Bioremediation Potential of Native Bacillus sp. Strains as a Sustainable Strategy for Cadmium Accumulation of Theobroma cacao in Amazonas Region

Overview

This study is a soil microbiology and bioremediation investigation, not a finished-food contamination study. It does not report cadmium concentrations in cocoa beans, powder, chocolate, or any food product intended for human consumption. Its relevance to the wiki is as context for pre-harvest mitigation pathways that may reduce cadmium uptake in cacao.

Arce-Inga and colleagues isolated 138 bacterial strains from the rhizosphere of Theobroma cacao cultivated in cadmium-contaminated soils in three districts of Bagua province, Amazonas department, Peru. Soil cadmium levels at the collection sites exceeded the Peruvian Ministry of Environment maximum allowable limit of 1.4 mg/kg. More than 88% of isolates (122 of 138) grew normally at cadmium concentrations up to 300 ppm CdCl2. Three hypertolerant isolates (surviving 400 ppm CdCl2) were identified by 16S rRNA sequencing: Bacillus sp. S1C2 (Cereus clade), Bacillus sp. R1C2 (Subtilis clade, related to B. pumilus), and Pseudomonas sp. V3C3 (P. putida complex). A fourth, low-tolerant strain, Peribacillus sp. T3C1, was included as a comparator. In vitro cadmium removal efficiency ranged from 29.29% to 46.04%. In a greenhouse pot trial with CCN-51 cacao seedlings, Bacillus strains S1C2 and R1C2 showed a negative trend in stem cadmium accumulation (reduction), although the difference did not reach statistical significance. Pseudomonas sp. V3C3 had no effect on stem cadmium, and the low-tolerant T3C1 appeared to increase cadmium accumulation. Unexpectedly, control seedlings grown in nominally cadmium-free sterilized soil showed 1.8 +/- 0.24 ppm cadmium (Cd) in stems, suggesting an endogenous cadmium source (possibly the seeds themselves).

Key numbers

Minimum inhibitory concentration (MIC) tolerance

The three hypertolerant bacteria (HCdB) surviving 400 ppm were S1C2, R1C2, and V3C3.

In vitro cadmium removal efficiency (RE%)

All strains showed RE values between 29.29% and 37.88% at 100 ppm with no statistically significant differences. At higher concentrations:

• Bacillus sp. R1C2: RE of 40.38% at 200 ppm CdCl2
• Pseudomonas sp. V3C3: RE of 44.03% at 200 ppm and 46.04% at 300 ppm CdCl2
• Bacillus sp. S1C2: lower RE at 200 and 300 ppm (significantly different from R1C2 and V3C3)

R1C2 showed approximately 4-fold increase in intracellular cadmium accumulation (CdA) with increasing Cd concentration. V3C3 showed non-significant CdA with increasing Cd, suggesting an extracellular detoxification mechanism (possibly EPS-mediated chelation) rather than intracellular accumulation.

Soil cadmium at collection sites

Collection sites in Copallin, Aramango, and La Peca had cadmium soil levels above 1.4 ppm (the Peruvian regulatory maximum for agricultural soils per Ministerio del Ambiente Resolucion Ministerial No. 307-2012-MINAM).

Greenhouse pot trial: stem cadmium (ppm, Cd in dry weight)

Control seedlings (no bacteria, no added Cd) showed 1.8 +/- 0.24 ppm Cd in stems. Seedlings inoculated with T3C1 showed 2.82 +/- 0.34 ppm (suggesting potential cadmium stimulation). S1C2 and R1C2 inoculated seedlings showed a negative trend in stem cadmium accumulation compared to the non-inoculated control, but differences were not statistically significant. At 1.4 and 3.5 ppm CdCl2 supplementation, no significant differences were found among treatments.

Soil-to-plant context (from cited literature)

The authors note that cocoa bean samples from Peruvian regions including Piura, Tumbes, and Huanuco have shown Cd values exceeding 0.96 ug/g, above the EU Commission Regulation (EU) No. 488/2014 maximum of 0.80 ug/g for cocoa-based products.

Evidence fitness

Rating: B-tier, context only. This is a primary peer-reviewed study with original experimental data, published in an MDPI open-access journal with peer review. It reports genuine measurements (MIC tolerance, in vitro cadmium removal, greenhouse pot trial), but none of those measurements are finished-food contamination data. The study contributes to the bioremediation and pre-harvest mitigation literature for cocoa cadmium, which is relevant background for understanding the cadmium supply-chain pathway from soil to bean. It does not support occurrence pooling, certification-limit setting, dietary-exposure assessment, or any food-concentration claim on ingredient or product pages. The greenhouse trial used a single cacao genotype (CCN-51) with 3 to 5 biological replicates per treatment; several comparisons did not reach statistical significance, and the unexpected baseline cadmium in control seedlings complicates interpretation.

Methods

Bacterial isolation: Rhizosphere soil from T. cacao at 20 cm depth in Bagua province (Copallin, Aramango, La Peca). Isolation on LB agar supplemented with 3 ppm CdCl2.

Identification: BOX-PCR fingerprinting for genetic uniqueness; 16S rRNA gene sequencing (Macrogen, Seoul); phylogenetic analysis by maximum likelihood (MEGA 11.0, Kimura-2-parameter + gamma distribution, 1000 bootstrap replicates). Sequences deposited in GenBank: S1C2 (ON479211), T3C1 (ON479212), R1C2 (ON479210), V3C3 (ON982449).

MIC tolerance: LB agar supplemented with 50, 100, 150, 200, 300, 350, and 400 ppm CdCl2, incubated at 28 C for 24 h.

In vitro cadmium removal and bioaccumulation: Strains grown in LB liquid medium supplemented with 100, 200, and 300 ppm CdCl2 for 24 h. Cadmium quantification by atomic absorption spectrophotometry (Agilent Technologies 4100 MP-AES) at the Soil Analysis Laboratory of INDES-CES, UNTRM.

Greenhouse pot trial: T. cacao genotype CCN-51 seeds germinated in sterile water, dipped in bacterial inoculum (approximately 10^7 CFU/mL) for 40 min, planted in sterilized native Amazonian soil in 1000 g pots, irrigated with 0, 1.4, and 3.5 ppm CdCl2 to field capacity. Completely randomized design, grown 12 weeks under semi-controlled greenhouse conditions in Bagua province. Stem and root cadmium determined by atomic absorption spectrophotometry after dry ashing at 450 C for 8 h and HCl extraction.

Statistics: One-way ANOVA with Tukey’s multiple-comparison test (Infostat software). Data transformations (Log and Normal Score) applied where residual homogeneity or normality departed.

Implications

Certification: This study provides no finished-food cadmium data and cannot be used for occurrence pooling or certification-limit setting for cocoa or chocolate products. Its value is as context for the pre-harvest mitigation pathway: it demonstrates that native rhizosphere bacteria from Amazonian cacao soils can reduce cadmium in vitro, though in vivo outcomes on cacao plant tissue are variable and often not statistically significant. This variability is itself an important finding for any future HMTc mitigation-credit or supply-chain-intervention framework.

Courses: Useful as a teaching example of (a) why in vitro bioremediation results do not reliably predict field outcomes for cadmium in cacao, (b) the complexity of plant-bacteria interactions relevant to cadmium uptake, and (c) the finding that cacao seeds may carry endogenous cadmium even when planted in nominally clean soil, which complicates controlled experiments and has implications for understanding the soil-to-bean cadmium pathway.

Provenance notes

Published 25 October 2022 in Microorganisms 10(11):2108 (MDPI, open access, CC BY 4.0). Authors affiliated with Instituto de Investigacion para el Desarrollo Sustentable de Ceja de Selva (INDES-CES), National University Toribio Rodriguez de Mendoza de Amazonas (UNTRM), Peru, and Universidad de los Lagos, Chile. Funded by CEINCACAO (SNIP NRO. 352641, MEF Peru), METACACAO (CONTRATO No. 008-2020-FONDECYT-BM), and GENCACAO (CONTRATO No. 30-2019-FONDECYT-BM-INC-INV). No conflicts of interest declared.

Related evidence

• Cocoa
• Cadmium

Update history

The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git.