Assessment of native cadmium-resistant bacteria in cacao ...Aug 06, 2021 · 1 Assessment of native...
Transcript of Assessment of native cadmium-resistant bacteria in cacao ...Aug 06, 2021 · 1 Assessment of native...
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Assessment of native cadmium-resistant bacteria in cacao (Theobroma cacao L.) - cultivated soils 1 2
Henry A. Cordoba-Novoa1 §, Jeimmy Cáceres-Zambrano2, §, Esperanza Torres-Rojas2* 3 4
1Faculty of Agricultural and Environmental Sciences, McGill University, Montreal, QC, Canada. 2Faculty of 5
Agricultural Sciences, Universidad Nacional de Colombia, Bogotá D.C., Colombia. §These authors 6
contributed equally. 7
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*Correspondence: 9
+57 3165000 ext. 19072 11
12 Abstract 13 Traces of cadmium (Cd) have been reported in some chocolate products due to soils with Cd and the high 14
ability of cacao plants to extract, transport, and accumulate it in their tissues. An agronomic strategy to 15
minimize the uptake of Cd by plants is the use of cadmium-resistant bacteria (Cd-RB). However, knowledge 16
about Cd-RB associated with cacao soils is scarce. This study was aimed to isolate and characterize Cd-RB 17
associated with cacao-cultivated soils in Colombia that may be used in the bioremediation of Cd-polluted 18
soils. Diversity of culturable Cd-RB, qualitative functional analysis related to nitrogen, phosphorous, carbon, 19
and Cd were performed. Thirty different Cd-RB morphotypes were isolated from soils with medium (NC, Y1, 20
Y2) and high (Y3) Cd concentrations using culture media with 6 mg Kg-1 Cd. Cd-RB were identified based on 21
morphological and molecular analyses. The most abundant morphotypes (90%) were gram-negative belong to 22
Phylum Proteobacteria and almost half of them showed the capacity to fix nitrogen, solubilize phosphates and 23
degrade cellulose. Unique morphotypes were isolated from Y3 soils where Burkholderia and Pseudomonas 24
were the dominant genera indicating their capacity to resist high Cd concentrations. P. putida GB78, P. 25
aeruginosa NB2, and Burkholderia sp. NB10 were the only morphotypes that grew on 18 up to 90 (GB78) 26
and 140 mg Kg-1 Cd (NB2-NB10); however, GB78 showed the highest Cd bioaccumulation (5.92 mg g-1). 27
This study provides novel information about culturable Cd-RB soil diversity with the potential to develop 28
biotechnology-based strategies. 29
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Key Words: Culturable Bacterial Diversity, cellulose, phosphorous, nitrogen, soil remediation, 31
bioaccumulation. 32
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Introduction 34
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Cadmium (Cd) is the most prevalent toxic agent to microorganisms, plants, and human beings. It could be 36
present in the environment naturally as a product of volcanic eruptions, weathering of bedrock, and burning of 37
vegetation (Cullen and Maldonado 2013); or as a result of anthropogenic activities such as mining, 38
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manufacturing of plastics, paint pigments, batteries, and others (Kirkham 2006). Cd enrichment in agricultural 39
soil is due to soil evolution (Gramlich et al. 2018) and rocks or mineral composition. Cd can be solubilized 40
from different types of minerals such as Greenockite (CdS), Otavite (CdCO3), Cadmoselite (CdSe), 41
Monteponite (CdO), and Metacinnabar Cd ([Hg, Cd]S) (Cullen and Maldonado 2013). The physical-chemical 42
properties (e.g. texture, pH, organic matter composition, effective cation exchange capacity, total metal 43
content), microbial community and climate conditions play also an important role (Amari et al. 2017). 44
Additionally, agricultural practices such as the application of phosphate-based fertilizers that could reach Cd 45
contents up to 130 mg Kg-1 (Siripornadulsil and Siripornadulsil 2013), crop irrigation with untreated 46
wastewater, and contaminated raw organic material recycling for the same crop (Maddela et al. 2020) may 47
increase Cd concentration and its mobility in soils. 48
The presence of Cd in plants is related to both the ability of the plant to extract, transport, and accumulate Cd 49
in its tissues; and the availability of Cd in soil (Taki 2013). Cd has been detected in vegetables such as lettuce, 50
swiss chard, spinach, fresh fruits, and some cereals (Quezada-Hinojosa et al. 2015). For cacao, which is 51
considered a traditional exportable crop with a global production of 4.7 million tons (in 2018/19), where 68% 52
comes from developing countries (ICCO, 2018; Maddela et al. 2020), Cd has also been reported (Mounicou et 53
al. 2003; Chavez et al. 2015; Gramlich et al. 2016, 2018; Arévalo-gardini et al. 2017). The European 54
Commission has regulated the maximum limits of permissible Cd for chocolate and other derived products 55
depending on the percentage of raw cacao present in the final product (Commission regulation (EU) No. 56
488/2014 2014) where the levels range from 0.1 – 0.8 mg Kg-1 Cd dry matter (Meter et al. 2019). 57
Concentrations of Cd in cacao-based chocolate and raw cacao beans above these critical levels have been 58
reported in some South and Centro American countries (Chavez et al. 2015; Bertoldi et al. 2016). This 59
situation represents a major concern because of the impact on food chain that compromises human health 60
(Clemens et al. 2013), food safety, and the cacao economy (Maddela et al. 2020). 61
62
It has been reported different agronomic strategies to reduce Cd uptake by plants that include the management 63
of pH, organic matter content, mineral nutrition, and replacement of contaminated fertilizers (Rai et al. 2019). 64
The reduction of Cd levels can also be achieved through the application of biological amendments. The use of 65
microorganisms with the ability to adsorb, bioaccumulate and biotransform Cd is an attractive approach to 66
carry out bioremediation processes of contaminated soils (Ashraf et al. 2017). Soil bacteria have several 67
resistance mechanisms to overcome toxic levels of Cd such as biosorption, intracellular sequestration, 68
extracellular binding, complexation, and removal by efflux systems. A reduction of Cd uptake by plants using 69
Cd resistant bacteria (Cd-RB) and Cd resistant plant growth-promoting rhizobacteria (PGPR) has been 70
reported in wheat and maize (Ahmad et al. 2014), rice (Treesubsuntorn et al. 2017), mustard (Sinha and 71
Mukherjee 2008), tomato (Madhaiyan et al. 2007), pea (Safronova et al. 2006), and soybean (Guo and Chi 72
2014). Among Cd-RB with the potential to reduce the availability of this heavy metal at the interchangeable 73
soil phase, Pseudomonas aeruginosa (Chakraborty and Das 2014), Pseudomonas putida (Li et al. 2014), 74
Serratia spp. (Sarma et al. 2016), Burkholderia spp. (Jin et al. 2013), Halomonas spp. (Asksonthong et al. 75
2016) and Enterobacter spp. (Chen et al. 2016) have been commonly reported. 76
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In Colombia, where the spatial distribution and concentration of Cd in cacao-cultivated soils has been mainly 78
associated with a geogenic origin (Rodríguez Albarrcín et al. 2019; Bravo and Benavides-Erazo 2020), Cd-79
RB belonging to genera Enterobacter sp., Burkholderia sp., Pseudomonas sp., and Methylobacterium sp. 80
from cacao-cultivated soils with Cd have been isolated (Bravo et al. 2018). However, the knowledge of the 81
taxonomic diversity, community structure, and functional activity of Cd-RB associated with cacao crop are 82
still scarce. The purpose of this study was to isolate and characterize culturable Cd-RB associated with cacao-83
cultivated soils with Cd, to contribute to the knowledge of this bacterial community that may be used in 84
integral soil management programs. 85
86
Materials and methods 87
Study area and sampling 88
89
Soil samples were obtained from two municipalities of Cundinamarca, Colombia: Nilo (NC) 4°18´25´´N - 90
74°37´12´O, and Yacopí (Y) 5°27´35´´N - 74°20´18´O (Rodríguez Albarrcín et al. 2019). These regions have 91
an average temperature of 26.5 °C and 21°C with annual precipitation of 1292 and 1500 mm, respectively. 92
Three cacao-producing farms were sampled in Y and one in NC (Fig 1). In each sampling site, three 93
producing trees were randomly selected, and four soil sub-samples (0.5 Kg each) were collected at 0.3 m deep 94
with 0.5 m from the trunk base. Sub-samples were mixed to get a composite sample per farm. These samples 95
were used for the determination of physical and chemical properties, and taxonomic classification 96
(Supplemental Table 1; Table 1). 97
98
Soil cadmium determination 99
100
Composite samples were used to quantify the total Cd and potentially available Cd. Total Cd was determined 101
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 102
acids were mixed using a single reaction chamber UltraWAVE (ECR; Milestone, Shelton, CT), the 103
temperature rose from 20 °C to 220 °C in 20 min and was maintained for 10 min for a total digestion time of 104
30 min. After digestion, 10 mL of deionized water was added, and samples were filtered using 0.2 µm x 47 105
mm sterile Swinnex filters (Merck-Millipore, Darmstadt, Germany). Potentially available Cd was 106
quantitatively determined with DTPA extraction solution [0.005 M diethylenetriaminepentaacetic acid 107
(DTPA), 0.01 M calcium chloride dihydrate (CaCl2 2H2O) and 0.1 M triethanolamine (TEA)] in a 1:2 108
proportion (w/v). 109
110
Determinations were performed using an Agilent 700 series Inductively Coupled Plasma Optical Emission 111
Spectroscopy (ICP-OES) spectrometer (Agilent Technologies, Inc., CA, USA) with five milliliters of the 112
digested sample and a running time of 90 s per sample. The ICP Expert II software was used to analyze raw 113
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ICP data. A standard curve was created using CdCl2 aliquots (Sigma-Aldrich Corp., 99% purity w/w, CA) in 114
eight concentrations ranging from 0 to 500 mg L-1; Supplemental Fig. 1). Additionally, aliquots of the 115
reference material WEPAL-ISE-997 (Sandy soil with 0.400 mg Kg-1 of Cd2+, obtained from the Wageningen 116
evaluating program for analytical laboratories, Wageningen Agricultural University, the Netherlands) were 117
included (Bravo et al. 2018). 118
119
In Colombia, neither reference values for soil Cd are available for agricultural production. In non-polluted 120
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 121
Pendias 2011). According to this, in this study Cd levels in the four locations were categorized as medium 122
(>1.1 to 5.0 mg Kg-1) and high (>5.0 mg Kg-1; Table 1). 123
124
125
Culturable Cd-RB isolation, morphological characterization, and molecular identification 126 127
Cd-RB were isolated by plating out serial dilutions of soil as described previously (Avellaneda-Torres et al. 128
2020) with slight modifications. 50 g of sifted soil (0.2 mm) were suspended in 450 mL of NaCl 0.85%, 129
mixed for 10 min and, 1 mL of the dilution was transferred to a new assay tube with 9 mL of NaCl and mixed 130
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, 10-7 131
were plated out on Mergeay agar (Mergeay 1995) pH 7 with 6 mg Kg-1 of Cd from CdCl2 and incubating at 132
37 °C for 3 days. Following incubation, the colony-forming unit (CFU) was calculated per gram of dry soil as 133
an indirect measure to determine total abundance by location (Bressan et al. 2015). Morphotypes with 134
differential morphological characteristics were subcultured in Mergeay agar with 6 mg Kg-1 of CdCl2 and 135
were preserved in glycerol stocks at - 70 °C. 136
137
Micro and macro morphological characterization were performed to each isolated assessing color using the 138
Pantone scale, shape, elevation, surface appearance, and colony consistency. Also, Gram-stain and endospore 139
stain with malachite green dye in contrast with safranin were performed. Molecular identification was made 140
by extracting DNA according to Chen y Kuo (Chen and Kuo 1993) and partial amplification of the 16S rRNA 141
gene with the universal primers 27F and 1492R as previously described (Avellaneda-Torres et al. 2020). All 142
sequences were submitted to the GenBank (NCBI). 143
144
Analysis of structural diversity and phylogeny of culturable Cd-RB 145
146
Cd-RB structural diversity was assessed by (i) richness and abundance; richness was considered as the 147
number of present genera per sample and the abundance as the number of isolated morphotypes for each 148
location (NC, Y1, Y2, Y3) (Lyngwi et al. 2013; dos Passos et al. 2014) and (2) phylogenetic analysis of the 149
16 rRNA genes from isolates. The phylogenetic tree was inferred using the Muscle multiple alignment 150
method and Neighbor-joining algorithm (Saitou and Nei 1987). The evolutionary distances were computed 151
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using the p-distance method and nodes are supported in the bootstrap method using 1000 pseudoreplicates 152
(Felsenstein 1985). Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). 153
154
Functional analysis of isolated Cd-BR morphotypes associated with C, P, and N 155
156
The cellulolytic potential of isolated morphotypes was assessed in LB agar with carboxymethyl cellulose 157
CMC, as the unique carbon source, and staining with Congo red (Avellaneda-Torres et al. 2014). Phosphate 158
solubilization activity was assessed in SMRS agar (Paul and Sundara 1971), and the solubilization factor (SF) 159
was calculated using bromocresol purple. Hydrolysis and solubilization factors establish the ratio of 160
degradation or solubilization diameter and colony growth diameter (Paul and Rao 1971). Finally, atmospheric 161
nitrogen fixation activity was observed on Rennie agar (Rennie 1981) with bromothymol blue as an indicator, 162
through growth and turning color of this medium that lacks N. Three replicates per morphotype were used for 163
each functional group. 164
Effect of Cd on bacterial growth, Cd bioaccumulation, and Minimum Inhibitory Concentration 165
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Morphotypes previously isolated on Mergeay agar with 6 mg Kg-1 of Cd were inoculated in LB agar with 12 167
and 18 mg Kg-1 of Cd. Bacterial growth curves were analyzed for bacteria resistant to 18 mg Kg-1 of Cd. LB 168
broth with (18 mg Kg-1) and without Cd was inoculated with 0.1% (v/v) of bacterial cultures of 18 h (Ivanova 169
et al. 2002; Cristani et al. 2012). Bacteria were incubated at 37 °C with shaking at 200 rpm. With three 170
replicates for each morphotype, optic density at 600 nm (OD600) was determined every 2 h during 36 h using 171
a NanoDropTM ONE spectrophotometer (ThermoFisher Scientific, USA). 172
173
Cd bioaccumulation test was made twice at different moments. The bacteria were inoculated into LB broth 174
containing 18 mg Kg-1 of Cd and incubated at 37 °C with shaking (200 rpm) with three replicates per bacteria. 175
The bacterial cells were harvested at 18 h after inoculation by centrifugation at 5000 rpm for 15 min and then 176
washed with sterile deionized water to remove free heavy metal ions. The supernatants were sterilized by 177
filtration with a 0.22 µm membrane filter (MILLIEX®GP Millipore Express®) and cell pellets were dried at 178
100 °C until constant weight. Media without adding Cd or inoculating bacteria were used as controls. The 179
concentration of Cd in supernatants was measured by Atomic Absorption Spectrophotometry (AAS; Perkin 180
Elmer AAnalyst 300, USA) and the metal bioaccumulation of bacteria was calculated as follows (Micheletti 181
et al. 2008): 182
183
� � ��� � ���
184
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where q (mg g-1) is the Cd bioaccumulation of bacterial cells, Ci is the initial concentration of heavy metal 185
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 186
V is the volume of the liquid medium (0.05 L in the experiments). 187
188
The bacteria resistant to 18 mg Kg-1 of Cd were grown in 15 mL conical bottom tubes with 7 mL of LB broth 189
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 190
culture of 18 h were inoculated and incubated at 37 °C with constant agitation at 200 rpm for 18 h. Following 191
incubation, turbidity was observed. The minimum Cd concentration at which bacterial isolate did not show 192
growth was considered as its Minimum inhibitory concentration (MIC). 193
Data analysis 194
195
Data from hydrolysis and solubilization factors and Cd bioaccumulation were analyzed using a completely 196
randomized design (CRD) with a one-way analysis of variance (ANOVA) in SAS v9.4 statistical software 197
(SAS Institute Inc., USA). Shapiro-Wilk normality test and Bartlett homogeneity of variance tests were made. 198
Media were compared using Tukey’s test (p<0.05). 199
Results 200
Culturable of Cd-RB isolation, morphological characterization, and molecular identification 201
202
A total of 30 Cd-RB morphotypes were isolated from the different soil samples in culture media with 6 mg 203
kg-1 Cd. One isolate of each morphotype was morphologically characterized and identified. The number of 204
CFU of Cd-RB was less than 106 CFU g-1 dry soil in all locations without significant differences 205
(Supplemental Fig. 2). The micro and macroscopic characterization are shown in Supplemental Table 2, and 206
the Gram analysis showed that 90% of the isolated (n=27) corresponded to Gram-negative bacteria and the 207
remaining 10% (n=3) to Gram-positive (Table 2). Based on the BLAST search results of the 16S rRNA gene, 208
it was identified eight genera: Burkholderia, Pseudomonas, Enterobacter, Serratia, Bacillus, Halomonas, 209
Herbaspirillum, and Rhodococcus (Supplemental Table 3). 210
211
Analysis of structural diversity and phylogeny of culturable Cd-RB 212
213
It also was found a greater abundance, unique morphotypes, and dominance of isolated Cd-RB belonging to 214
the genera Burkholderia and Pseudomonas in soil with the highest natural concentration of Cd (location Y3; 215
Fig. 2). Additionally, Y1 and Y2 shown the greatest similarity according to the isolated morphotypes (Fig. 2). 216
217
The phylogenetic analysis showed that 90% (n=27) of the isolated bacteria belong to the phylum 218
Proteobacteria, 6.7% to Firmicutes (n=2), and 3.3% to Actinobacteria (n=1) (Fig. 3; Supplemental Table 3). 219
In the phylogenetic tree, Rhodococcus sp. GB17, Bacillus flexus YB22, and Bacillus sp. GB18 were used as 220
an outgroup due to their relationship with the other isolated morphotypes. Within the phylum proteobacteria, 221
two classes stand out, Gamma and Beta Proteobacteria. Gammaproteobacteria were represented by the genera 222
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Serratia (6 morphotypes), Enterobacter (4 morphotypes), Pseudomonas (7 morphotypes), and Halomonas (1 223
morphotype) belonging to the families Enterobacteriaceae (Enterobacteriales), Pseudomonadaceae 224
(Pseudomonadales), and Halomonadaceae (Oceanospirillales), respectively. Beta Proteobacteria had the 225
genera Burkholderia, which presented the highest number of morphotypes found (8), and Herbaspirillum (1); 226
belonging to the family Burkholderiaceae and Oxalobacteraceae, respectively, and both to order 227
Burkholderiales. The genera Bacillus (2 morphotypes) and Rhodococcus (1) were the least represented, 228
belonging to the Bacillaceae (Bacillales) and Nocardiaceae (Actinomycetales) class, respectively. 229
230
Functional analysis of isolated morphotypes associated with C, P, and N 231
232
Some isolated bacteria presented functional activities related to C, P, and N in addition to their Cd resistance. 233
The 46.7% (n=14) of the isolated morphotypes showed the ability to degrade cellulose, highlighting strains 234
such as Enterobacter sp. YB11, Pseudomonas sp. GB86, Herbaspirillun sp. GB90 and Burkholderia sp. 235
GB68 with hydrolysis factors above 2.5 (Table 2). Regarding P, a higher percentage of morphotypes were 236
able to solubilize phosphates (53%, n=16) compared with other functional groups analyzed. Bacteria from 237
genera Enterobacter, Burkholderia, and Serratia showed high solubilization factors ranging from 1.56 to 4.39 238
(Table 2). Finally, atmospheric nitrogen fixation ability was observed in half of the isolated morphotypes. 239
240
Effect of Cd on bacterial growth, Cd bioaccumulation, and Minimum Inhibitory Concentration 241
242
Among the 30 isolated morphotypes only Pseudomonas sp. GB73, Burkholderia sp. NB10, P. aeruginosa 243
NB2, P. putida GB78 grew on media with 18 mg Kg-1 Cd, and the last three were considered for further 244
analysis. The effect of Cd on the growth curve varied among the assessed strains. In presence of 18 mg Kg-1, 245
P. aeruginosa NB2 showed an average reduction of more than one unit of OD600 during the log phase from 14 246
h after inoculation compared to control without Cd (Fig. 4A). In contrast, P. putida GB78 did not show any 247
difference in OD600 with and without Cd during all growth phases (Fig. 4B). Burkholderia sp. NB10 had a 248
slight reduction of OD600 with Cd from 2 to 20 h post-inoculation, however, from 20 h the OD600 of bacteria 249
grown with Cd reached similar magnitudes to culture without Cd. 250
251
Regarding Cd bioaccumulation (q), P. putida GB78 accumulated 5.92 mg g-1, which was significantly higher 252
than P. aeruginosa NB2 and Burkholderia sp. NB10 accumulation (around 1 mg g-1; Fig. 5). Finally, it was 253
found that Burkholderia sp. NB10 and P. aeruginosa NB2 showed a MIC of 140 mg Kg-1, which is 1.5 times 254
higher than the MIC recorded for P. putida GB78 (90 mg Kg-1). 255
256
Discussion 257
Culturable of Cd-RB isolation, morphological characterization, and molecular identification 258
259
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The Log CFU g-1 of dry soil is an indirect measure of the abundance of microorganisms present in soil and for 260
agricultural soils, values between 108 – 1010 cells per gram have been reported (Marteinsson et al. 2015). In 261
this study, the bacterial abundances for all studied locations were considered low (below 106), which could be 262
attributed to the presence of Cd as a selection factor in the isolation culture media with 6 mg Kg-1 Cd (Torsvik 263
et al. 2002; Itävaara et al. 2016). Although it has been found that heavy metals affect adversely the 264
composition of the microbial community (Etesami 2018), in this study, it was selected cacao-cultivated soil 265
and culture media with a high concentration of Cd to isolated bacteria, due to these conditions would allow a 266
better selection of morphotypes with an enhanced capacity to survive under Cd stress condition in vitro. 267
268
The morphological characterization showed that 90% of the isolated morphotypes corresponded to Gram-269
negative bacteria. Gram-negative and Gram-positive bacteria respond differently to the presence of heavy 270
metals (Bruins et al. 2000; Limcharoensuk et al. 2015). Bruins et al. (Bruins et al. 2000) assert that some 271
Gram-negative bacteria (genus Pseudomonas) can tolerate 5 to 30 times more Cd than Gram-positive bacteria 272
(Staphylococcus aureus, S. faecium, Bacillus subtilis). In this regard, Wu et al. (Wu et al. 2009) reported that 273
Azotobacter chroococum, a Gram-negative bacteria, had a greater capacity for binding to Cd than Bacillus 274
megaterium, a Gram-positive one. This differential response can be explained due to the composition of the 275
cell wall and the resistance mechanisms used. 276
277
Gram-positive bacteria have a thick layer of peptidoglycan (20-80 nm) in which they fix acids, proteins, and 278
polysaccharides. This layer forms a barrier to avoid the entry of heavy metals into the cell, prevailing passive 279
resistance mechanisms such as adsorption (Bruins et al. 2000; Mounaouer et al. 2014). In contrast, Gram-280
negative bacteria have thinner walls (5-10 nm) that allow the influx and efflux of heavy elements and force 281
the bacteria to develop active mechanisms or flow systems and to have a higher protein synthesis in presence 282
of Cd (Bruins et al. 2000). Nevertheless, both types of bacteria can present passive and active resistance 283
mechanisms to Cd and other heavy metals at the same time (Sharma and Archana 2016; Etesami 2018). This 284
indicates that resistance depends also on the other factors such as type of strain, type, and concentration of the 285
heavy metals, physicochemical properties of soil, and environmental conditions (Sharma and Archana 2016). 286
In this regard, Lima et al. (Lima e Silva et al. 2012) observed a greater presence of Gram-positive Cr resistant 287
bacteria, while Hg and Ag resistant bacteria were mostly Gram-negative. This demonstrates that the pollutant 288
also determines the type of bacteria that survive in contaminated environments. 289
290
Burkholderia, Pseudomonas, Enterobacter, and Serratia were the most frequent isolated genera. These genera 291
have been reported in the management of cultivated soils. Strains of Burkholderia spp. have the potential to 292
promote plant growth, biocontrol pathogens, and biodegrade toxic molecules (Castanheira et al. 2016). 293
Enterobacter spp. can improve bioremediation processes of soils contaminated with heavy metals (Qiu et al. 294
2014; Marteinsson et al. 2015) and within the genus Pseudomonas, growth-promoting strains have also been 295
reported with the ability to mitigate stress in plants caused by the presence of heavy metals such as Cd and Pb, 296
and metals such as Zn that are toxic at high levels (Lin et al. 2016; Rojjanateeranaj et al. 2017). Serratia sp. 297
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has been reported as a bacteria that can confer tolerance to heavy metal stress in cereals (Ahmad et al. 2014). 298
Additionally, genera found in less frequency such as Halomonas and Herbaspirillum, have been involved in 299
bioremediation processes of Cr and Sr in symbiosis with algae, and in free life (Achal et al. 2012; Murugavelh 300
and Mohanty 2012; Gupta and Diwan 2017), with the potential to fix nitrogen, and chelate heavy metals such 301
As, Pb, and Cu (Govarthanan et al. 2014; Li et al. 2018). The bacteria isolated in this study, constitute a 302
potential resource of native microorganisms to manage Cd in cacao cultivated soils. However, further studies 303
to characterize and evaluate the performance of bacteria in the field and their interaction with plants, are 304
needed. 305
306
Analysis of structural diversity and phylogeny of culturable Cd-RB 307
308
The structure of microbial communities can be affected by the presence of heavy elements. In this study, 309
results showed that there was not a relationship between the richness of isolated morphotypes and the level of 310
Cd present in the soil. However, the presence of unique morphotypes and the dominance of Burkholderia and 311
Pseudomonas genera (Fig. 2), show that the presence of Cd in soils may alter the uniformity of the 312
community of Cd-RB present in the studied soil. It has been reported that more sensitive microorganisms to 313
heavy metals are suppressed by the toxic element and replaced by more resistant with adaptive characteristics, 314
occupying their ecological niche (Azarbad et al. 2015). The presence of unique morphotypes isolated from Y3 315
soils agrees with the results reported by Tipayno et al. (Tipayno et al. 2018) who studied sites with different 316
levels of Cd and other heavy metals (As and Pb) and found that the site with the highest concentration of Cd 317
also presented the highest number of unique morphotypes. 318
319
Most of the isolated morphotypes (90%) in this study corresponded to the Proteobacteria phylum. This 320
phylum represents the largest and most diverse group of prokaryotes and contemplates the vast majority of 321
Gram-negative bacteria studied to date (Spain et al. 2009). Also, it is usually reported as the most frequent in 322
studies related to heavy metals, so strains belonging to several genera have been isolated, characterized, and 323
reported by their resistance (Xiao et al. 2019). Additionally, Chen et al. (Chen et al. 2018) highlight that the 324
Proteobacteria phylum together with Bacteroidetes and Firmicutes harbor the largest set of heavy metal 325
resistance genes. 326
327
Functional analysis of isolated Cd-RB morphotypes associated with C, P, and N 328
329
Cellulolytic potential, free nitrogen fixation, and phosphate solubilization were determined in all isolated 330
morphotypes using selective media to promote an integrated crop cacao management. The use of these 331
bacteria could enhance nutrient cycling, decrease the use of chemical fertilizers, and increase the 332
environmental sustainability of the crop. In agroforestry production systems such as cacao, some interactions 333
and processes favor nutrient cycling, as a result of the decomposition of organic matter from crop residues 334
and pruning such as litter in the crop, stems, among others (Aikpokpodion 2010). The decomposition of 335
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10
cellulose increases the levels of organic matter, and carbon can be used as a source of energy for edaphic 336
processes and plant physiology (Gupta et al. 2012). Organic matter is a fraction to which heavy metals such as 337
Cd can bind and be adsorbed by chelates or other substances. Therefore, when levels of organic matter 338
increase in soil, the bioavailability of Cd for plants can be reduced (Gadd 2000). 339
340
The solubilization of phosphates and the promotion of plant growth have been reported for the genus 341
Enterobacter isolated from soil (Beltrán 2014). The main cellular mechanism through which bacteria 342
solubilize phosphates is the production of organic acids such as gluconic acid, 2-ketogluconic acid, lactic 343
acid, isovaleric acid, isobutyric acid, and acetic acid (Beltrán 2014). These molecules have a negative charge 344
and therefore can form complexes with metal cations in solution and be involved in the chelation of Cd (Yang 345
et al. 2018). The bacteria involved in the atmospheric nitrogen fixation (ANF-B) have also played an 346
important role in the remediation of Cd polluted soils (Chen et al. 2020). Ivishina et al. (Ivshina et al. 2014) 347
showed that ANF-B can secrete extracellular polymeric substances (EPS) and various organic acids and 348
amino acids that may alter the Cd availability in the soil by adsorbing and trapping metals due to the presence 349
of many anionic functional groups. 350
351
The management of Cd-polluted agricultural soils is a complex issue where the Cd-RB could have an 352
important function not only to decrease the Cd availability but also to improve soil fertility. The presence of 353
activity associated with the cycling of C, P, and N in Cd-RB is an advantage within holistic crop soil 354
management strategies. In this study, 11 from 30 isolated morphotypes showed the ability to degrade 355
cellulose, solubilize phosphate and, fix atmospheric nitrogen, simultaneously. It would be valuable additional 356
analyses that quantify the activity of involved enzymes and determine the effect of bacteria on plant growth 357
and physiology. 358
359
Effect of Cd on bacterial growth, Cd bioaccumulation, and Minimum Inhibitory Concentration 360
361
The effect of Cd on bacterial growth varies depending on bacterial strain. Heavy metals affect bacterial 362
growth by causing multiple metabolic alterations such as DNA damage, decreasing protein synthesis, 363
disruption of respiration and cell division, and amino acid biosynthesis (Chen et al. 2016). These effects 364
explain the growth decrease of P. aeruginosa NB2 at 18 mg Kg-1 of Cd along with the growth phases (Fig. 365
4A). In Burkholderia sp. NB10, a reduction of the growth was also detected in the lag and log phases (Fig. 366
4C); however, the cells were able to adapt and resume growth at the stationary phase. It can be explained by 367
the repair of cadmium-mediated cellular damage and adjustment of the cell physiology to limit the 368
distribution of toxic ions in the cell (Mohamed and Abo-Amer, 2006). 369
370
Despite Cd effects, bacteria have different Cd resistance strategies dependent and independent on intracellular 371
accumulation (Ashraf et al. 2017; Etesami 2018). Mechanisms of resistance include metal efflux systems, 372
transportation, precipitation, transformation, and sequestration by metallothionein proteins and thiol 373
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11
compounds such as glutathione (Nies 2003). The results showed that resistance mechanisms of P. putida 374
GB78 are efficient enough to resist 18 mg Kg-1 of Cd without affecting its growth. These results can also be 375
explained based on the difference between the efficiency of resistance mechanisms of strains. Chen et al. 376
(Chen et al. 2014) suggest that bacteria can activate resistance mechanisms such as metal efflux bombs at 377
particular moments which results in a depletion of intracellular Cd content. 378
379
The evaluation of Cd bioaccumulation (q) is a measure of the potential of microorganisms to be used in 380
bioremediation processes and management of toxic concentrations of heavy metal in agricultural soils 381
(Cánovas et al. 2003). A higher q detected in P. putida GB78 suggests that this strain favors intracellular 382
accumulation processes (Fig. 5). In contrast, Burkholderia sp. NB10 and P. aeruginosa NB2 showed a lower 383
q, which may be attributed to greater efficiency in extracellular Cd outflow mechanisms. 384
385
The MIC is an indication of the resistance capacity that a microorganism has to grow in the presence of Cd 386
and highlights its potential for applications at different heavy metal concentrations in soils (Xu et al. 2012). 387
Results showed a higher resistance in Burkholderia sp. NB10 and P. aeruginosa NB2 (140 mg Kg-1) 388
compared to P. putida GB78 (90 mg Kg-1). Different Pseudomonas and Burkholderia species have been 389
widely reported for their resistance to Cd, other heavy metals such as Cu and Pb, and high concentrations of 390
Zn (Naik and Dubey 2011; Jin et al. 2013). It has been reported MICs from 100 to 190 mg Kg-1 for P. putida 391
(Lee et al. 2001; Leedjärv et al. 2008), up 900 mg Kg-1 for P. aeruginosa (Ghaima et al. 2017), and 200 mg 392
Kg-1 for Burkholderia sp. (Jin et al. 2013). These values show the wide variation that exists in the level of 393
resistance in bacteria. Overall, the bacterial growth and the MIC results suggest that 18 mg Kg-1 of Cd is not a 394
limiting concentration for P. putida GB78. However, it can resist up to 90 mg Kg-1 of Cd by favoring 395
intracellular bioaccumulation. On the contrary, Burkholderia sp. NB10 and P. aeruginosa NB2, with a MIC 396
of 140 mg Kg-1 of Cd, showed growth rate reductions that may be an acclimatization strategy to maintain 397
viability for a longer time and activate resistance mechanisms such as efflux systems (lower q). 398
399
Conclusions 400
Cacao-cultivated soils harbor a diversity of bacteria that exhibit resistance to Cd and functional activity 401
associated with the cycling of C, P, and N. The structural diversity of culturable Cd-RB bacterial community 402
is affected by the presence of Cd in soil samples where high levels of Cd enhance the selection of unique and 403
dominant morphotypes such as Burkholderia spp. and Pseudomonas spp. These genera can resist up to 90 to 404
140 Kg-1 of Cd showing their potential to use in soil bioremediation. In this study, multifunctional Cd-RB 405
morphotypes were identified with the potential to develop integrative soil management of Cd-polluted soils. 406
407
408
Supplementary Information 409
The online version contains supplementary material available at 410
411
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12
Acknowledgements The authors thank the cacao farmers for letting them to collect samples. The authors 412
thank the funding organization and the Universidad Nacional de Colombia for supporting the research group. 413
414
Authors’ contributions ETR, HCN, and JCZ conceived the project and designed the experiments. ETR, 415
HCN and JCZ collected the samples. HCN and JCZ performed the experiments and analyzed the data. ETR 416
supervised the research and analyzed the data. All authors wrote, and read and approved the final manuscript. 417
418
Funding This research was supported by the Cundinamarca-Colombia government (Contract No. 2 419
Framework Derivative Agreement of Agro-industrial Technological Corridor (No. 395 - 2012), and 420
Universidad Nacional de Colombia internal grant for research projects. This work was conducted under 421
Ministerio de Ambiente, Vivienda y Desarrollo Territorial (MAVDT) collection permit 0255, March 14th, 422
2014. 423
424
Data availability The datasets used and/or analyzed during the current study are available from the 425
corresponding author on reasonable request. The 16S rRNA sequencing are available at NCBI according to 426
GenBank accession numbers. 427
428
Declarations 429
430
Ethics approval and consent to participate This work was conducted under Ministerio de Ambiente, 431
Vivienda y Desarrollo Territorial (MAVDT) collection permit 0255, March 14th, 2014. Not applicable for 432
animal or human data or tissue. 433
434
Consent for publication: Not applicable. 435
436
Competing interest: The authors declare that they have no competing interests. 437
438
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Fig. 1 Location of sampling sites. Each dot represents a cacao farm where previously soil cadmium levels 680 have been documented 681 682
Fig. 2 Venn diagram of the culturable Cd-RB morphotypes isolated from locations studied. Each gray circle 683 represents the location, referred to with a label. Black circles represent the isolated morphotypes, the different 684 morphotypes belonging to the same genera are represented by the red contour circle. The morphotypes were 685 isolated from cacao cultivated soils with medium (>1.0 to 5.0 mg Kg-1; CN, Y1 and Y2) and high (>5.0 mg 686 Kg-1; Y3) total Cd levels 687 688
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Fig. 3 Phylogenetic tree of strains isolated in Mergeay agar with 6 mg kg-1 of Cd from cacao culture soil. The 689 evolutionary history was inferred using the neighbor-joining method. The bootstrap test was inferred with 690 1000 replicates and the analysis was performed with 36 sequences. Positions containing gaps and missing 691 data were eliminated. The evolutionary distances were computed with the p-distance method. Evolutionary 692 analyses were performed in MEGA 7. Sequences indicated with black squares were used as a template 693
694
Fig. 4 Growth of A) P. aeruginosa NB2, B) P. putida GB78 and C) Burkholderia sp. NB10 in the presence 695 and absence of 18 mg Kg-18 of Cd. The bars represent the standard error (n=6) 696
697
Fig. 5 Bioaccumulation of Cd in bacteria at 18 h of growth on media with 18 mg Kg-1 of Cd. The bars 698 represent the typical error (n=3). Means with different letters indicate significant differences by Tukey’s test 699 (p<0.05) 700
701
Table 1 Location, soil taxonomy, and total and potentially available Cd concentrations in sampling sites in 702
Nilo (NC) and Yacopí (Y). Medium (>1.0 to 5.0 mg Kg-1) and high (>5.0 mg Kg-1) total Cd level 703
704
Table 2 Cellulose hydrolysis and phosphate solubilization factors, and free nitrogen-fixing activity of Cd-RB. 705
Letters in the same columns represent significant differences (p<0.05) between strains according to Tukey’s 706
multiple comparison test. + and – indicate the presence or absence of growth/fixation on Rennie agar at 3 days 707
post-inoculation (dpi) 708
709
710
711
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712 713
714
715
716
717
718
719
720
721
722
723
724
725
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726 727
728
729
730
731
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732 733
734
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22
735
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736 737
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753
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Location Natural region Soil Taxonomy Cd level Total Cd
(mg Kg-1) Available Cd
(mg Kg-1)
NC Dry Tropical
Forest Dystric Eutrudepts Medium 1.68 1.28
Y1 Tropical
Premontane Moist Forest
Typic Udorthents Medium 1.74 0.20
Y2 Typic Udorthents Medium 4.24 2.12
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
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25
No. ID NCBI ID Loc. Morphotype Growth at Cellulose
hydrolysis factor
P solubilization
factor
N fixation
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. + - - - -
30 GB90 MN719065.1 Y3 Herbaspirillum
sp. + - 2.77 ± 0.21 abc - +
785
786
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