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

Immobilization of Pseudomonas putida PT in resistant matrices to environmental stresses: a... 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 CaCO 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 2+ 2+ stresses (pH and temperature). The results showed that PT strain was resistant up to 10 mM Pb and 8 mM Cd . 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–CaCO and rice bran showed the highest metal 2+ 2+ removal efficiency for Pb as 75% and Cd as 96.7%, respectively. Metal adsorbents were reusable, and the highest 2+ 2+ removal efficiency in the second cycle was observed in inoculated alginate–PVA–CaCO (79.5% Pb and 45% Cd ). 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. . . . . Keywords Heavy metals Biosorption Bioaccumulation Microbe immobilization Viability Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1402-7) contains supplementary In recent years, environmental pollution by heavy metals has material, which is available to authorized users. become a great concern worldwide. Heavy metals establish adverse effects on diversity and activity of rhizosphere micro- * Zahra Etemadifar z.etemadifar@sci.ui.ac.ir; zetemadifar@gmail.com bial population and reduce growth and quality of crops (Oves et al. 2010). Also, heavy metals arrive into human food chain through contaminated sea foods and crops (Bi et al. 2018; Department of Biology, Faculty of Sciences, University of Isfahan, Chen et al. 2018; Tchounwou et al. 2012). These elements, P.O. Box: 81746-73441, Isfahan, Iran even at low concentration, are toxic and threaten seriously to Environmental Research Institute, University of Isfahan, human health (Tchounwou et al. 2012). Isfahan, Iran 3 Unlike other pollutants, the toxicity of heavy metals does Department of Soil and Water Research, Agricultural Research, not disappear gradually because they are non-biodegradable Educating and Extension Organization, AREEO, Isfahan, Iran 932 Ann Microbiol (2018) 68:931–942 and have tendency to accumulate in the environment. Materials and methods Therefore, environmental pollution with heavy metals is a global concern and reduction heavy metals burden of soil Source of bacterial strain, maintenance, and growth and water is essential to keep healthy life. Using of chemical condition and physical methods for remediation of contaminated zones is expensive, because they require a lot of energy Bacterial strain was previously isolated from agricultural soils and reagent. Other deficiencies of these techniques are in- contaminated with heavy metals and identified as P. putida PT complete removal of metals and generation of toxic com- based on 16S rRNA gene sequence analysis (GenBank acces- pounds (Krishnani et al. 2008). Microbial cells which pres- sion number: KX963368). This strain was saved on Tryptic ent in heavy metals contaminated sites are powerful agents Soy Broth (TSB) (Merck, Germany) medium supplemented for bioremediation because they are resistant to metals tox- with glycerol at − 80 °C. Pseudomonas putida PT was icity greatly. Microbial removal is a low cost and eco- reactivated by transferring to TSB and Tryptic Soy Agar friendly approach to prevent the metals accumulation in (TSA) (Merck, Germany) media followed by incubation at the environment (Özdemir et al. 2012). However, due to 30 °C for 24 h. instability of biological systems under environmental stress- es like extreme temperature and pH, this approach is inef- Detection of MIC fectiveness (Pires et al. 2011). To overcome this limitation, bacteria must be immobilized in a nontoxic matrix. Bacterial cell growth was evaluated in the presence of differ- Providing high bacterial biomass, easy transposition, regen- ent concentrations of cadmium chloride (Sigma, Germany) eration of bacteria, reusability, providing a suitable micro- and lead nitrate (Sigma, Germany) by microtiter plate method. environment for protecting of cells against harsh conditions Briefly, 100 μl of TSB medium, 50 μl of different concentra- and toxic compounds are the advantages of immobilized tions of each metal prepared in deionized water, and 50 μlof cells in comparison with free cells (Bayat et al. 2015). bacterial culture adjusted to 0.5 Mcfarland were added to each A suitable matrix should be cost-effective and easy to well. The experiment was done in triplicate. Growth was mea- handle. Also, the type of used matrix for immobilization sured by ELISA reader (Stat ax-2100, Germany) at 630 nm affects metabolism, physiology, activity, and viability of after 24 h incubation at 30 °C (Giti et al. 2005). Wells without the cells during storage and transportation (Das and metals were considered as control. Adholeya 2015). So, evaluating different matrices is neces- sary to find the best matrix which maintains the survival and FTIR analysis activity of microbial population. There are reports on reme- diation of heavy metals using immobilized microbial cells FTIR was used to identify major chemical functional groups (Pires et al. 2011; Rani et al. 2010) while there is no report involved in biosorption of metals by P. putida PT. Briefly, the regarding the viability of the bioremediation agents under bacterial cells were grown in the presence and absence of environmental stresses. Therefore, the aim of this study was metal ions for 24 h at 30 °C. The bacterial cells were harvested to produce effective technological tools for bioremediation by centrifugation at 3000 rpm for 10 min and washed several of heavy metals under extreme environmental conditions. times with normal saline (0.9% sodium chloride). The bacte- To achieve this objective, (1) the resistance of P. putida rial pellets were dried at 50 °C. Then, the bacterial biomass PT to heavy metals (i.e., lead and cadmium) and extracellu- was powdered and analyzed with a spectrometer in the region lar (biosorption) and intracellular (bioaccumulation) mech- −1 4000–400 cm . anisms that may be involved in metal tolerance were inves- tigated to confirm the ability of P. putida PT as an efficient metal removal agent. (2) Pseudomonas putida PT was Molecular characterization of cadA immobilized in carboxymethyl cellulose (CMC), rice bran, and a new composite based on alginate to prepare the sus- The primers cadAF (5′-TCCCCGTGGACAAGCAACC-3′) tainable metal adsorbents during a prolonged period of stor- and cadAR (5′-CGCGGCCAATCATGTTGCTC-3′)were de- age and deleterious environmental factors. (3) Heavy metal signed by using Oligo7 software to amplify an internal frag- removal efficiency and the reusability of immobilized ment (690 bp) of cadA gene. The reaction mixture (25 μl) P. putida PT in various matrices were compared. (4) The contained 0.5 μlofdNTPs,1 μlofeachprimer, 3 μlof shelf life of immobilized cells and the impact of environ- DNA template, 0.5 μlof MgCl ,2.5 μl of PCR buffer, and mental stresses (pH and temperature) on cells survival were 0.2 μl of Taq DNA polymerase. Amplification was carried out assayed. by heating the mixture for 5 min at 94 °C, followed by Ann Microbiol (2018) 68:931–942 933 30 cycles of 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C, and Morphology of heavy metal adsorbents finally an extension cycle for 5 min at 72 °C. Morphology of external surfaces and inner sections of sam- Biomass preparation ples and cells distribution in each matrix were observed by scanning electron microscope (Philips XL 30) with an accel- Avolume of 500 μl overnight bacterial culture was inoculated erating voltage of 10 kV after coating the samples with gold. to 50 ml of TSB medium and incubated at 30 °C on a rotatory shaker at 120 rpm. Bacterial cells were harvested in exponen- Heavy metal removal analysis tial growth phase (~ 2.5 × 10 CFU/ml) by centrifugation (5000 rpm for 15 min), washed twice and pellet was resus- In metal uptake batch experiments, 0.3 g of each adsorbent pended in normal saline. was added to 10 ml of metal ion solutions (60 mg/l of CdCl · 2H Oand 400mg/l of Pb(NO ) ). In control flasks, blank 2 3 2 adsorbents (matrices without bacterial cells) were used. After Preparation of heavy metal adsorbent 24-h incubation at 25 °C on a rotatory shaker at 120 rpm, the by immobilization of P. putida PT in different adsorbents were separated from each metal solution by a matrices strainer. Finally, the residual metal concentration was ana- lyzed by atomic absorption spectrophotometer (Perkin-Elmer Modified alginate (alginate–PVA–CaCO composite) 3030, USA). The metal removal efficiency R (%) was calcu- lated for each experiment by using following formula: Polyvinyl alcohol (3%) (Sigma, Germany) and sodium algi- RðÞ % ¼ C –C =C  100 i f i nate (5%) (Merck, Germany) were dissolved in distilled water and sterilized at 121 °C for 15 min. After cooling, CaCO where C and C are initial and final metal concentrations in i f (3%) was added to this solution (ratio 1:3 v/v) and mixed the solution, respectively. carefully. Then, the bacterial suspension and alginate–PVA– CaCO composite (1:1 v/v) were mixed thoroughly. This mix- Evaluation of continuous metal removal efficiency ture was extruded through a sterile syringe into CaCl (2%) and boric acid (7%) solution to form the spherical beads. The Residual metal adsorbents from previous cycle were washed beads were placed at 4 °C for 24 h to enhance the mechanical with sterile distilled water (Pal et al. 2013) and transferred to stability. Finally, the beads were collected by sieving, washed 10 ml of metal ion solutions (60 mg/l of CdCl ·2H O and 2 2 several times with sterile distilled water to remove the excess 400 mg/l of Pb (NO ) ). After 24-h incubation at 25 °C on a 3 2 amounts of boric acid and CaCl from beads surface. rotatory shaker at 120 rpm, metal adsorbents were separated Immobilized cells were stored at 4 °C in sterile distilled water. and residual metal was analyzed as described above. Carboxyl methyl cellulose Determination the viability and regeneration Bacterial suspension was mixed with an equal volume of of immobilized P. putida PT during storage CMC solution (2%) (Sigma, Germany) and extruded to FeCl solution (0.05 M) by a sterile syringe (Chandran and Survival of bacterial cells was measured at 7 days intervals for Das 2011). To increase the mechanical stability, the formed 4 weeks. Immobilized cells were released from alginate– beads were placed at 4 °C for 24 h. Thereafter, beads were PVA–CaCO and CMC beads by immersing in 10 ml of so- collected by sieving, washed several times with sterile dis- dium citrate (60 mg/l, pH 8.5) and 10 ml of NaHCO (4%) tilled water, and stored at 4 °C in sterile distilled water. solutions, respectively. Rice bran was added to 10 ml of nor- mal saline, sonicated for 2 min, followed by vortexing for Rice bran 10 min. Thereafter, bacterial cells were stained with Rhodamine-123 (Rho-123). For this purpose, Rho-123 was Rice bran was washed in ethanol (70%) followed by distilled made up (20 mg/ml in absolute ethanol) and kept at − 20 °C water to remove surface impurities. After sterilization (at as stock solution. On the day of experiment, working solution 121 °C for 15 min), bacterial suspension was added to this of Rho-123 was prepared (1 mg/ml) in phosphate-buffered matrix (1 ml bacterial suspension in each gram of rice bran), saline (PBS). Then, 100 μl of bacterial cells was added to incubated at room temperature for 5 days and then stored at 400 μl of Rho-123 working solution and placed at dark for 4 °C (Hazaimeh et al. 2014). 10 min (Baatout et al. 2006). Finally, the viable cells were 934 Ann Microbiol (2018) 68:931–942 counted by a flow cytometer through the green channel of a FTIR analysis florescence detector (FL1, 525 nm). Each experiment was done in duplicate. Some bacteria have devised various ways to withstand the heavy metal toxicity such as biosorption which can be utilized in the bioremediation of metal polluted sites (Maitra 2016). Effect of some environmental stresses on immobilized Metal biosorption by the microbial biomass happens through bacterial cells viability the metals binding to functional groups of the bacterial cell wall. The FTIR analysis of unloaded and metal loaded Effect of pH −1 P. putida PT dried biomass in the range 400–4000 cm was performed to identify the functional groups that could be in- Experiment was carried out by using flasks which contained volved in the biosorption process (Fig. 1). The peaks at 2700– 10 ml of sterile normal saline with different pH values (4, 7, −1 3400 cm belonged to OH bonds and increased absorbance and 10) at 25 °C. After 1 week, the cells were released from 2+ 2+ (%) in this region (in presence of Cd and Pb )represented each matrices and viability was measured by flow cytometry. heavy metals attachment through H-bonding onto the biomass All of the experiments were done in duplicate. and enhanced hydration of bacterial cells. The peaks at ~ −1 3300 cm indicated the presence of amide groups and the Effect of temperature shift in these peaks was due to chemical interaction of metals −1 with proteins. The bands at ~ 1240 cm confirmed the pres- Immobilized cells in various matrices were added to saline ence of asymmetric phosphate moieties in the PT strain and (pH 7) and placed at different temperatures (25 °C and the shift in these peaks represented increased hydration of 45 °C) for 1 week. Then, viability was measured by flow 2+ phosphate groups due to Pb binding. Absorbance in the cytometry. Each experiment was performed in duplicate. −1 −1 region about 1460–1440 cm and 1150–1000 cm repre- sented various C–H, C–O–Cand C–C–O vibration modes in polyesters and increased absorbance in these regions reflected Results and discussion the accumulation of polyester compounds (poly-3- hydroxybutyrate and probably phospholipids) in bacterial bio- 2+ 2+ Detection of minimal inhibitory concentration mass in presence of Cd and Pb . Absorbance change in −1 fingerprinting zone (> 1000 cm ) was related to interaction Microbes can only tolerate a confined range of pollutants, and of metals with phosphates and sulfur groups (Oves et al. extra amount of metals will cause adverse effects within the 2013). Data is shown in Table 1. According to these results, microbial cell. These adverse changes will lead to a significant metals binding to bacterial surface functional groups lead to reduction of bioremediation efficiency. To overcome this lim- stimulate of metabolic responses like hydration and polyester itation, there is a need to identify the microbes which can accumulation in this strain. The formation of varying spectra tolerate high concentrations of heavy metals (Liaqat 2017). following adsorption of the metal ions on bacterial biomass To determine the resistance of P. putida PT to lead and cad- validated the contribution of biosorption mechanism in resis- mium, concentration of each metal in bacterial culture medi- tance to toxic metals and metal ions uptake in P. putida PT. um was increased gradually until minimal inhibitory concen- 2+ 2+ tration (MIC) was achieved. The MICs of Cd and Pb were Molecular characterization of cadA 8 mM and 10 mM, respectively. Results showed that P. putida 2+ 2+ PT is more vulnerable to Cd than Pb . It was also reported Bioaccumulation is another mechanism to cope with the 2+ 2+ that Cd is more lethal than Pb for Pseudomonas heavy metal toxicity which can be used in the process of metal aeruginosa, Acinetobacter resistance, and Proteus vulgaris uptake by bacteria (Maitra 2016). Heavy metals translocating (Marzan et al. 2017). In previous studies, Enterobacter sp. p-type ATPases, which located in inner membrane of some and avirulent Bacillus anthracis PS2010 had been reported bacteria, play an important role in intracellular accumulation as heavy metal-resistant microbes which could be used for of metals. CadA protein is a member of this group involved in remediation of metal ions. These strains were resistant against transporting lead and cadmium (Hynninen 2010;Nies 2003). 2+ Pb with MIC of 3 mM. Also, Enterobacter sp. and avirulent Sulfate-reducing bacteria possessing cadA gene have been 2+ Bacillus anthracis PS2010 were resistant against Cd with reported as effective agents in the removal of toxic metals MIC of 0.4 mM and 0.6 mM, respectively. These values are from the environment (Naz et al. 2005). This gene has been lower than those shown in present work. Thus, P. putida PT reported as a good target for the screening and selection 2+ 2+ can serve as an effective bioremediation agent. biosorbent agents of Pb /Cd (Icgen and Yilmaz 2016). Ann Microbiol (2018) 68:931–942 935 2+ 2+ Fig. 1 FTIR spectra of P. putida PT in absence and presence of Pb and Cd Also, inhibition of this carrier in heavy metal-resistant bacte- efficiency. In this study, immobilization of P. putida PT was rium, Cupriavidus metallidurans CH34, reduced significantly performed in three various matrices to preserve the viability of metal uptake in this strain (Shamim et al. 2014). PCR was bioremediation agents in harsh conditions. After bacterial cell carried out to amplify an internal fragment of cadAgene from immobilization on rice bran, no change was observed in initial genomic DNA of P. putida PT by using specific primers de- shape of this matrix (Fig. 3a). Rice bran, the by-product of the signed in this study. The amplification of cadAgene (a690-bp rice milling industry, is produced in large amounts as a waste, band; Fig. 2) confirmed the presence of this carrier in P. putida causing environmental problems (Krishnani et al. 2008). This PT, as a critical factor involved in resistance as well as bioac- matrix has an insoluble structure in water with high stability cumulation of lead and cadmium in this strain. and mechanical strength. So, we used rice bran as a natural, available, and economical matrix for P. putida PT immobili- zation. According to the scanning electron microscope pic- Preparation of heavy metal adsorbent tures, the structure of rice bran fibers is angular. These angles by immobilization of P. putida PT in different matrices can provide an appropriate surface for binding the cells and In practical terms, the formulation and method of application increase efficiency of immobilization process. Scanning elec- tron microscope images indicated the binding of bacterial cells of microbial inoculants determine the potential success of bio- remediation. In general, shortly after inoculation of bacteria at the angles of this matrix (Fig. 4). Hydrogel beads based on CMC and alginate were the other into the environment without a proper carrier, the bacteria population declines rapidly (Bashan et al. 2014). This phe- adsorbents produced in this study. Hydrogel is a three dimen- sionally cross-linked network polymer with insoluble nomenon will lead to a significant reduction of bioremediation Table 1 Changes in vibrational Spectroscopic changes induced by heavy metals Vibrational modes spectra of P. putida PT strain 2+ 2+ induced by Cd and Pb −1 Increase absorption in the region 3400–2700 cm ν (OH) −1 −1 Shift at ~ 3300 cm to lower frequencies (~ 15–40 cm ) ν (NH) −1 Increase absorption in the region ~ 1460–1440 cm Various δ(C–H) modes −1 −1 2 − Shift at ~ 1240 cm to lower frequencies (~ 6 to 10 cm ) νas (PO ) −1 Increase absorption in the region ~ 1150–1000 cm Various C–O–C and C–C–O vibrations −1 Change in lower frequences than 1000 cm Phosphates and sulfur groups (Bfingerprint^ zone) 936 Ann Microbiol (2018) 68:931–942 prepare of CMC hydrogel. Scanning electron microscope im- ages represented the porous structure of the CMC beads and the position of bacterial cells (Fig. 5). The penetration of sub- strates or contaminants into the CMC beads and adsorption of these elements by immobilized cells can enhance trough these porous structure. The round white beads with diameters about 1.7 mm were synthesized after immobilization of P. putida PT in alginate/PVA/CaCO composite (Fig. 3c). Alginate is one of the most known polymers used for live cells immobili- zation. High production cost, decrease in the mechanical strength, and leakage of the entrapped cells in contact with −3 + environmental ions such as (PO ) and Na are the disad- vantages of alginate (Cruz et al. 2013;Wuetal. 2012). The number of bacterial cells transferred to the target site and penetration of contaminants into the immobilized cells de- pend on the porosity of the alginate hydrogel (Hedlund et al. 2015). Wen et al. (2018) immobilized endogenous Bacillus licheniformis on magnetic PVA-alginate compos- ite for lead ions removal. A great lead ion adsorption abil- ity by the immobilized bacterial cells was indicated by them. In this work, we tried to enhance physicochemical features of alginate beads. For the first time, we proposed alginate–PVA–CaCO composite as a new matrix for the Fig. 2 Agarose gel electrophoresis. (A) Ten kilobase pair DNA marker cells immobilization. In this composite, mechanical and (B) PCR product of cadA gene (a 690-bp band) strength and porosity of the alginate beads were improved by adding PVA as physicochemical strength enhancer structure in water. Chemical and mechanical stabilities, poros- agent and CaCO as pore forming agent (Fig. 6). Also, ity, size, and shape of particles are the most important param- these additives decreased the alginate bead production cost. eters which can effect on the heavy metal removal efficiency by hydrogels beads (Yang et al. 2011). Heavy metal removal analysis In the case of CMC, the spherical orange beads with diam- eters about 2 mm were formed (Fig. 3b). CMC is a polymer The ability of immobilized bacterial cells in various matrices with favorable physicochemical properties such as biocompat- to adsorb heavy metals was evaluated and compared. Among ibility and biodegradability. The cross-linked network forma- the blank adsorbents (matrices without bacterial cell), rice tion between CMC and iron is a simple and rapid technique to bran removed a significant amount of each metal (66.7% lead Fig. 3 Macroscopic appearance of prepared heavy metal adsorbents in different matrices. a Rice bran. b CMC beads. c Alginate–PVA–CaCO beads 3 Ann Microbiol (2018) 68:931–942 937 Fig. 4 Scanning electron microscope images of rice bran inoculated with P. putida PT. Arrows indicate the position of bacterial cells in rice bran fibers. (a, b), and (c) show different magnifications from surface of the rice bran (low to high) Fig. 5 Scanning electron microscope photographs of porous surface (a, b) and inside (c) of CMC beads. Arrow shows the distribution of immobilized bacterial cells in the internal gel matrix and 40% cadmium) followed by hydrogels of alginate–PVA– metal removal efficiency in all matrices was increased after 2+ 2+ 2+ CaCO (60% Pb and 20% Cd )and CMC(3% Pb and immobilization of Pseudomonas putida PT. This point indi- 2+ Cd 5%) (Fig. 7). Angular surfaces and numerous functional cated successful immobilization and the ability of this strain in groups (such as carboxyl and silanol), due to the presence of metal adsorption (Fig. 7). In agreement to our findings, previ- cellulose, hemicellulose, lignin, and protein in the rice bran ous study has also reported similar results (Pires et al. 2011). structure, cause high metal adsorption capacity in this matrix As shown in Fig. 7, the adsorbents had different performance (Ahmaruzzaman and Gupta 2011; Krishnani et al. 2008). against each metal. The removal efficiency was increased to 2+ 2+ 2+ 2+ Irregular and angular surfaces are represented in Fig. 4.The 96.7% Pb /40% Cd ,75.5% Pb /75% Cd , and 32% Fig. 6 Scanning electron microscope pictures of surface (a) and inner section (b, c)ofalginate–PVA–CaCO beads. Arrows indicate the position of immobilized bacterial cells in granules 938 Ann Microbiol (2018) 68:931–942 Fig. 7 Metal removal efficiency (%) by different adsorbents with initial metal concentration 2+ 400 mg/l of Pb (a) and 60 mg/l 2+ of Cd (b) at 25 °C and pH 7 after 24 2+ 2+ Pb /15% Cd by inoculated rice bran, alginate–PVA– reusable and could remove metals but with slight decrease CaCO , and CMC, respectively. except for inoculated alginate–PVA–CaCO that its effi- 3 3 ciency was increased in case of lead (79.5%). In the second Evaluation of continuous metal removal efficiency cycle, metal removal efficiency was as follows (data is shown in Fig. 8): To investigate the potential of immobilized P. putida PT Inoculated alginate–PVA–CaCO for continuous metal removal, the inoculated adsorbents 3 were separated from the residual metal solutions and used > inoculated rice bran immediately in the next cycle. In the second cycle, the > inoculated CMC: adsorbents retained their initial form. Also, they were Ann Microbiol (2018) 68:931–942 939 Fig. 8 Continuous metal removal efficiency by P. putida PT immobilized in various matrices Determination the viability and regeneration cases. One of the key factors for bioremediation by of immobilized P. putida PT during storage immobilized cell is maintaining high population of alive and active bacterial cells (Bayat et al. 2015). According The effect of matrix type on shelf life of immobilized bac- to present results, the selected matrices in this study are terial cells was analyzed during storage at 4 °C for 4 weeks suitable tools for protecting and proliferating cells. by flow cytometry. Flow cytometry is a very rapid (Berninger et al. 2018) and accurate method for evaluating the survival of immobilized bacteria compared with con- Effect of some environmental stresses on immobilized ventional methods such as plate counting or optical density bacterial cells viability measurement (Baatout et al. 2006). Rho-123 is a fluores- cent cationic dye widely used to determine the viability by Effect of environmental pH flow cytometry. In living cells, the inner part of the cell is negatively charged, in comparison with the outer part of Extreme pHs (acidic or alkaline pHs) are one of the environ- the cell due to transport systems activity. Rho-123 as a mental stresses which can effect on bacterial cells viability and viability marker can cross the cytoplasmic membrane but reduce the bioremediation efficiency. To solve this problem, is only held inside the cells that have a negative inside bacteria must be immobilized in matrices resistant to extreme membrane potential. So, the fluorescent density represents pHs (Bayatetal. 2015). In the present work, the viability was the number of living cells which can accumulate Rho-123 assessed at different pHs (4, 7, and 10) by flow cytometry to (Baatout et al. 2006). After 7 days storage at 4 °C, there compare protective effect of matrices from cells. All adsor- were significant differences between the various matrices bents retained their initial shapes in alkaline (pH 10) or acidic under study (p ≤ 0.05). At this time, viability was about (pH 4) environments, and no disruption in the adsorbents 77.65% for immobilized cells in alginate–PVA–CaCO forms or cell leakage was observed. After 1 week, more than followed by immobilized cells in rice bran (76.96%) and 96% viability was observed at neutral pH, followed by alka- CMC (68.78%). After 14 days, an increase in bacterial line (more than 86%) and acidic pH (more than 85%). There number was observed in all cases and the viability reached were no significant differences between the viability in vari- to > 95% (Fig. 9) (Fig. A at supplementary data). Increase ous matrices at the same pH (Fig. 10)(Fig. B at supplementary in the percentage of living cells (about 20%) in the second data). According to these results, immobilization matrices week may be due to the providing appropriate conditions used in this study can be protected from the cells against by the matrices for proliferation of the cells. In the last two environmental undesirable pHs and produced metal adsor- storage weeks, the number of viable cells remained con- bents can be used for treatment of aqueous solutions contam- stant and more than 97% viability was observed in all inated with heavy metals in acidic or alkaline pHs. 940 Ann Microbiol (2018) 68:931–942 Fig. 9 Immobilized bacterial cells survival in various matrices during storage. Different letters (a, b) on each curve show significant differences between matrices within the same time (P ≤ 0.05) Effect of environmental temperature 25 °C and at 4 °C (refer to previous sections), the heavy metal adsorbents could easily be stored at room temperature without High temperature is another kind of environmental stress require to additional costs to provide special temperature con- which has negative effect on viability. The number of viable ditions during storage or transposition. By increasing temper- cells was evaluated at different temperatures (25 and 45 °C) by ature from 25 °C to 45 °C, viability was decreased about 10% flow cytometry. The viability was about 98% at 25 °C, and in all cases and there were no significant differences between there were no significant differences between immobilized various matrices. Nevertheless, at 45 °C, a considerable part of bacterial cells in various matrices (Fig. 11)(Fig. C at the cells (more than 87%) survived (Fig. C). Recent results supplementary data). Since the viability nearly was same at indicated that low efficiency of metal removal due to Fig. 10 The effect of pH on immobilized cells viability. Similar letters on each bar indicate no significant difference between matrices within the same pH (P ≤ 0.05) Ann Microbiol (2018) 68:931–942 941 Fig. 11 The effect of temperature on viability. Similar letters on each bar denote no significant difference between matrices within thesametemperature(P ≤ 0.05) reduction of viable cells number especially at high tempera- matrices in this study could protect cells from undesirable ture can be enhanced by using the produced metal adsorbents conditions. Thus, prepared adsorbents in this study can be in this study. used for treatment aqueous solutions contaminated with heavy metals in broad range of environmental stresses like high or low temperatures and acidic or alkaline pH. Conclusion Funding This study was funded by the University of Isfahan and Soil and Water Research Institute of Iran. This study focused on the preparation of efficient bio-agents 2+ 2+ which can remove Cd and Pb ions from aqueous solution Compliance with ethical standards under environmental stresses. The two main aspects dominat- ing the success of bioremediation are the effectiveness of the Conflict of interest The authors declare that they have no conflict of bacterial isolate and the proper application technology. So, in interest. the first step, we endeavored to introduce P. putida strain PTas a heavy metal-resistant bacterium which could remove these Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. toxic elements from the aqueous solution by biosorption and bioaccumulation mechanisms. In the next step, for improve- Informed consent Informed consent was obtained from all individual ment of the sustainability of this strain in the contaminated participants included in the study. sites, P. putida PT was immobilized in CMC, rice bran, and a new composite based on alginate. The heavy metal removal efficiency by P. putida PT was affected by the type of immo- References bilization matrix. Immobilized P. putida PT in the modified alginate composite and rice bran removed remarkable quanti- Ahmaruzzaman M, Gupta VK (2011) Rice husk and its ash as low-cost ties of metals in two successive cycles from the medium. The adsorbents in water and wastewater treatment. Ind Eng Chem Res efficiency of heavy metal adsorption by immobilized cells is 50:13589–13613 Baatout S, De Boever P, Mergeay M (2006) Physiological changes in- strongly influenced by the number of viable and active cells. duced in four bacterial strains following oxidative stress. 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Immobilization of Pseudomonas putida PT in resistant matrices to environmental stresses: a strategy for continuous removal of heavy metals under extreme conditions

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Springer Journals
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Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-018-1402-7
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Abstract

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 CaCO 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 2+ 2+ stresses (pH and temperature). The results showed that PT strain was resistant up to 10 mM Pb and 8 mM Cd . 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–CaCO and rice bran showed the highest metal 2+ 2+ removal efficiency for Pb as 75% and Cd as 96.7%, respectively. Metal adsorbents were reusable, and the highest 2+ 2+ removal efficiency in the second cycle was observed in inoculated alginate–PVA–CaCO (79.5% Pb and 45% Cd ). 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. . . . . Keywords Heavy metals Biosorption Bioaccumulation Microbe immobilization Viability Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1402-7) contains supplementary In recent years, environmental pollution by heavy metals has material, which is available to authorized users. become a great concern worldwide. Heavy metals establish adverse effects on diversity and activity of rhizosphere micro- * Zahra Etemadifar z.etemadifar@sci.ui.ac.ir; zetemadifar@gmail.com bial population and reduce growth and quality of crops (Oves et al. 2010). Also, heavy metals arrive into human food chain through contaminated sea foods and crops (Bi et al. 2018; Department of Biology, Faculty of Sciences, University of Isfahan, Chen et al. 2018; Tchounwou et al. 2012). These elements, P.O. Box: 81746-73441, Isfahan, Iran even at low concentration, are toxic and threaten seriously to Environmental Research Institute, University of Isfahan, human health (Tchounwou et al. 2012). Isfahan, Iran 3 Unlike other pollutants, the toxicity of heavy metals does Department of Soil and Water Research, Agricultural Research, not disappear gradually because they are non-biodegradable Educating and Extension Organization, AREEO, Isfahan, Iran 932 Ann Microbiol (2018) 68:931–942 and have tendency to accumulate in the environment. Materials and methods Therefore, environmental pollution with heavy metals is a global concern and reduction heavy metals burden of soil Source of bacterial strain, maintenance, and growth and water is essential to keep healthy life. Using of chemical condition and physical methods for remediation of contaminated zones is expensive, because they require a lot of energy Bacterial strain was previously isolated from agricultural soils and reagent. Other deficiencies of these techniques are in- contaminated with heavy metals and identified as P. putida PT complete removal of metals and generation of toxic com- based on 16S rRNA gene sequence analysis (GenBank acces- pounds (Krishnani et al. 2008). Microbial cells which pres- sion number: KX963368). This strain was saved on Tryptic ent in heavy metals contaminated sites are powerful agents Soy Broth (TSB) (Merck, Germany) medium supplemented for bioremediation because they are resistant to metals tox- with glycerol at − 80 °C. Pseudomonas putida PT was icity greatly. Microbial removal is a low cost and eco- reactivated by transferring to TSB and Tryptic Soy Agar friendly approach to prevent the metals accumulation in (TSA) (Merck, Germany) media followed by incubation at the environment (Özdemir et al. 2012). However, due to 30 °C for 24 h. instability of biological systems under environmental stress- es like extreme temperature and pH, this approach is inef- Detection of MIC fectiveness (Pires et al. 2011). To overcome this limitation, bacteria must be immobilized in a nontoxic matrix. Bacterial cell growth was evaluated in the presence of differ- Providing high bacterial biomass, easy transposition, regen- ent concentrations of cadmium chloride (Sigma, Germany) eration of bacteria, reusability, providing a suitable micro- and lead nitrate (Sigma, Germany) by microtiter plate method. environment for protecting of cells against harsh conditions Briefly, 100 μl of TSB medium, 50 μl of different concentra- and toxic compounds are the advantages of immobilized tions of each metal prepared in deionized water, and 50 μlof cells in comparison with free cells (Bayat et al. 2015). bacterial culture adjusted to 0.5 Mcfarland were added to each A suitable matrix should be cost-effective and easy to well. The experiment was done in triplicate. Growth was mea- handle. Also, the type of used matrix for immobilization sured by ELISA reader (Stat ax-2100, Germany) at 630 nm affects metabolism, physiology, activity, and viability of after 24 h incubation at 30 °C (Giti et al. 2005). Wells without the cells during storage and transportation (Das and metals were considered as control. Adholeya 2015). So, evaluating different matrices is neces- sary to find the best matrix which maintains the survival and FTIR analysis activity of microbial population. There are reports on reme- diation of heavy metals using immobilized microbial cells FTIR was used to identify major chemical functional groups (Pires et al. 2011; Rani et al. 2010) while there is no report involved in biosorption of metals by P. putida PT. Briefly, the regarding the viability of the bioremediation agents under bacterial cells were grown in the presence and absence of environmental stresses. Therefore, the aim of this study was metal ions for 24 h at 30 °C. The bacterial cells were harvested to produce effective technological tools for bioremediation by centrifugation at 3000 rpm for 10 min and washed several of heavy metals under extreme environmental conditions. times with normal saline (0.9% sodium chloride). The bacte- To achieve this objective, (1) the resistance of P. putida rial pellets were dried at 50 °C. Then, the bacterial biomass PT to heavy metals (i.e., lead and cadmium) and extracellu- was powdered and analyzed with a spectrometer in the region lar (biosorption) and intracellular (bioaccumulation) mech- −1 4000–400 cm . anisms that may be involved in metal tolerance were inves- tigated to confirm the ability of P. putida PT as an efficient metal removal agent. (2) Pseudomonas putida PT was Molecular characterization of cadA immobilized in carboxymethyl cellulose (CMC), rice bran, and a new composite based on alginate to prepare the sus- The primers cadAF (5′-TCCCCGTGGACAAGCAACC-3′) tainable metal adsorbents during a prolonged period of stor- and cadAR (5′-CGCGGCCAATCATGTTGCTC-3′)were de- age and deleterious environmental factors. (3) Heavy metal signed by using Oligo7 software to amplify an internal frag- removal efficiency and the reusability of immobilized ment (690 bp) of cadA gene. The reaction mixture (25 μl) P. putida PT in various matrices were compared. (4) The contained 0.5 μlofdNTPs,1 μlofeachprimer, 3 μlof shelf life of immobilized cells and the impact of environ- DNA template, 0.5 μlof MgCl ,2.5 μl of PCR buffer, and mental stresses (pH and temperature) on cells survival were 0.2 μl of Taq DNA polymerase. Amplification was carried out assayed. by heating the mixture for 5 min at 94 °C, followed by Ann Microbiol (2018) 68:931–942 933 30 cycles of 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C, and Morphology of heavy metal adsorbents finally an extension cycle for 5 min at 72 °C. Morphology of external surfaces and inner sections of sam- Biomass preparation ples and cells distribution in each matrix were observed by scanning electron microscope (Philips XL 30) with an accel- Avolume of 500 μl overnight bacterial culture was inoculated erating voltage of 10 kV after coating the samples with gold. to 50 ml of TSB medium and incubated at 30 °C on a rotatory shaker at 120 rpm. Bacterial cells were harvested in exponen- Heavy metal removal analysis tial growth phase (~ 2.5 × 10 CFU/ml) by centrifugation (5000 rpm for 15 min), washed twice and pellet was resus- In metal uptake batch experiments, 0.3 g of each adsorbent pended in normal saline. was added to 10 ml of metal ion solutions (60 mg/l of CdCl · 2H Oand 400mg/l of Pb(NO ) ). In control flasks, blank 2 3 2 adsorbents (matrices without bacterial cells) were used. After Preparation of heavy metal adsorbent 24-h incubation at 25 °C on a rotatory shaker at 120 rpm, the by immobilization of P. putida PT in different adsorbents were separated from each metal solution by a matrices strainer. Finally, the residual metal concentration was ana- lyzed by atomic absorption spectrophotometer (Perkin-Elmer Modified alginate (alginate–PVA–CaCO composite) 3030, USA). The metal removal efficiency R (%) was calcu- lated for each experiment by using following formula: Polyvinyl alcohol (3%) (Sigma, Germany) and sodium algi- RðÞ % ¼ C –C =C  100 i f i nate (5%) (Merck, Germany) were dissolved in distilled water and sterilized at 121 °C for 15 min. After cooling, CaCO where C and C are initial and final metal concentrations in i f (3%) was added to this solution (ratio 1:3 v/v) and mixed the solution, respectively. carefully. Then, the bacterial suspension and alginate–PVA– CaCO composite (1:1 v/v) were mixed thoroughly. This mix- Evaluation of continuous metal removal efficiency ture was extruded through a sterile syringe into CaCl (2%) and boric acid (7%) solution to form the spherical beads. The Residual metal adsorbents from previous cycle were washed beads were placed at 4 °C for 24 h to enhance the mechanical with sterile distilled water (Pal et al. 2013) and transferred to stability. Finally, the beads were collected by sieving, washed 10 ml of metal ion solutions (60 mg/l of CdCl ·2H O and 2 2 several times with sterile distilled water to remove the excess 400 mg/l of Pb (NO ) ). After 24-h incubation at 25 °C on a 3 2 amounts of boric acid and CaCl from beads surface. rotatory shaker at 120 rpm, metal adsorbents were separated Immobilized cells were stored at 4 °C in sterile distilled water. and residual metal was analyzed as described above. Carboxyl methyl cellulose Determination the viability and regeneration Bacterial suspension was mixed with an equal volume of of immobilized P. putida PT during storage CMC solution (2%) (Sigma, Germany) and extruded to FeCl solution (0.05 M) by a sterile syringe (Chandran and Survival of bacterial cells was measured at 7 days intervals for Das 2011). To increase the mechanical stability, the formed 4 weeks. Immobilized cells were released from alginate– beads were placed at 4 °C for 24 h. Thereafter, beads were PVA–CaCO and CMC beads by immersing in 10 ml of so- collected by sieving, washed several times with sterile dis- dium citrate (60 mg/l, pH 8.5) and 10 ml of NaHCO (4%) tilled water, and stored at 4 °C in sterile distilled water. solutions, respectively. Rice bran was added to 10 ml of nor- mal saline, sonicated for 2 min, followed by vortexing for Rice bran 10 min. Thereafter, bacterial cells were stained with Rhodamine-123 (Rho-123). For this purpose, Rho-123 was Rice bran was washed in ethanol (70%) followed by distilled made up (20 mg/ml in absolute ethanol) and kept at − 20 °C water to remove surface impurities. After sterilization (at as stock solution. On the day of experiment, working solution 121 °C for 15 min), bacterial suspension was added to this of Rho-123 was prepared (1 mg/ml) in phosphate-buffered matrix (1 ml bacterial suspension in each gram of rice bran), saline (PBS). Then, 100 μl of bacterial cells was added to incubated at room temperature for 5 days and then stored at 400 μl of Rho-123 working solution and placed at dark for 4 °C (Hazaimeh et al. 2014). 10 min (Baatout et al. 2006). Finally, the viable cells were 934 Ann Microbiol (2018) 68:931–942 counted by a flow cytometer through the green channel of a FTIR analysis florescence detector (FL1, 525 nm). Each experiment was done in duplicate. Some bacteria have devised various ways to withstand the heavy metal toxicity such as biosorption which can be utilized in the bioremediation of metal polluted sites (Maitra 2016). Effect of some environmental stresses on immobilized Metal biosorption by the microbial biomass happens through bacterial cells viability the metals binding to functional groups of the bacterial cell wall. The FTIR analysis of unloaded and metal loaded Effect of pH −1 P. putida PT dried biomass in the range 400–4000 cm was performed to identify the functional groups that could be in- Experiment was carried out by using flasks which contained volved in the biosorption process (Fig. 1). The peaks at 2700– 10 ml of sterile normal saline with different pH values (4, 7, −1 3400 cm belonged to OH bonds and increased absorbance and 10) at 25 °C. After 1 week, the cells were released from 2+ 2+ (%) in this region (in presence of Cd and Pb )represented each matrices and viability was measured by flow cytometry. heavy metals attachment through H-bonding onto the biomass All of the experiments were done in duplicate. and enhanced hydration of bacterial cells. The peaks at ~ −1 3300 cm indicated the presence of amide groups and the Effect of temperature shift in these peaks was due to chemical interaction of metals −1 with proteins. The bands at ~ 1240 cm confirmed the pres- Immobilized cells in various matrices were added to saline ence of asymmetric phosphate moieties in the PT strain and (pH 7) and placed at different temperatures (25 °C and the shift in these peaks represented increased hydration of 45 °C) for 1 week. Then, viability was measured by flow 2+ phosphate groups due to Pb binding. Absorbance in the cytometry. Each experiment was performed in duplicate. −1 −1 region about 1460–1440 cm and 1150–1000 cm repre- sented various C–H, C–O–Cand C–C–O vibration modes in polyesters and increased absorbance in these regions reflected Results and discussion the accumulation of polyester compounds (poly-3- hydroxybutyrate and probably phospholipids) in bacterial bio- 2+ 2+ Detection of minimal inhibitory concentration mass in presence of Cd and Pb . Absorbance change in −1 fingerprinting zone (> 1000 cm ) was related to interaction Microbes can only tolerate a confined range of pollutants, and of metals with phosphates and sulfur groups (Oves et al. extra amount of metals will cause adverse effects within the 2013). Data is shown in Table 1. According to these results, microbial cell. These adverse changes will lead to a significant metals binding to bacterial surface functional groups lead to reduction of bioremediation efficiency. To overcome this lim- stimulate of metabolic responses like hydration and polyester itation, there is a need to identify the microbes which can accumulation in this strain. The formation of varying spectra tolerate high concentrations of heavy metals (Liaqat 2017). following adsorption of the metal ions on bacterial biomass To determine the resistance of P. putida PT to lead and cad- validated the contribution of biosorption mechanism in resis- mium, concentration of each metal in bacterial culture medi- tance to toxic metals and metal ions uptake in P. putida PT. um was increased gradually until minimal inhibitory concen- 2+ 2+ tration (MIC) was achieved. The MICs of Cd and Pb were Molecular characterization of cadA 8 mM and 10 mM, respectively. Results showed that P. putida 2+ 2+ PT is more vulnerable to Cd than Pb . It was also reported Bioaccumulation is another mechanism to cope with the 2+ 2+ that Cd is more lethal than Pb for Pseudomonas heavy metal toxicity which can be used in the process of metal aeruginosa, Acinetobacter resistance, and Proteus vulgaris uptake by bacteria (Maitra 2016). Heavy metals translocating (Marzan et al. 2017). In previous studies, Enterobacter sp. p-type ATPases, which located in inner membrane of some and avirulent Bacillus anthracis PS2010 had been reported bacteria, play an important role in intracellular accumulation as heavy metal-resistant microbes which could be used for of metals. CadA protein is a member of this group involved in remediation of metal ions. These strains were resistant against transporting lead and cadmium (Hynninen 2010;Nies 2003). 2+ Pb with MIC of 3 mM. Also, Enterobacter sp. and avirulent Sulfate-reducing bacteria possessing cadA gene have been 2+ Bacillus anthracis PS2010 were resistant against Cd with reported as effective agents in the removal of toxic metals MIC of 0.4 mM and 0.6 mM, respectively. These values are from the environment (Naz et al. 2005). This gene has been lower than those shown in present work. Thus, P. putida PT reported as a good target for the screening and selection 2+ 2+ can serve as an effective bioremediation agent. biosorbent agents of Pb /Cd (Icgen and Yilmaz 2016). Ann Microbiol (2018) 68:931–942 935 2+ 2+ Fig. 1 FTIR spectra of P. putida PT in absence and presence of Pb and Cd Also, inhibition of this carrier in heavy metal-resistant bacte- efficiency. In this study, immobilization of P. putida PT was rium, Cupriavidus metallidurans CH34, reduced significantly performed in three various matrices to preserve the viability of metal uptake in this strain (Shamim et al. 2014). PCR was bioremediation agents in harsh conditions. After bacterial cell carried out to amplify an internal fragment of cadAgene from immobilization on rice bran, no change was observed in initial genomic DNA of P. putida PT by using specific primers de- shape of this matrix (Fig. 3a). Rice bran, the by-product of the signed in this study. The amplification of cadAgene (a690-bp rice milling industry, is produced in large amounts as a waste, band; Fig. 2) confirmed the presence of this carrier in P. putida causing environmental problems (Krishnani et al. 2008). This PT, as a critical factor involved in resistance as well as bioac- matrix has an insoluble structure in water with high stability cumulation of lead and cadmium in this strain. and mechanical strength. So, we used rice bran as a natural, available, and economical matrix for P. putida PT immobili- zation. According to the scanning electron microscope pic- Preparation of heavy metal adsorbent tures, the structure of rice bran fibers is angular. These angles by immobilization of P. putida PT in different matrices can provide an appropriate surface for binding the cells and In practical terms, the formulation and method of application increase efficiency of immobilization process. Scanning elec- tron microscope images indicated the binding of bacterial cells of microbial inoculants determine the potential success of bio- remediation. In general, shortly after inoculation of bacteria at the angles of this matrix (Fig. 4). Hydrogel beads based on CMC and alginate were the other into the environment without a proper carrier, the bacteria population declines rapidly (Bashan et al. 2014). This phe- adsorbents produced in this study. Hydrogel is a three dimen- sionally cross-linked network polymer with insoluble nomenon will lead to a significant reduction of bioremediation Table 1 Changes in vibrational Spectroscopic changes induced by heavy metals Vibrational modes spectra of P. putida PT strain 2+ 2+ induced by Cd and Pb −1 Increase absorption in the region 3400–2700 cm ν (OH) −1 −1 Shift at ~ 3300 cm to lower frequencies (~ 15–40 cm ) ν (NH) −1 Increase absorption in the region ~ 1460–1440 cm Various δ(C–H) modes −1 −1 2 − Shift at ~ 1240 cm to lower frequencies (~ 6 to 10 cm ) νas (PO ) −1 Increase absorption in the region ~ 1150–1000 cm Various C–O–C and C–C–O vibrations −1 Change in lower frequences than 1000 cm Phosphates and sulfur groups (Bfingerprint^ zone) 936 Ann Microbiol (2018) 68:931–942 prepare of CMC hydrogel. Scanning electron microscope im- ages represented the porous structure of the CMC beads and the position of bacterial cells (Fig. 5). The penetration of sub- strates or contaminants into the CMC beads and adsorption of these elements by immobilized cells can enhance trough these porous structure. The round white beads with diameters about 1.7 mm were synthesized after immobilization of P. putida PT in alginate/PVA/CaCO composite (Fig. 3c). Alginate is one of the most known polymers used for live cells immobili- zation. High production cost, decrease in the mechanical strength, and leakage of the entrapped cells in contact with −3 + environmental ions such as (PO ) and Na are the disad- vantages of alginate (Cruz et al. 2013;Wuetal. 2012). The number of bacterial cells transferred to the target site and penetration of contaminants into the immobilized cells de- pend on the porosity of the alginate hydrogel (Hedlund et al. 2015). Wen et al. (2018) immobilized endogenous Bacillus licheniformis on magnetic PVA-alginate compos- ite for lead ions removal. A great lead ion adsorption abil- ity by the immobilized bacterial cells was indicated by them. In this work, we tried to enhance physicochemical features of alginate beads. For the first time, we proposed alginate–PVA–CaCO composite as a new matrix for the Fig. 2 Agarose gel electrophoresis. (A) Ten kilobase pair DNA marker cells immobilization. In this composite, mechanical and (B) PCR product of cadA gene (a 690-bp band) strength and porosity of the alginate beads were improved by adding PVA as physicochemical strength enhancer structure in water. Chemical and mechanical stabilities, poros- agent and CaCO as pore forming agent (Fig. 6). Also, ity, size, and shape of particles are the most important param- these additives decreased the alginate bead production cost. eters which can effect on the heavy metal removal efficiency by hydrogels beads (Yang et al. 2011). Heavy metal removal analysis In the case of CMC, the spherical orange beads with diam- eters about 2 mm were formed (Fig. 3b). CMC is a polymer The ability of immobilized bacterial cells in various matrices with favorable physicochemical properties such as biocompat- to adsorb heavy metals was evaluated and compared. Among ibility and biodegradability. The cross-linked network forma- the blank adsorbents (matrices without bacterial cell), rice tion between CMC and iron is a simple and rapid technique to bran removed a significant amount of each metal (66.7% lead Fig. 3 Macroscopic appearance of prepared heavy metal adsorbents in different matrices. a Rice bran. b CMC beads. c Alginate–PVA–CaCO beads 3 Ann Microbiol (2018) 68:931–942 937 Fig. 4 Scanning electron microscope images of rice bran inoculated with P. putida PT. Arrows indicate the position of bacterial cells in rice bran fibers. (a, b), and (c) show different magnifications from surface of the rice bran (low to high) Fig. 5 Scanning electron microscope photographs of porous surface (a, b) and inside (c) of CMC beads. Arrow shows the distribution of immobilized bacterial cells in the internal gel matrix and 40% cadmium) followed by hydrogels of alginate–PVA– metal removal efficiency in all matrices was increased after 2+ 2+ 2+ CaCO (60% Pb and 20% Cd )and CMC(3% Pb and immobilization of Pseudomonas putida PT. This point indi- 2+ Cd 5%) (Fig. 7). Angular surfaces and numerous functional cated successful immobilization and the ability of this strain in groups (such as carboxyl and silanol), due to the presence of metal adsorption (Fig. 7). In agreement to our findings, previ- cellulose, hemicellulose, lignin, and protein in the rice bran ous study has also reported similar results (Pires et al. 2011). structure, cause high metal adsorption capacity in this matrix As shown in Fig. 7, the adsorbents had different performance (Ahmaruzzaman and Gupta 2011; Krishnani et al. 2008). against each metal. The removal efficiency was increased to 2+ 2+ 2+ 2+ Irregular and angular surfaces are represented in Fig. 4.The 96.7% Pb /40% Cd ,75.5% Pb /75% Cd , and 32% Fig. 6 Scanning electron microscope pictures of surface (a) and inner section (b, c)ofalginate–PVA–CaCO beads. Arrows indicate the position of immobilized bacterial cells in granules 938 Ann Microbiol (2018) 68:931–942 Fig. 7 Metal removal efficiency (%) by different adsorbents with initial metal concentration 2+ 400 mg/l of Pb (a) and 60 mg/l 2+ of Cd (b) at 25 °C and pH 7 after 24 2+ 2+ Pb /15% Cd by inoculated rice bran, alginate–PVA– reusable and could remove metals but with slight decrease CaCO , and CMC, respectively. except for inoculated alginate–PVA–CaCO that its effi- 3 3 ciency was increased in case of lead (79.5%). In the second Evaluation of continuous metal removal efficiency cycle, metal removal efficiency was as follows (data is shown in Fig. 8): To investigate the potential of immobilized P. putida PT Inoculated alginate–PVA–CaCO for continuous metal removal, the inoculated adsorbents 3 were separated from the residual metal solutions and used > inoculated rice bran immediately in the next cycle. In the second cycle, the > inoculated CMC: adsorbents retained their initial form. Also, they were Ann Microbiol (2018) 68:931–942 939 Fig. 8 Continuous metal removal efficiency by P. putida PT immobilized in various matrices Determination the viability and regeneration cases. One of the key factors for bioremediation by of immobilized P. putida PT during storage immobilized cell is maintaining high population of alive and active bacterial cells (Bayat et al. 2015). According The effect of matrix type on shelf life of immobilized bac- to present results, the selected matrices in this study are terial cells was analyzed during storage at 4 °C for 4 weeks suitable tools for protecting and proliferating cells. by flow cytometry. Flow cytometry is a very rapid (Berninger et al. 2018) and accurate method for evaluating the survival of immobilized bacteria compared with con- Effect of some environmental stresses on immobilized ventional methods such as plate counting or optical density bacterial cells viability measurement (Baatout et al. 2006). Rho-123 is a fluores- cent cationic dye widely used to determine the viability by Effect of environmental pH flow cytometry. In living cells, the inner part of the cell is negatively charged, in comparison with the outer part of Extreme pHs (acidic or alkaline pHs) are one of the environ- the cell due to transport systems activity. Rho-123 as a mental stresses which can effect on bacterial cells viability and viability marker can cross the cytoplasmic membrane but reduce the bioremediation efficiency. To solve this problem, is only held inside the cells that have a negative inside bacteria must be immobilized in matrices resistant to extreme membrane potential. So, the fluorescent density represents pHs (Bayatetal. 2015). In the present work, the viability was the number of living cells which can accumulate Rho-123 assessed at different pHs (4, 7, and 10) by flow cytometry to (Baatout et al. 2006). After 7 days storage at 4 °C, there compare protective effect of matrices from cells. All adsor- were significant differences between the various matrices bents retained their initial shapes in alkaline (pH 10) or acidic under study (p ≤ 0.05). At this time, viability was about (pH 4) environments, and no disruption in the adsorbents 77.65% for immobilized cells in alginate–PVA–CaCO forms or cell leakage was observed. After 1 week, more than followed by immobilized cells in rice bran (76.96%) and 96% viability was observed at neutral pH, followed by alka- CMC (68.78%). After 14 days, an increase in bacterial line (more than 86%) and acidic pH (more than 85%). There number was observed in all cases and the viability reached were no significant differences between the viability in vari- to > 95% (Fig. 9) (Fig. A at supplementary data). Increase ous matrices at the same pH (Fig. 10)(Fig. B at supplementary in the percentage of living cells (about 20%) in the second data). According to these results, immobilization matrices week may be due to the providing appropriate conditions used in this study can be protected from the cells against by the matrices for proliferation of the cells. In the last two environmental undesirable pHs and produced metal adsor- storage weeks, the number of viable cells remained con- bents can be used for treatment of aqueous solutions contam- stant and more than 97% viability was observed in all inated with heavy metals in acidic or alkaline pHs. 940 Ann Microbiol (2018) 68:931–942 Fig. 9 Immobilized bacterial cells survival in various matrices during storage. Different letters (a, b) on each curve show significant differences between matrices within the same time (P ≤ 0.05) Effect of environmental temperature 25 °C and at 4 °C (refer to previous sections), the heavy metal adsorbents could easily be stored at room temperature without High temperature is another kind of environmental stress require to additional costs to provide special temperature con- which has negative effect on viability. The number of viable ditions during storage or transposition. By increasing temper- cells was evaluated at different temperatures (25 and 45 °C) by ature from 25 °C to 45 °C, viability was decreased about 10% flow cytometry. The viability was about 98% at 25 °C, and in all cases and there were no significant differences between there were no significant differences between immobilized various matrices. Nevertheless, at 45 °C, a considerable part of bacterial cells in various matrices (Fig. 11)(Fig. C at the cells (more than 87%) survived (Fig. C). Recent results supplementary data). Since the viability nearly was same at indicated that low efficiency of metal removal due to Fig. 10 The effect of pH on immobilized cells viability. Similar letters on each bar indicate no significant difference between matrices within the same pH (P ≤ 0.05) Ann Microbiol (2018) 68:931–942 941 Fig. 11 The effect of temperature on viability. Similar letters on each bar denote no significant difference between matrices within thesametemperature(P ≤ 0.05) reduction of viable cells number especially at high tempera- matrices in this study could protect cells from undesirable ture can be enhanced by using the produced metal adsorbents conditions. Thus, prepared adsorbents in this study can be in this study. used for treatment aqueous solutions contaminated with heavy metals in broad range of environmental stresses like high or low temperatures and acidic or alkaline pH. Conclusion Funding This study was funded by the University of Isfahan and Soil and Water Research Institute of Iran. This study focused on the preparation of efficient bio-agents 2+ 2+ which can remove Cd and Pb ions from aqueous solution Compliance with ethical standards under environmental stresses. The two main aspects dominat- ing the success of bioremediation are the effectiveness of the Conflict of interest The authors declare that they have no conflict of bacterial isolate and the proper application technology. So, in interest. the first step, we endeavored to introduce P. putida strain PTas a heavy metal-resistant bacterium which could remove these Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. toxic elements from the aqueous solution by biosorption and bioaccumulation mechanisms. 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Journal

Annals of Microbiology – Springer Journals

Published: Nov 12, 2018

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