Annals of Microbiology

, Volume 68, Issue 12, pp 931–942 | Cite as

Immobilization of Pseudomonas putida PT in resistant matrices to environmental stresses: a strategy for continuous removal of heavy metals under extreme conditions

  • Sanaz Khashei
  • Zahra EtemadifarEmail author
  • Hamid Reza Rahmani
Original Article


The purpose of this study was to investigate the potential of immobilized lead- and cadmium-resistant Pseudomonas putida strain PT to remove heavy metals from aqueous medium under extreme conditions. The tolerance and accumulation of cadmium and lead ions by strain PT were investigated by minimal inhibitory concentration (MIC) determination and polymerase chain reaction (PCR) of cadA gene, respectively. The surface chemical functional groups of P. putida PT involved in the metal biosorption were identified by Fourier transform infrared (FTIR). Pseudomonas putida PT was immobilized in three matrices include carboxy-methyl cellulose (CMC), rice bran, and a new composite made of alginate, polyvinyl alcohol (PVA), and CaCO3 to prepare heavy metal adsorbent. The biosorbents were analyzed by SEM, and their metal removal capability was assayed in two consecutive cycles by atomic absorption spectroscopy. The viability of immobilized bacterial cells was determined by flow cytometry during storage at 4 °C and exposure to the environmental stresses (pH and temperature). The results showed that PT strain was resistant up to 10 mM Pb2+ and 8 mM Cd2+. FTIR analysis revealed that alcohol, sulfur, phosphate, esters, and amide groups played important roles in metal biosorption process and, also change in metabolic reactions like hydration and polyesters accumulation was observed after metal biosorption. The presence of cadA gene, a heavy metal translocating pump-coding gene, indicated the ability of metals bioaccumulation by the PT strain. Immobilized cells in alginate–PVA–CaCO3 and rice bran showed the highest metal removal efficiency for Pb2+ as 75% and Cd2+ as 96.7%, respectively. Metal adsorbents were reusable, and the highest removal efficiency in the second cycle was observed in inoculated alginate–PVA–CaCO3 (79.5% Pb2+ and 45% Cd2+). Flow cytometric analysis represented that the immobilized cell viability was retained (< 97%) after 4 weeks storage at 4 °C. Viability under two environmental stresses in all matrices was as follows: < 96% at 25 °C, < 87% at 45 °C, < 85% at pH 4, < 96% at pH 7, and < 89% at pH 11. The results signify that these metal adsorbents are efficient technological tools for bioremediation even in harsh environmental conditions.


Heavy metals Biosorption Bioaccumulation Microbe immobilization Viability 



This study was funded by the University of Isfahan and Soil and Water Research Institute of Iran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

13213_2018_1402_MOESM1_ESM.jpg (683 kb)
Fig. A Viability of immobilized P. putida PT during storage at 4 °C. Bacterial cell samples were stained with Rho - 123 and analyzed by flow cytometer (FL1, 525 nm). a, b, c and d indicate the viability of immobilized bacterial cells on rice bran, e, f, g and h represent the viability of immobilized bacterial cells in CMC and i, j, k and l show the viability of immobilized bacterial cells in alginate-PVA-CaCO3 from week 1 to week 4 (JPG 683 kb)
13213_2018_1402_MOESM2_ESM.jpg (622 kb)
Fig. B Flow cytometric analysis of P. putida PT at different pHs. a, b and c indicate the viability of immobilized bacterial cells on rice bran, e, f and g represent the viability of immobilized bacterial cells in CMC and i, j and k show the viability of immobilized bacterial cells in alginate-PVA-CaCO3 at pH 4, 7 and 10, respectively (JPG 621 kb)
13213_2018_1402_MOESM3_ESM.jpg (430 kb)
Fig. C Flow cytometric analysis of P. putida PT at different temperatures. a and d indicate the viability of immobilized bacterial cells on rice bran, b and e represent the viability of immobilized bacterial cells in CMC and c and f show the viability of immobilized bacterial cells in alginate-PVA-CaCO3 at 25 °C and 45 °C, respectively (JPG 429 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Authors and Affiliations

  1. 1.Department of Biology, Faculty of SciencesUniversity of IsfahanIsfahanIran
  2. 2.Environmental Research InstituteUniversity of IsfahanIsfahanIran
  3. 3.Department of Soil and Water Research, Agricultural Research, Educating and Extension Organization, AREEOIsfahanIran

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