Mercury Biodecontamination from Milk by using L. acidophilus ATCC 4356

Overview

Massoud and colleagues at the Iran Standard Organization and Shahid Beheshti University of Medical Sciences ran a laboratory biosorption experiment to test whether Lactobacillus acidophilus ATCC 4356 can remove inorganic mercury (Hg(NO3)2) added to sterile dairy milk at low (µg/L) concentrations representative of reported milk contamination. The work uses Plackett-Burman screening followed by central-composite response surface methodology to identify Hg concentration, bacterial biomass, and contact time as the dominant variables, then fits Langmuir and Freundlich isotherm models to the equilibrium data. Maximum removal was 72% at 80 µg/L initial Hg, 1×10^12 CFU L. acidophilus, day 4, with the Langmuir model (R² = 0.9121) fitting better than Freundlich (R² = 0.8747). The paper is a process-intervention study, not a contamination survey: the milk-Hg values it cites for real-world supply (China, Iran, Codex permitted limit) are second-hand citations, not original measurements.

Key numbers

All concentrations from the paper text and Tables 1-4. Hg analysis was by ICP-MS (model 4500a, England) on supernatants after centrifugation; values are total Hg in the residual liquid phase, with no Hg speciation between Hg(II) ions and Hg-bacterial complexes (only the supernatant is measured; biomass-bound Hg is inferred by mass balance).

Experimental Hg spiking levels used in the biosorption runs:

• Plackett-Burman screening: two levels, 40 µg/L and 100 µg/L initial Hg in milk (Table 1).
• Central-composite RSM: five levels (-α, -1, 0, +1, +α) at 40, 50, 70, 90, 100 µg/L initial Hg (Table 2).
• Isotherm characterization: 20, 40, 60, 80, 100 µg/L initial Hg (Table 4).

L. acidophilus biomass levels: 1×10^10, 10×10^11, 1×10^12, 10×10^13, 10×10^14 CFU (Table 2, central-composite design).

Other process variables:

• Contact time: 0, 1, 2, 3, 4 days (Table 2).
• Inoculation temperature held at 25 °C and shaking at 50 rpm during RSM runs (after Plackett-Burman screening showed temperature and shaking-rate effects were non-significant).
• Plackett-Burman ANOVA p-values (Table 3): shaking rate p = 0.4125, Mercury concentration p = 0.0542, L. acidophilus concentration p = 0.0712, contact time p = 0.0688, inoculation temperature p = 0.3202. The authors describe Mercury concentration, biomass, and contact time as the “significant” main effects despite all three p-values exceeding the conventional 0.05 cutoff.

Bioremoval outcome:

• Maximum removal efficiency: 72% at 80 µg/L initial Hg, 1×10^12 CFU L. acidophilus, day 4 (abstract and Conclusion). The abstract and Conclusion give the maximum-removal day as “the 4th day”; the discussion paragraph instead reports “the 3rd day” for the same maximum, an internal inconsistency reproduced here verbatim.
• Removal efficiency rose with increasing contact time from day 1 to day 4 (Figure 2 contour plot) and with rising initial Hg concentration from 40 to 100 µg/L (Figure 3 contour plot).
• Removal efficiency rose with biomass up to 1×10^12 CFU, then declined slightly at higher biomass — the authors attribute the decline to bacterial aggregation reducing surface-site availability.

Isotherm-model parameters (Table 4, Hg(II) in milk after 4-day exposure to 10^12 CFU/mL L. acidophilus):

Where Ce = equilibrium Hg concentration in milk supernatant, Qe = amount of Hg bound to biomass at equilibrium. Note that the Table 4 column labelled “Qe (µg/L)” is presented in volume-normalised units in the source; mechanistically Qe is the metal loaded per mass of biomass, so the original units may be a printing error. The values are preserved verbatim.

Linear regression coefficients (Figure 4):

• Langmuir (Ce/Qe vs Ce): y = −0.0774x + 2.7541, R² = 0.9121. The negative slope is mathematically inconsistent with the Langmuir equation Ce/Qe = 1/(K·Qmax) + Ce/Qmax (which should yield a positive slope 1/Qmax), so the reported regression statistic is reproduced verbatim and flagged.
• Freundlich (Ln Qe vs Ln Ce): y = 2.4779x − 4.4005, R² = 0.8747.
• The authors conclude that the Langmuir model fits better (higher R²) and that “Mercury absorptions obey Langmuir isotherm model.”

Real-world milk-Hg values cited from prior literature (introduction, p.2314, not measured in this study):

• Codex standard for contaminants and toxins in food: maximum permissible Hg in milk “less than 0.05 µg/L” (per the authors’ citation of Codex Stan 193, 2009; the Codex CXS 193 General Standard does not state a 0.05 µg/L milk-Hg limit in the public 1995/2009 versions and this citation is likely a transcription or jurisdictional-attribution error — see Verification notes).
• China milk Hg: 0.08 µg/L (cited as Wang MQ et al. 2002, J Food Hyg).
• Iran raw cow and ewe milk Hg: 0.07 µg/L (cited as Najarnezhad and Akbarabadi 2013, Food Add Contamin).

These three numbers are not original measurements in this paper; they are quoted from other authors to motivate the biodecontamination study.

Methods (brief)

Bacteria: L. acidophilus ATCC 4356 supplied by Tak Gen Zist Company (Tehran). Inoculated in MRS broth, incubated at 37 °C for 48 h, enumerated by total plate count on MRS agar and plate count agar.

Milk model: Raw milk → standardization → homogenization → pasteurization (75 °C, 15 s) → cooling (40 °C) → adding L. acidophilus (Figure 1 schematic). Hg added as Hg(NO3)2 standard solution (1000 mg/L, Merck, Spain) at 20-100 µg/L, before or after pasteurization per the schematic. Milk species (cow vs other) is not stated; the HTST pasteurization parameters and the absence of any species-specific qualifier are consistent with generic dairy milk. The sterile milk had 20 minutes rest after biomass addition, then Hg spike, then shaker, then 1- or 4-day contact, then 8000 × g for 20 min centrifugation, then supernatant analysis. All biosorption experiments in triplicate.

Hg analysis: ICP-MS model 4500a (England) with cross-flow nebuliser and Peltier-cooled quartz spray chamber, multi-element aqueous tuning before each run. Microwave digestion in segmented-rotor MPR-600 instrument at up to 35 bar and 260 °C before ICP-MS injection. No CRM, LOD, LOQ, recovery, or precision values are reported; this is a notable methodological gap relative to the surveys this paper cites for comparison.

Hg removal formula: %Removal = 100 × [(C0 − C1) / C0] where C0 is initial Hg added and C1 is residual supernatant Hg after the contact period.

Experimental design: Plackett-Burman in 8 runs at 2 levels per variable (Table 1) screened five candidate factors (biomass, temperature, contact time, Hg, shaking). Surviving three factors entered a central-composite RSM in 5 levels (Table 2), analysed in Design-Expert 7.1.5 (Stat-Ease Inc., USA). Statistics in Minitab 14, ANOVA-based, p < 0.05 cutoff.

Isotherm fitting: linear Langmuir form Ce/Qe = 1/(K·Qmax) + Ce/Qmax and linear Freundlich form Ln Qe = Ln Kf + (1/n) Ln Ce, both fit to the 5-point (Hg-initial) dataset at 10^12 CFU/mL biomass on day 4.

The authors do not analyse the milk for background Hg before spiking, do not characterise the milk fat/protein/casein content, do not assess Hg recovery via spike controls, and do not test alternative pH or temperature ranges representative of dairy supply.

Implications

Certification: This paper does not contribute occurrence data for HMTc threshold-setting. The 0.05, 0.07, and 0.08 µg/L milk-Hg values cited in the introduction are secondary citations, not measurements; using them as occurrence inputs would double-count the underlying primary sources (Wang 2002 for China; Najarnezhad and Akbarabadi 2013 for Iran; Codex Stan 193 for the regulatory limit, with the caveat that the 0.05 µg/L attribution appears to be incorrect). The paper’s contribution is to the mitigation/treatment evidence base: 72% Hg removal at 80 µg/L by 10^12 CFU L. acidophilus is a single-lab benchmark for LAB-based milk decontamination at concentrations roughly 1000× higher than reported real-world milk contamination.

Courses: Useful as a worked example of response surface methodology (Plackett-Burman → central composite) applied to a food-decontamination process, and of Langmuir/Freundlich isotherm model selection from linear regression coefficients. Also a teachable case study in methodological-gap recognition: the lack of CRM, recovery, LOD reporting; the experimental Hg spike levels being 400-1600× the cited real-world milk Hg values; and the day-3 vs day-4 internal inconsistency illustrate why mechanism/process papers cannot stand alone as evidence for food-supply contamination levels.

App: Not directly informative for milk-and-dairy contamination_profile values. The biosorption capacity numbers are biomass-loading parameters, not ingredient occurrence priors. The cited Wang 2002 (China 0.08 µg/L) and Najarnezhad 2013 (Iran 0.07 µg/L) milk Hg values should be sourced from the underlying primary papers if Hg priors for cow milk are needed.

Microbiome (Part 24): The paper documents L. acidophilus ATCC 4356 as a Hg(II)-binding probiotic in a milk matrix, with surface functional groups (carboxyl, hydroxyl, phosphate on teichoic acid and peptidoglycan) attributed as the binding moieties. This is relevant context for any future synthesis on probiotic-mediated luminal heavy-metal sequestration in dairy and dairy-derived foods.

Related evidence

• Milk and Dairy
• Mercury, Total

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