036.1 NC P. aeruginosa + + - - -

2 NB10 MN719037.1 NC Burkholderia sp. + + 2.04 ± 0.28 abcd 3.31 ± 0.55 abc +

3 NB40 MN719038.1 NC Halomonas sp. + - - - -

4 NB58 MN719039.1 NC Enterobacter sp. + - - - -

5 NB59 MN719040.1 NC Serratia sp. + - - - -

6 NB61 MN719041.1 NC Serratia sp. + - - < 1.00 -

7 NB80 MN719042.1 NC Serratia sp. + - 1.92 ± 0.23 bcd < 1.00 -

8 YB11 MN719043.1 Y1 Enterobacter sp. + - 3.25 ± 0.28 a 3.83 ± 0.48 ab +

9 YB30 MN719044.1 Y1 S. marcescens + - - 4.39 ± 0.49 a +

10 YB42 MN719045.1 Y1 S. marcescens + - - - -

11 YB43 MN719046.1 Y1 Serratia sp. + - 0.99 ± 0.01 d < 1.00 +

12 YB5 MN719047.1 Y2 Enterobacter sp. + - 1.53 ± 0.30 cd 1.56 ± 0.21 c +

13 YB13 MN719048.1 Y2 Pseudomonas sp. + - - - -

14 YB22 MN719049.1 Y2 Bacillus flexus - - - - -

15 YB48 MN719050.1 Y2 Enterobacter sp. + - 2.00 ± 0.08 bcd 2.02 ± 0.18 bc +

16 GB13 MN719051.1 Y3 Burkholderia sp. + - - < 1.00 +

17 GB16 MN719051.1 Y3 Burkholderia sp. + - 1.04 ± 0.13 d < 1.00 +

18 GB17 MN719053.1 Y3 Rhodococcus sp. + - 1.01 ± 0.04 d < 1.00 +

19 GB18 MN719054.1 Y3 Bacillus sp. + - - - -

20 GB58 MN719055.1 Y3 Burkholderia sp. + - 1.07 ± 0.11 d - -

21 GB66 MN719056.1 Y3 Burkholderia sp. + - 1.20 ± 0.02 d < 1.00 +

22 GB67 MN719057.1 Y3 Burkholderia sp. + - - - -

23 GB68 MN719058.1 Y3 Burkholderia sp. + - 2.73 ± 0.58 abc 3.46 ± 0.46 abc +

24 GB71 MN719059.1 Y3 Pseudomonas sp. + - - < 1.00 +

25 GB73 MN719060.1 Y3 Pseudomonas sp. + + - - -

26 GB78 MN719061.1 Y3 P. putida + + 1.18 ± 0.01 d < 1.00 +

27 GB82 MN719062.1 Y3 Burkholderia sp. + - - - -

28 GB86 MN719063.1 Y3 Pseudomonas sp. + - 3.12 ± 0.43 ab < 1.00 +

29 GB88 MN719064.1 Y3 Pseudomonas sp. + - - - -

Herbaspirillum 30 GB90 MN719065.1 Y3 + - 2.77 ± 0.21 abc - + sp. 785 786