THE EF FECT OF GLUCONIC ACID SE CRE TION
BY PHOS PHATE-SOLUBILIZING PSEU DO MO NAS PUTIDA
BAC TE RIA ON DIS SO LU TION OF PYROMORPHITE Pb
5
(PO
4
)
3
Cl
AND Pb REMOBILIZATION
Justyna TOPOLSKA1, Pawel BOROWICZ2, Maciej MANECKI1, Tomasz BAJDA1, Stefan KASCHABEK3 & Broder J. MER KEL4
1
AGH Uni ver sity of Sci ence and Tech nol ogy, Fac ulty of Ge ol ogy, Geo phys ics and En vi ron men tal Pro tec tion, De part ment of Min er al ogy, Pe trog ra phy and Geo chem is try, al. Mickiewicza 30, 30-059 Kraków, Po land;
e-mail: jm.topolska@gmail.com 2
Fac ulty of Bio chem is try, Bio phys ics and Bio tech nol ogy, Jagiellonian Uni ver sity,Gronostajowa Str. 7, 30-387 Kraków, Po land
3
In sti tute of Bio sci ences, TU Bergakademie Freiberg, Leipziger Str. 29, D-09596 Freiberg, Ger many 4
In sti tute of Ge ol ogy, TU Bergakademie Freiberg, Gustav Zeuner Str. 12, D-09596 Freiberg, Ger many
Topolska, J., Borowicz, P., Manecki, M., Bajda, T., Kaschabek, S. & Mer kel, B. J., 2013. The ef fect of gluconic acid se cre tion by phos phate-solubilizing Pseu do mo nas putida bac te ria on dis so lu tion of pyromorphite Pb5(PO4)3Cl and
Pb remobilization. Annales Societatis Geologorum Poloniae, 83: 343–351.
Ab stract: The pur pose of this study was to in ves ti gate the ef fect of bac te ri ally pro duced gluconic acid on the
dis so lu tion of pyromorphite and Pb remobilization. Pyromorphite Pb5(PO4)3Cl is formed as a prod uct of the phos
-phate-in duced treat ment of Pb-con tam i nated sites. This very sta ble min eral greatly de creases the bioavailability of Pb. In this study, bac te rial and abiotic batch ex per i ments on the dis so lu tion of pyromorphite were car ried out. In the mi cro bial ex per i ments, the min eral was dis solved in the pres ence of the phos phate–solubilizing soil bac te rium, Pseu do mo nas putida. The bac te rial growth me dium was sup ple mented with glu cose, which un der nat u ral con di -tions can be sup plied to mi crobes via sym bi o sis with plants. P. putida ac quired P from pyromorphite and en hanced its dis so lu tion. El e vated Pb con cen tra tions were ob served in the sus pen sions with bac te ria. The bac te rial se cre tion of 16.5 mM gluconic acid played a sig nif i cant role in Pb remobilization; the pH of the so lu tion dropped down from an ini tial 7.4 to 3.5. In the abiotic ex per i ments, pyromorphite was dis solved at sev eral con cen tra tions of gluconic acid and at an acidic to neu tral pH range. Both acid i fi ca tion and for ma tion of sta ble Pb-gluconate lig ands en hanced the dis so lu tion of pyromorphite and caused Pb remobilization.
Key words: Pyromorphite, Pseu do mo nas putida, gluconic acid, Pb remobilization, P-in duced method.
Manu script re ceived 28 July 2013, ac cepted 27 December 2013
IN TRO DUC TION
Pyromorphite is lead ap a tite that com monly oc curs inna ture as a sec ond ary min eral phase in the ox i da tion zones of ore de pos its (Nakamoto et al., 1969). There are sev eral meth ods of syn the siz ing pyromorphite and the prop er ties of this min eral have been stud ied ex ten sively (Baker, 1966; Dai and Hughes, 1989; Manecki, 2007; Xie and Giammar, 2007). Pyromorphite forms a con tin u ous solid so lu tion with vanadinite [Pb5(VO4)3Cl] and mimetite [Pb5(AsO4)3Cl]
(Flis et al., 2010). The high ther mo dy namic sta bil ity of these min er als is the rea son, for which they are con sid ered as metal se ques tra tion agents in ar eas con tam i nated by lead and ar senic (e.g., Bajda, 2010; Flis et al., 2011 and the lit er -a ture therein). Pyromorphite (log Ksp = -79, af ter Flis et al.,
2011) is one of the most sta ble min er als in the en vi ron ment and the pre cip i ta tion of Pb in this form greatly de creases the
bioavailability of the metal (Manecki et al., 2006). Hence,
in situ im mo bi li za tion with the use of phos phate amend
-ments is now a days one of the best stud ied and recommen-ded treat ments for sites con tam i nated by Pb (USEPA, 2005). In this treat ment method, the dis so lu tion of ap a tite re sults in phos phate re lease. The PO43- ions com bine with
Pb and pre cip i tate as nanoparticles of the min eral pyromor-phite, Pb5(PO4)3Cl (e.g., Ma et al., 1993, 1994a, b, 1995;
Ruby et al., 1994; Cot ter-Howells, 1996; Cot ter-Howells and Caporn, 1996).
Re cently, a con tro ver sial op ti mi za tion of the method was pre sented by Park et al. (2011a, b). They pro pose to in -oc u late the phos phate amend ments with a live strain of phos phate-solubilizing bac te ria (PSB). It is as sumed that the mi crobes en hance the dis so lu tion of min eral P, pro mot
-ing its trans for ma tion into pyromorphite. The con tro versy of the method stems from the need to in tro duce a liv ing, ex tra ne ous strain of bac te ria into an un con trolled en vi ron -ment, al though the long-term ef fect of such a treat ment is un known. Re cently, the pres ent au thors showed that PSB can af fect the sta bil ity of pyromorphite and that the ef fec tive ness of the pro cess de pends on the avail abil ity of dis solved phos phates in so lu tion (Topolska et al., 2014). How -ever, the in ter ac tion be tween mi crobes and min er als are com plex and some as pects of the po ten tial in volve ment of PSB in remediation treat ments re main un clear.
There is some ev i dence that var i ous or ganic com pounds, mi cro bial me tab o lites and plant ac tiv ity may in crease the dis so lu tion of pyromorphite and cause a sec ond -ary Pb re lease (e.g., Sayer et al., 1997; Formina et al., 2004; Manecki, 2007; Manecki and Maurice, 2008; Debela et al., 2010, 2013). The se cre tion of or ganic ac ids is of ten in di -cated as the ac tive bac te rial strat egy for solubilizing min eral nu tri ents (e.g., Vyas and Gulati, 2009; Debela et al., 2010). In these terms, ef fi ciency de pends, among other things, on the avail abil ity of a source of or ganic car bon for the mi -crobes and glu cose seems to be par tic u larly ef fec tive (Nautiyal, 1999). If the bac te ria uti lize glu cose, they ex crete con -sid er able amounts of gluconic acid, which greatly en hances the dis so lu tion of min eral phos phates (Lin et al., 2006; Buch et al., 2008). Some bac te ria, in clud ing var i ous spe cies of the PSB, can live in the rhizosphere in sym bi o sis with the roots of plants, which sup ply nu tri ents, such as glu cose, to a sym bi otic mi cro bial strain (e.g., Espinosa-Urgel et al., 2002; Rosas et al., 2006; Buch et al., 2008; Vyas and Gu-lati, 2009). Hence, the sym bi otic bac te ria ex hibit par tic u lar ca pa bil i ties of dis solv ing the min eral forms of phos phates via or ganic acid pro duc tion, which makes the nu tri ent P more avail able for the plants.
The ef fect of glu cose as a sub strate for the bac te ria on pyromorphite sta bil ity is un known and this is sue is in ter est -ing for at least two rea sons. The first one is re lated to the fact that the gluconic acid is se creted by bac te ria via sym bi -o sis with plants’ r-o-ots and this is likely t-o -oc cur in the rhizosphere, if the PSB are in tro duced into the Pbcon tam i nated soil. The sec ond rea son is that glu cose is a rec om mended car bon source for bac te rial growthme dia, as a sub strate sup port ing mi cro bial ca pa bil i ties for solubilizing min -eral phos phates (Nautiyal, 1999). Hence, there is a strong pos si bil ity that the PSB strain, pre pared in the lab o ra tory for remediation treat ment with the use of phos phate amend -ments, will be sup plied with glu cose and that the gluconic acid will be se creted as soon as the bac te ria are in the soil.
To fill this gap in knowl edge and clar ify the vague is -sues, re lated to bac te rial im pact on pyromorphite sta bil ity, the au thors con ducted mi cro bial and abiotic batch ex per i -ments, in which they in ves ti gated (i) the growth of the PSB
Pseu do mo nas putida in the pres ence of glu cose and
pyro-morphite as a phos phate source; (ii) the ef fect of glu cose as a car bon source for the PSB P. putida on the sta bil ity of pyromorphite and Pb remobilization; and (iii) the ef fect of gluconic acid on pyromorphite sta bil ity to un der stand the mech a nisms be hind point (ii). The bac te rium Pseu do mo nas
putida was se lected for this pro ject for be ing com mon, rather than for be ing unique or un usual. These mi crobes are
ubiq ui tous aer o bic, gramneg a tive bac te ria, com monly oc -cur ring in the en vi ron ment. Pseu do mo nas can be found in un pol luted soils as well as in sites con tam i nated with heavy met als (Roane, 1999; Leung et al., 2001; Rugierro et al., 2005; Matlakowska et al., 2008). P. putida has been re-ported to ex hibit the ca pa bil ity of solubilizing the min eral forms of phos phates via sym bi o sis with plants (e.g., EspinosaUrgel et al., 2002) and it of ten serves as a model or gan ism for en vi ron men tal, ge netic and bio en gi neer ing ex -per i ments (Reva et al., 2006).
EX PER I MEN TAL METH ODS
Ma te ri als
Syn the sis of pyromorphite (C)
A syn the sized pyromorphite Pb5(PO4)3Cl was used in
this study. The syn the sis fol lows a method pre vi ously de -scribed by Flis et al. (2010) and Topolska et al. (2014). A combination of 0.3M Pb(NO3)2, 0.14M K2HPO4 and
0.05M NaCl so lu tions was used. Equal vol umes of the so lu -tions (500mL) were si mul ta neously in tro duced, us ing a pe-ristaltic pump (flow rate 1.5 mL min-1) into a glass beaker filled partly with 1 li ter of dis tilled deionized wa ter and stir-red with a mag netic stir bar. The pre cip i tate was aged in the sus pen sion for 24 hours, as de scribed by Scheckel and Ryan (2002). Then, the pre cip i tate was washed thor oughly on a pa per fil ter (Whatman) with DDIW, air dried, and kept in a des ic ca tor un til use.
The pyromorphite was iden ti fied, us ing a HITACHI S4700 field emis sion scan ning elec tron mi cro scope cou -pled with NORAN Van tage en ergy dispersive spec trom e ter (SEM/EDS), at the In sti tute of Geo log i cal Sci ences, Jagiel-lonian Uni ver sity, Kraków.
The X-ray pow der dif frac tion (XRD) pat terns of the syn the sized sol ids were col lected with a Philips PW 3020 X’Pert-APD Diffractometer sys tem (with a Cu an ode and a graph ite mono chro ma tor), us ing a step scan mode at a step size of 0.02 2Q and a rate of 1s per step.
Prior to the bac te rial batch dis so lu tion ex per i ments, 0.05 g por tions of the min eral were ster il ized in a heater at 180°C for 3 hours. Prior to the abiotic batch dis so lu tion ex -per i ments, 0.05 g por tions of pyromorphite were prewea-thered in dis tilled deionized wa ter for 24 h and cen tri fuged. The ap plied ster il iza tion and ag ing pro ce dures did not al ter the prop er ties of the min eral.
Bac te ria
The Pseu do mo nas putida strain (IBPRS KKP 1136), used in this study, was ob tained from the com mer cially avail able col lec tion of the In sti tute of Ag ri cul tural and Food Bio tech nol ogy in War saw, Po land. For the batch dis so lu tion ex per i ments, the bac te ria were pre pared, as in the pre -vi ous study by Topolska et al. (2014): the mi crobes were grown in a phos pho rusrich me dium (MP), as de scribed be -low, un til an op ti cal den sity at 600 nm (OD600) of 0.8 was
reached (mid-log a rith mic growth); then they were pelleted by centrifugation, re sus pend ed in the ex per i men tal so lu -tions and in oc u lated 1:100 into flasks.
Batch dis so lu tion ex per i ments (B)
Bac te rial ex per i ments (C)
Three types of so lu tion were used in the bac te rial ex per i ments: a phos pho rusrich so lu tion (MP), a phos pho rusde fi cient, succinate sup ple mented so lu tion (MS), and a phos -pho rus-de fi cient, glu cose-sup ple mented so lu tion (MG). These were cre ated with the con stit u ents nec es sary for bac -te rial growth. The MP so lu tion con tained the fol low ing in-gredients per li ter: succinic acid di sod ium salt an hy drous, 5 g; K2HPO4, 0.5 g; NH4Cl, 1 g; MgSO4×7H2O, 0.2 g;
CaCl2, 0.05 g; KCl, 0.5 g; FeEDTA, 30 mM; glyc erol 6.5 g
and 0.125 mL of trace el e ments (MnSO4×H2O, 0.005 g;
CoSO4×7H2O, 0.0065 g; CuSO4, 0.0023 g; ZnSO4, 0.0033 g;
MoO3, 0.0024 g per 100 mL of wa ter). The MS and MG so
-lu tions were iden ti cal to MP, ex cept for the ab sence of K2HPO4 and glu cose in tro duced, in stead of succinate into
the MG so lu tion. The pH of the so lu tions was ad justed to ~7.3 prior to autoclaving. Re agent grade chem i cals and ul -tra-pure, dis tilled 18 MWcm-1
wa ter (Milli-Q, Millipore) were used through out.
In the ex per i ments, pyromorphite was dis solved in the pres ence of Pseu do mo nas putida bac te ria in so lu tions, con -tain ing glu cose (MG) or succinate salt (MS) as a car bon source for the mi crobes. The sus pen sions were de void of aque ous phos phate ions and the min eral pyromorphite was the only source of P for the bac te ria. The op ti cal den sity of the bac te rial sus pen sions (OD600 – to mon i tor the cul ture
growth) and the time evo lu tion of Pb con cen tra tion and pH were in ves ti gated. When the pH of the so lu tions sta bi lized (af ter 24 hours), the sus pen sions were an a lyzed for the gluconic acid con cen tra tion. To en sure that P. putida can ac quire phos pho rus from pyromorphite, the strain was also grown in MG and MS so lu tions with out any P source. To ob serve the po ten tial ef fect of glu cose on the sol u bil ity of pyromorphite, dis so lu tion of the min eral in ster ile MG and MS so lu tions were com pared.
A sum mary of the ex per i men tal con di tions is pre sented in Ta ble 1. The ex per i ments were run in trip li cate in 500 mL flasks, con tain ing 100 mL of the so lu tion. Prep a ra tion of the min eral par ti cles and of the bac te ria for the ex per i ments is de scribed in the “Ma te ri als” sec tion. The flasks were in cu -bated at room tem per a ture (21°C ± 1°C) on a gyratory sha-ker (100 rpm) for 160 h, un til a full bac te rial growth cy cle
was ob served in the flask with the MG so lu tion. The aer a tion speed al lowed the min eral par ti cles to set tle to the bot -tom of the flask, so that they did not in ter fere with absor-bance read ings; a ster ile MP so lu tion amended with pyro-morphite pre cip i tate was used as a blank. Liq uid growth cul tures were mon i tored for con tam i na tion by streak ing agar plates and ex am in ing the mor phol ogy of the re sult ing col o nies. Any sign of con tam i na tion re sulted in ter mi na tion of the ex per i ment.
Abiotic ex per i ments
Three abiotic ex per i ments (marked as E1, E2 and E3) on the dis so lu tion of pyromorphite were car ried out in the pres ence of gluconic acid; a de tailed sum mary of the ex per i -men tal setup is pre sented in Ta ble 2. In Ex per i -ment 1 (E1) – in ves ti gat ing the to tal ef fect of gluconic acid on pyromor-phite sol u bil ity – the min eral was dis solved in so lu tions with sev eral gluconic acid con cen tra tions. The pH of the so -lu tions equil i brated spon ta ne ously. The Ex per i ment 2 (E2) de ter mined the role of metal-che lat ing ca pa bil i ties of the acid in dis so lu tion of the min eral. Hence, the pH of all gluconic acid so lu tions was ad justed to 3.5 with the use of HNO3 and KOH. In the Ex per i ment 3 (E3), the dis so lu tion
re ac tion was per formed un der acidic to neu tral con di tions, which are likely to oc cur at Pb-con tam i nated sites.
In all ex per i ments, 0.05 g por tions of preweathered syn thetic pyromorphite were in tro duced to poly propy lene bot -tles, con tain ing 100 mL of the sol vent; 0.05 M KNO3 was
used as a back ground elec tro lyte. The flasks were in cu bated on a gyratory shaker (100 rpm), at 21°C ± 1°C for 2 months. For this time, the so lu tions were sam pled reg u larly for anal -y sis of the con cen tra tion of dis solved Pb and P, and for pH mea sure ments. To re move the sus pended sol ids, the sam -ples were fil tered, us ing 0.2 µm polycarbonate fil ters. Af ter the ex per i ments, the re sid ual par ti cles were sep a rated from the so lu tions by centrifugation. The ex per i ments were per -formed in trip li cate.
Ta ble 1 The ex per i men tal set – bac te rial batch dis so lu tion
ex per i ments
Experiment Solution Mineral Bacteria Time/Temp. Analysis Bacterial MG Pyromorphite (0.05 g) Pseudo-monas putida 160 h/22°C OD600, pH, Pb, gluconic acid Control 1 MS Control 2 MS – Control 3 MG – Control 4 MG Pyromorphite (0.05 g) – Control 5 MS
(MS – succinate sup ple mented so lu tion; MG – glu cose sup ple mented so lu -tion)
Ta ble 2 The ex per i men tal set – abiotic batch dis so lu tion
ex per i ments Experi-ment Solution with gluconic acid [mM] Mineral pH adjustment Time/ Temp. Analysis E1 0 0.1 1.0 10 50 Pyromorphite (0.05 g) – 1400 h/ 22°C pH, Pb, P E2 0 0.1 1.0 10 50 Pyromorphite (0.05 g) 3.5 E3 0 10 Pyromorphite (0.05 g) 2.0 3.0 4.0 5.0 6.0
An a lyt i cal meth ods
Ex per i men tal re sid ual par ti cles were char ac ter ized, us -ing a HITACHI S-4700 field emis sion scan n-ing elec tron microscope cou pled with NORAN Van tage en ergy disper-sive spec trom e ter (SEM/EDS), at the In sti tute of Geo log i cal Sci ences, Jagiellonian Uni ver sity, Kraków, and a FEI QUANTA 200 FEG at 15 kV at the Fac ulty of Ge ol ogy Geophys ics and En vi ron men tal Pro tec tion, AGH Uni ver -sity of Sci ence and Tech nol ogy, Kraków.
The op ti cal den sity of the bac te rial sus pen sions was de -ter mined by absorbance mea sure ments at 600 nm, with a use of a Cary 50 Bio UVvis i ble spectrophotometer. A ster -ile re ac tion so lu tion with pyromorphite par ti cles was used as a blank.
To tal Pb con cen tra tion was an a lyzed with a Thermo Sci en tific X Se ries ICP-MS. Prior to the el e ment anal y sis, the sam ples were cen tri fuged and fil tered us ing 0.2 µm polycarbonate fil ters to re move the bac te rial cells if needed. Then, the supernatants were di luted in 0.01M HCl and spiked with the in ter nal stan dards of Ge and Re. To tal P(V) was mea sured us ing colorimetry, the molybdene blue method of Dhar et al. (2004), us ing a Cary 50 Bio UVvis i -ble spectrophotometer. The se creted gluconic acid was
quan ti fied, us ing HPLC RP-18 col umns, ac cord ing to the method de scribed by Buch et al. (2008).
RE SULTS AND DIS CUS SION
Char ac ter iza tion of sol ids
The syn the sis, car ried out as a part of this study, re -sulted in a white ho mo ge neous pre cip i tate. It was iden ti fied as pure pyromorphite by means of XRD anal y sis. Ex am i na -tion by SEM/EDS yielded par ti cles, rang ing from 0.1 to 2 µm in size and con tain ing Pb, P, O, Cl as ma jor con stit u -ents (Fig. 1A). The re sults of the anal y sis were de scribed by Topolska et al. (2014) with ref er ence to the same ma te rial. The meth od ol ogy of syn the sis al lowed for pre cip i ta tion of small par ti cles, sim i lar to those formed in the rhizosphere at Pb-con tam i nated sites (Traina and Laperche, 1999).
Ex am i na tion of ex per i men tal re sid u als did not yield sig -nif i cant changes in the mor phol ogy of the par ti cles (Fig. 1B). Crys tals par tially lost their shape, be com ing more rounded. The el e men tal com po si tion of the re sid u als was un changed, as in di cated by EDS anal y sis, and no sec ond ary phase was found.
Bac te rial ex per i ments
The bac te rial ex per i ment was ter mi nated af ter 160 h, when the mi cro bial ex tinc tion in the me dium with glu cose was ob served – the OD600 of the sus pen sion dropped by
25% (Fig. 2). In the flasks with the min eral, growth of P.
putida was ap par ent, whereas both con trol ex per i ments, to
-tally de void of phos pho rus, did not yield bac te rial cul ture evo lu tion; af ter the ini tial growth, sys tem atic and slow die off of the mi cro bial pop u la tion oc curred. Phos pho rus is an es sen tial el e ment nec es sary for life, thus, the ap par ent mi -cro bial growth in the me dium with pyromorphite in di cated that the min eral had ef fec tively served as a phos phate source for the bac te ria. The growth of the cul ture was sig -nif i cantly greater, when glu cose in stead of succinate served as a car bon source for the bac te ria. Dur ing the 160 h of ex -per i men tal time, a full growth cy cle was ob served in the flask with the MG and pyromorphite, whereas the num ber of the bac te ria cul ti vated on succinate and the min eral was only slightly greater than in the flask with out phos pho rus. How ever, the dif fer ence be tween the last two was more ap -par ent with time.
Acid i fi ca tion of the so lu tion sup ple mented with glu -cose and in oc u lated with P. putida (Fig. 3A) was ap par ent. The de crease in pH from the ini tial 7.3 to the fi nal 3.6 was rel a tively rapid; it started be tween the 3rd and the 5th h and lasted for a fur ther 20 h (Fig. 3A). This was not ob served for the other flasks. The pH of the abiotic con trols, in which py-romorphite was dis solved in the ster ile MG or MS so lu tion, re mained at the ini tial level (Fig. 3A). In the con trol with P.
putida, MS and pyromorphite, the pH in creased slightly from
7.3 to ~9 by the end of the ex per i ment (Fig. 3A).
The sig nif i cant acid i fi ca tion in the flask with the MG so -lu tion ob vi ously in hib ited the growth of the bac te ria (Fig. 2). The op ti cal den sity of the sus pen sion with MS, kept in creas -ing af ter the ex per i men tal 160 h (data not shown), reach -ing
Fig. 1. Scan ning elec tron mi cro scope im ages of syn the sized pyromorphite used in the ex per i ments (up per im age) and re sid u als from the abiotic ex per i ments on dis so lu tion of pyromorphite in 50 mM gluconic acid (lower im age)
the max i mum absorbance of 3.5. How ever, it is worth men -tion ing here, that the cul ture ex tinc -tion ob served for the MG flask is un likely to oc cur in na ture. In the rhizosphere, the bac te ria are ex posed to dif fer ent kinds of physicochemical stress (Reva et al., 2006 and the lit er a ture therein). In re -sponse, the mi crobe con trols the flu id ity and per me abil ity of its mem brane by chang ing the length, de gree of sat u ra tion, and the cis/trans ra tio of fatty ac ids. In this way, it sur vives dif fi cult con di tions, in clud ing a low (~4) pH (Ramos et al., 1997). In the work by Reva et al. (2006), it was re ported, that in the acidic en vi ron ment, the growth of P. putida is sup pressed, when the bac te ria are ex posed to high ox y gen ten sion and a ho mo ge neous con cen tra tion of me tab o lites. In other words, it is likely that the ag i ta tion and lab o ra tory con di tions have hin dered a nat u ral pro tec tive mech a nism of the bac te ria and as a re sult the con sid er able rapid ex tinc tion of the cul ture was ob served (Fig. 2). The pro duc tion of or -ganic ac ids via a glu cose met a bolic path way is un likely to in hibit the growth of P. putida in its nat u ral hab i tat and the bac te ria will grow as long as car bon, ox y gen, and phos pho -rus sources are avail able.
The au thors at trib ute the acid i fi ca tion of the MG so lu -tion to the bac te rial se cre -tion of gluconic acid. The measurements in di cated that af ter sta bi li za tion of the pH, the con -cen tra tion of the gluconic acid in the flask with pyromor-phite, P. putida and MG was 16.5 ± 0.24 mM (HPLC). These find ings are in agree ment with ob ser va tions re ported by Lin et al. (2006) and Buch et al. (2008). They in di cated that the bac te rial met a bolic path way of glu cose re sults in the se cre tion of sig nif i cant amounts of gluconic acid and that the pro cess is fol lowed by rel a tively rapid and sig nif i cant acid i fi ca tion of the mi cro bial mi lieu. Lin et al. (2006) cor re -lated a de crease in so lu tion pH from an ini tial 8 to 3, with 16.3 mM of gluconic acid and 3.8 mM 2-keto-gluconic acid un der lab o ra tory con di tions. The in crease in the pH in the con trol so lu tion with succinate salt from ini tial 7.3 to fi nal ~9 was a typ i cal re sult of mi cro bial ac tiv ity in this kind of me dium and is due to so dium hy dro ly sis. This was ob served and de scribed e.g. by Dehner et al. (2010), when they were working with the same growth medium.
Dis solv ing pyromorphite re leased Pb ions into the so lu -tions (Fig. 3B). In the flasks with P. putida and pyromor-phite, the Pb con cen tra tion suc ces sively in creased with time. At the end of the ex per i ment, in both cases (MS and MG so lu tions), the Pb con cen tra tion was sig nif i cantly higher than in the ster ile con trols. Thus, pyromorphite dis so lu -tion was en hanced by the bac te rial ac tiv ity. The rate of the re ac tion was sig nif i cantly dif fer ent for the so lu tion, in which the bac te ria were sup ple mented with glu cose, as com pared with the so lu tion with succinate. At the end of the ex per i ment, the amount of Pb in the so lu tion with glu cose was 20 times higher than in the abiotic con trols, whereas in the so lu tion with succinate, it was only 5 times higher. The fi nal con cen tra tion of Pb dis solved in the sus pen sions with MG and MS was 20 ± 3 mM and 5 ± 0.4 mM, re spec tively. In the ster ile so lu tion, there was no ef fect of the pres ence of glu cose on the sol u bil ity of pyromorphite and the con cen -tra tion of dis solved Pb. In both abiotic con trols with MG and MS, pyromorphite dissolved at statistically the same level.
The re lease of Pb cor re lated with the num ber of the bac -te rial cells in the sus pen sions; the more bac -te ria, the grea-ter prog ress in the dis so lu tion. How ever, re gard less of the num ber of bac te ria, the strain sup ple mented with glu cose was par tic u larly ef fi cient in the solubilization pro cess. The
Fig. 2. Growth of Pseu do mo nas putida, ex pressed as the op ti cal den sity of the bac te rial sus pen sions. Er ror bars rep re sent stan -dard de vi a tion of trip li cates
Fig. 3. Re sults of the bac te rial ex per i ments. A. Time change of pH in ex per i men tal flasks con tain ing pyromorphite as sole source of phos phates in so lu tion. Er ror bars are de picted within the marks.
B. Con cen tra tion of Pb as a func tion of time in bac te rial ex per i
-ments and their abiotic con trols. Pb was dis solved from pyro-morphite. Er ror bars rep re sent stan dard de vi a tion of trip li cates
amount of Pb re leased dur ing the bac te rial ex per i ments was nor mal ized to the num ber of the mi crobes in the sus pen -sions. The nor mal iza tion was per formed for the cul tures in midlog a rith mic growth, when all of the en ergy of the or gan isms was con verted into di vi sion of cells, and the mi -crobes ex hibit sta tis ti cally iden ti cal fea tures. For the flask with MG, the con cen tra tion of Pb was 0.55 ± 0.07 mmol per cell *10-6, while for the flask with MS it was only 0.05 ± 0.005 mmol per cell *10-6. Ap par ently, a P. putida bac te -rium can dis solve sig nif i cantly more pyromorphite, when its metabolism is supported by glucose.
Abiotic ex per i ments
In the abiotic ex per i ment 1 (E1), pyromorphite was dis solved in the pres ence of sev eral gluconic acid con cen tra -tions and in KNO3 as a con trol. The pH of the so lu tions was
al lowed to equilibrate spon ta ne ously and at the end of the ex per i ment it was: 4.15 for the KNO3 ex per i ment, 3.78 for
the 0.1 mM gluconic acid so lu tion, 3.05 for 1mM, 2.38 for 10 mM and 2.00 for 50 mM gluconic acid. Even a small amount of the gluconic acid caused a sig nif i cant de crease in
the pH of the so lu tion. Hence, the bac te ria that base their me tab o lism on glu cose are prone to acid ify their mi lieu. In terms of dis solved Pb, in the con trol-KNO3 ex per i ment,
steady-state con di tions were reached within 24 h (Fig. 4A). The more gluconic acid in the so lu tion, the later the pla teau in the plot was ob served. For ex am ple, in the 50 mM gluconic acid so lu tion, the Pb con cen tra tion reached a max -i mum at 150 h of the ex per -i men tal t-ime, and sta b-i l-ized af ter 350 hours. Pyromorphite sol u bil ity in creased grad u ally with the gluconic acid con cen tra tion in the so lu tion. At the end of the ex per i ment, in the con trol-KNO3 so lu tion, the amount
of dis solved Pb was al most 50 times lower than in the 50 mM gluconic acid so lu tion. As stated by Drever and Stil-lings (1997), there are at least three mech a nisms, in which or ganic ac ids can in duce min eral weath er ing. De crease of pH along with an abil ity to che late met als are the most fre -quently re ported (Shen et al., 1996; Welch and Ullman, 1996; Jones, 1998; Banfield et al., 1999; Strobel, 2001; Ja-rosz-Wilkolazka and Gadd, 2003; Jones et al., 2003; Gadd 2004; Debela et al., 2010; Bajda, 2011). The protonation of a sur face and the for ma tion of or ganic ligandmetal com -plexes are com ple men tary and ad di tive pro cesses. The role of gluconic acid as a metal-che lat ing agent in the dis so lu tion of pyromorphite can be dis cussed on the ba sis of Fig. 4B.
In the abiotic ex per i ment 2 (E2), the pH of the so lu tions was ad justed to 3.5 us ing HNO3 or KOH. The ex per i men tal
pH was cho sen on the ba sis of the re sults of the mi cro bial ex per i ments, in which bac te rial ac tiv ity acid i fied the sus -pen sions to pH = 3.5. In all ex per i ments with gluconic acid, the steady-state con di tions were reached af ter 150 h, but in the con trol-KNO3 so lu tion, the pla teau of the Pb evo lu tion
pat tern ap peared af ter 350 h (Fig. 4B). In gen eral, the con -cen tra tions of Pb in the so lu tions from the Ex per i ment 2 were lower than in the Ex per i ment 1. How ever, the amount of dis solved Pb in creased grad u ally with gluconic acid con -cen tra tion in the re ac tors. The pH of the so lu tions in abiotic ex per i ment 2 was con stant. Thus the dif fer ences in Pb con -cen tra tion be tween par tic u lar flasks were the ef fects of the for ma tion of gluconate-metal com plexes. Even the low est con cen tra tion of gluconic acid in the so lu tion (0.1 mM) re -sulted in en hanced pyromorphite dis so lu tion. The amount of Pb dis solved through complexation, ex pressed as a per -cent age of the to tal Pb re leased from pyromorphite, was: 19% for 0.1 mM gluconic acid solution, 21% for 1 mM, 37% for 10 mM and 52% for 50 mM gluconic acid solution.
The sta bil ity con stant (lgK1:1) re ported for 1:1
Pb-glu-conate che lates var ies be tween 2.49 (Escandar et al., 1996) and 2.13 (Vicedomini, 1983). This puts gluconic acid among che lat ing agents of me dium strength. How ever, in the con text of pyromorphite dis so lu tion, the Pb-gluconate com plexes seem to be more sta ble than, for ex am ple, ox a -late, ac e tate, ci trate or malate. In their pa per on pyromor-phite dis so lu tion in 0.1 mM low mo lec u lar weight or ganic ac ids (LMWOA), Debela et al. (2010) ob served that the Pbox a late, ac e tate, ci trate or malate che lates had dis in te -grated within the first 100 hours of the ex per i ment. This was not the case in the pres ent study: the con cen tra tion of Pb in -creased with time and af ter steady state con di tions had been reached, the amount of Pb in the so lu tions was sta ble till the end of the ex per i ment (Fig. 4). An in ter est ing is sue is that
Fig. 4. Evo lu tion of the Pb con cen tra tion in so lu tions of the abiotic ex per i ment. A. Con cen tra tion of Pb as a func tion of time in the so lu tions from abiotic dis so lu tion ex per i ments of pyromorphite in pres ence of gluconic acid. The pH of the so lu tions was ad justed spon ta ne ously dur ing the re ac tions. B. The ef fect of for ma -tion of che lates be tween gluconate and Pb on con cen tra -tion of Pb dis solved from pyromorphite. The pH of the so lu tions was ad -justed to 3.5. The so lu tion with KNO3 was a con trol. Er ror bars
al though in the bac te rial ex per i ments, the pH of the sus pen sion with GM and pyromorphite was 3.5 and the con cen tra -tion of gluconic acid was 16.5 mM, the to tal dis solved Pb con cen tra tion was for the en tire time of the ex per i ment much lower than in any of the so lu tions from abiotic ex per i -ment 2 (com pare Figs 3B, 4B). The dif fer ence is prob a bly due to the biosorption of Pb ions on the bac te rial sur face. Con sid er ing the fact that the ex per i men tal con di tions (solid to so lu tion ra tio, vol ume of sol vent, aer a tion speed, etc.) were the same for the bac te rial and abiotic ex per i ments and the SEM anal y sis of re sid ual par ti cles did not yield any pha- ses other than pyromorphite, there is no other ra tio nal rea -son for such a dif fer ence. The prob lem of biosorption, in the con text of pyromorphite dis so lu tion in the pres ence of P.
putida, was dis cussed by Topolska et al. (2014). Some
-times, the pro cess of biosorption is mis tak enly as so ci ated with non-me tab o liz ing bac te ria and for this rea son neglec-ted dur ing in ter pre ta tion of in-vivo ex per i ments. Considering that the bac te ri ally bound metal may go back into so -lu tion with time, the pro cess should be ap pre ci ated to avoid mis lead ing findings.
The ca pa bil ity of gluconic acid to bind Pb ions var ies with the pH of the so lu tion. The con cen tra tion of Pb, re -leased by pyromorphite dur ing its 6-weeks dis so lu tion in 10 mM gluconic acid and a pH range from 2 to 6, is shown in Fig. 5A (abiotic ex per i ment 3). Ex per i ments in KNO3 were
car ried out si mul ta neously as a con trol. In terms of dis -solved Pb, the sol u bil ity of pyromorphite de creased with the pH of the so lu tions. As men tioned be fore, dis so lu tion of pyromorphite strongly de pends on the acid ity of the en vi ron ment (Manecki and Maurice, 2008). For the en tire ex -per i men tal pH range, the ef fect of for ma tion of Pb-gluco-nate che lates on dis so lu tion of pyromorphite was ap par ent: the Pb con cen tra tion was higher in the 10 mM gluconic acid so lu tions than in the KNO3 con trols (Fig. 5A). How ever,
the dif fer ence was more sig nif i cant at pH val ues above 3.0; such conditions are likely to occur in the rhizosphere.
In the so lu tions with an ini tial pH of 2.0, the mo lar Pb/P ra tio was iden ti cal, within the lim its of ex per i men tal er ror, with the stoichiometric ra tio of these el e ments in the pyromorphite’s struc ture (Fig. 5B). Fur ther more, at this pH, there was no sig nif i cant dif fer ence be tween the so lu tion with gluconic acid and the KNO3 con trol (Fig. 5B). Un der
these con di tions, the dis so lu tion of pyromorphite was con gru ent. This is in agree ment with pre vi ously re ported find ings (Flis et al., 2011). The fi nal Pb/P ra tio in the ex per i -men tal so lu tions de creased with in creas ing pH. The au thors at trib ute this mainly to readsorption of Pb cat ions on the mi- neral crys tals. The pro cess might oc cur pref er en tially in so -lu tions that are weaker pro ton do nors. A pos si ble readsorp-tion of Pb also was in di cated by Bajda (2010), as a third step of dis so lu tion of mimetite in or ganic ac ids. Mimetite, Pb5(AsO4)3Cl, is an ar senic an a logue of pyromorphite and
ex hib its sim i lar ther mo dy namic prop er ties. It is also pos si -ble, that at pH = 5.0 and 6.0, mi nor pre cip i ta tion of some sec ond ary Pb phases, such as Pb(OH)2, oc curred (Manecki
and Maurice, 2008). How ever, the ex am i na tion of the ex -per i men tal res i due, did not yield the pres ence of any phases other than pyromorphite. At a pH above 3.0, the Pb/P ra tio was sig nif i cantly higher in the gluconic acid so lu tion than in
the KNO3 con trol. We at trib ute this to the for ma tion of
Pb-gluconate com plexes, which as dis cussed above at pH > 3.0 play a sig nif i cant role in the dis so lu tion of pyromorphite. Seem ingly, the for ma tion of che lates in hib its to some ex tent the readsorbtion of Pb ions on the min eral sur face. Lang and Kaupenjohann (2003) and Hashimoto et al. (2009) have shown that the for ma tion of pyromorphite in soil can be in -hib ited by organo-metal complexes. The property of glu-conic acid of forming stable chelates with Pb might also hinder the reprecipitation of the dissolved Pb.
The pro cesses de ter min ing the bioavailability of dissol- ved Pb and P at sites remediated by the phos phate-in duced method are com plex. It is likely that the au thors’ find ings do not ad dress all as pects of the prob lem. How ever, the com -ple men tary sets of ex per i ments, in volv ing in-vivo and abio- tic sys tems, yielded re sults, which shed new light on the use of a phos phate-solubilizing bac te rium (PSB) on sites conta-minated by Pb.
The neg a tive im pact of the soil bac te ria on the sta bil ity of pyromorphite is ap par ent. Thus it can not be ruled out that at sites con tam i nated by Pb, the autochthonous mi cro or gan isms might af fect the Pin duced remediation treat ment. In -tro duc ing an ex tra ne ous strain of P-solubilizing bac te ria, as pro posed by Park et al. (2011a, b), to op ti mize this Pbim mo bi li za tion method might bring the op po site re sult. Nev er -the less, if one de cides on such a remediation treat ment, -the au thors rec om mend against the use of bac te ria, which en ter
Fig. 5. Ef fect of pH on con cen tra tion of Pb dis solved from pyromorphite (A) and on stoichiometry of the so lu tions (B) in the ex -per i ments on dis so lu tion of pyromorphite in 10 mM gluconic acid and KNO3 (con trol). Er ror bars rep re sent stan dard de vi a tion of
into sym bi o sis with na tive plants, as these might in hibit the for ma tion of pyromorphite. The au thors also sug gest that glu cose not be used as a sub strate for growth and prep a ra -tion of the po ten tial PSB strain; oth er wise the bac te ria will pro duce gluconic acid, which is an effective solvent of pyromorphite.
CON CLU SIONS
Pyromorphite, which is a sta ble (log Ksp – 79; Flis et
al., 2011) form of Pb, con sid ered not to be bioavailable, ef
fec tively served as a phos phate source for the soil bac te -rium, Pseu do mo nas putida IBPRS KKP 1136 strain, which is a com mon phos phatesolubilizing mi cro or gan ism and en -hanced the dis so lu tion of pyromorphite. The pro cess was par tic u larly ef fi cient, when the bac te rial sus pen sion was sup ple mented with glu cose. As a re sult of the me tab o lism of glu cose, P. putida se creted gluconic acid in the amount of 16.5 ± 0.24 mM. This caused a sig nif i cant acid i fi ca tion of the bac te rial mi lieu and in creased the dis solved Pb con cen -tra tion in so lu tion. Fur ther more, the ca pa bil ity of gluconic acid to form com plexes with di va lent cat ions, in clud ing Pb, played a sig nif i cant role in the dis so lu tion of pyromorphite, es pe cially at pH > 3. The Pb-gluconate che lates are relati-vely sta ble and they seem to im pede the readsorbtion of Pb ions on min eral sur faces. The pro cesses de scribed here might have con sid er able con se quences in the environment at Pb-contaminated sites, remediated by the P-induced method.
Ac knowl edge ments
The au thors grate fully ac knowl edge sup port of the MNiSW through grant N N307 771540 and AGH Uni ver sity of Sci ence and Tech nol ogy through stat u tory grant 11.11.140.319. We thank the VNG-Verbundnetz Gas AG Foun da tion for the sup port of J. To-polska through a re search grant. Maciej Manecki was par tially funded by MNiSW grant N N307 101535. We thank the Cen ter for En vi ron men tal Sci ence and Tech nol ogy (CEST) at the Uni ver sity of No tre Dame for the use of the HPLC in stru ment and Nicolai Kummer, Janosch Gröning and Beate Erler from TUBA Freiberg for help dur ing lab o ra tory work and anal y ses.
REF ER ENCES
Bajda, T., 2010. Sol u bil ity of mimetite Pb5(AsO4)3Cl at 5–55°C.
En vi ron men tal Chem is try, 7: 268–278.
Bajda, T., 2011. Dis so lu tion of mimetite Pb5(AsO4)3Cl in
low-mo lec u lar-weight or ganic ac ids and EDTA. Chelow-mosphere, 83: 1493–1501.
Baker, W. E., 1966 An Xray dif frac tion study of syn thetic mem -bers of the pyromorphite se ries. Amer i can Min er al o gists, 51: 1712–1721.
Banfield, J. F., Baker, W. W., Welch, S. A. & Tauton A., 1999. Bi o log i cal im pact on min eral dis so lu tion: ap pli ca tion of the li -chen model to un der stand ing min eral weath er ing in the rhizo- sphere. Pro ceed ings of the Na tional Acad emy of Sci ences of the United States of Amer ica, 96: 3404–3411.
Buch, A., Archana, G. & Kumar, G. N., 2008. Met a bolic chan nel -ing of glu cose to wards gluconate in phos phate-solubiliz-ing
Pseu do mo nas aeruginosa P4 un der phos pho rus de fi ciency. Re search in Mi cro bi ol ogy, 159: 635–642.
Cot terHowells, J., 1996. Lead phos phate for ma tion in soils. En vi -ron men tal Pol lu tion, 93: 9–16.
Cot terHowells, J. & Caporn, S., 1996. Remediation of con tam i -nated land by for ma tion of heavy metal phos phates. Ap plied Geo chem is try, 11: 335–342.
Crundwell, F. K., 2003. How do bac te ria in ter act with min er als? Hydrometalurgy, 71: 75–81.
Debela, F., Arocena, J. M., Thring, R. W. & Withcombe, T., 2010. Or ganic acid-in duced re lease of lead from pyromorphite and its rel e vance to rec la ma tion of Pb-con tam i nated soils. Che-mosphere, 80: 450–456.
Debela, F., Arocena, J. M., Thring, R. W. & Withcombe, T., 2013. Or ganic ac ids in hibit the for ma tion of pyromorphite and Zn-phos phate in Zn-phos pho rous amended Pb- and Zn-con tam i nated soil. Jour nal of En vi ron men tal Man age ment, 116: 156–162. Dehner, C. A., Barton, L., Maurice, P. A. & DuBois, J. L., 2010.
Size-de pend ent bioavailability of he ma tite (a-Fe2O3)
nanopar ti cles to a com mon aer o bic bac te rium. En vi ron men tal Sci -ence and Tech nol ogy, 45: 977–983.
Drever, J. I. & Stillings, L. L., 1997. The role of or ganic ac ids in min eral weath er ing. Colloids Sur face A 120: 167–181. Dhar, R. K., Zheng, Y., Rubenstone, J. & van Geen, A., 2004. A
rapid colorimetric method for mea sur ing ar senic con cen tra -tions in ground wa ter. Analytica Chimica Acta, 526: 203–209. Escandar, G. M., Peregrin, J. M., Si erra, M. G., Martino, D.,
Santoro, M., Frutos A. A., Gar cia, S. I., Labadie, G. & Sala, L. F., 1996. In ter ac tion of di va lent metal ions with Dgluconic acid in the solid phase and aque ous so lu tion. Poly he -dron, 15: 2251–2261.
EspinosaUrgel, M., Kolter, R. & Ramos, J. L., 2002. Root col o ni za tion by Pseu do mo nas putida: love at first sight. Mi cro bi ol -ogy, 148: 341–343.
Flis, J., Borkiewicz, O., Bajda, T., Manecki, M. & Klasa, J., 2010. Syn chro tron–based X-ray dif frac tion of the lead ap a tite se ries Pb10(PO4)6Cl2 – Pb10(AsO4)6Cl2. Jour nal of Syn chro tron
Radiation, 17: 207–214.
Flis, J., Manecki, M. & Bajda, T., 2011. Sol u bil ity of pyromor-phite Pb5(PO4)3Cl – mimetite Pb5(AsO4)3Cl solid so lu tion
se ries. Geochimica et Cosmochimica Acta, 75: 1858–1868. Formina, M., Al ex an der, I. J., Hill ier, S. & Gadd, G. M., 2004.
Zinc phos phate and pyromorphite solubilization by soil plant- sym bi otic fungi. Geomicrobiology Jour nal, 21: 351–366. Gadd, G. M., 2004. Mi cro bial in flu ence on metal mo bil ity and ap
-pli ca tion for bioremediation. Geoderma, 122: 109–119. Hashimoto, Y. M., Takaoka, M., Oshita, K. & Tanida, H, 2009. In
-com plete trans for ma tions of Pb to pyromorphite by phos -phate-in duced im mo bi li za tion in ves ti gated by X-ray absorp-tion fine struc ture (XAFS) spec tros copy. Chemosphere, 76: 616–622.
Illmer, P. & Schinner, F., 1995. Solubilization of in or ganic cal -cium phos phates–solubilization mech a nisms. Soil Bi ol ogy and Bio chem is try, 27: 257–263.
Jarosz-Wilkolazka, A. & Gadd, G. F., 2003. Ox a late pro duc tion by woodrot ting fungi grow ing in toxic metalamended me -dium. Chemosphere, 52: 541–547.
Jones, D. L., 1998. Or ganic ac ids in the rhizosphere – a crit i cal re -view. Plant Soil, 205: 25–44.
Jones, D. L., Den nis, P. G., Owen, A. G. & van Hees, P. A. W., 2003. Or ganic acid be hav ior in soils-mis con cep tions and kno-ledge gaps. Plant Soil, 248: 31–41.
Lang, F. & Kaupenjohann, M., 2003. Ef fect of dis solved or ganic mat ter on pre cip i ta tion and mo bil ity of lead com pound chloropyromorphite in so lu tion. Eu ro pean Jour nal of Soil Sci
-ence, 54: 139–148.
Leung, W. C., Chua, H. & Lo, W., 2001. Biosorption of heavy met als by bac te ria iso lated from ac ti vated sludge. Ap plied Bio chem is try and Bio tech nol ogy, 91–93: 171–184.
Lin, T., Huang, H., Shen, F. & Young, C., 2006. The pro tons of gluconic acid are the ma jor fac tor re spon si ble for the dis so lu -tion of tricalcium phos phate by Burkholderia cepacia CC-A174. Bioresource Tech nol ogy, 97: 957–960.
Ma, Q. Y., Traina, S. J., Lo gan, T. J., Ryan, J. A., 1993. In-situ lead im mo bi li za tion by ap a tite. En vi ron men tal Sci ence and Tech nol ogy, 27: 1803–1810.
Ma, Q. Y., Traina, S. J., Lo gan, T. J. & Ryan, J. A., 1994a. Ef fects of aque ous Al, Cd, Cu, Fe(II), Ni, and Zn on Pb im mo bi li za tion by hydroxyapatite. En vi ron men tal Sci ence and Tech nol -ogy, 28: 1219–1228.
Ma, Q. Y., Traina, S. J., Lo gan, T. J. & Ryan, J. A., 1994b. Ef fects of NO3-, Cl-, F-, SO42-, and CO32- on Pb2+ im mo bi li za tion by
hydroxyapatite. En vi ron men tal Sci ence and Tech nol ogy, 28: 408–418.
Ma, Q. I., Lo gan, T. J. & Traina, S. J., 1995. Lead im mo bi li za tion from aque ous so lu tions and con tam i nated soils us ing phos -phate rocks. En vi ron men tal Sci ence and Tech nol ogy, 29: 1118–1126.
Manecki, M., 2009. Rola i dynamika przemian piromorfitu Pb5(PO4)3Cl w œrodowisku. Rozprawy i Monografie, 176, 91
pp. UWND AGH, Kraków, [In Pol ish].
Manecki, M. & Maurice, P. A., 2008. Siderophore pro moted dis -so lu tion of pyromorphite. Soil Sci ence, 173: 821–830. Manecki, M., Bogucka, A., Bajda, T. & Borkiewicz, O., 2006. De
-crease of Pb bioavailability in soils by ad di tion of phos phate ions. En vi ron men tal Chem is try Let ters, 3: 178–181.
Matlakowska, R., Drewniak, L. & Sklodowska, A., 2008. Ar -senic-hypertolerant Pseu do mo nas iso lated from an cient gold and cop per-bear ing black shale de pos its. Geomicrobiology Jour nal, 25: 357–362.
Nakamoto, A., Urasima, Z., Sugiura, S., Nakano, H., Yachi, T. & Tadokoro, K., 1969. Pyromorphite-mimetite min er als from the OtaruMatsukura bar ite mine in Hokkaido, Ja pan. Min er -al og i c-al Jour n-al, 6: 85–101.
Nautiyal, C. S., 1999. An ef fi cient mirobiological growth me dium for screen ing phos phate solubilizing mi cro or gan isms. FEMS Mi cro bi ol ogy Let ters, 170: 265–270.
Park, J. H., Bolan, N., Megharaj, M. & Naidu, R., 2011a. Iso la tion of phos phate solubilizing bac te ria and their po ten tial for lead im mo bi li za tion in soil. Jour nal of Haz ard ous Ma te rial, 185: 829–836.
Park, J. H., Bolan, N., Merharaj, M. & Naidu, R. 2011b. Com par a -tive value of phos phate sources on the im mo bi li za tion of lead and leach ing of lead and phos pho rus in lead con tam i nated soils. Sci ence of the To tal En vi ron ment, 409: 853–860. Ramos, J. L., Duque, E., Ro dri guez-Herva, J. J., Godoy, P.,
Haidour, A., Reyes, F. & FernandezBarrero A., 1997. Mech a -nisms for sol vent tol er ance in bac te ria. Jour nal of Bi o log i cal Chem is try, 272: 3887–3890.
Reva, O. N., Weinel, Ch., Weinel, M., Böhm, K., Stjepandic, D., Hoheisel, J. D. & Burkhard, T., 2006. Func tional genomics of stress re sponse in Pseu do mo nas putida KT2440. Jour nal of Bac te ri ol ogy, 188: 4079–4092.
Roane, T. M., 1999. Lead re sis tance in two bac te rial iso lates from heavy metal–con tam i nated soils. Mi cro bial Ecol ogy, 37: 218–224.
Ro dri guez, H. & Fraga, R., 1999. Phos phate solubilizing bac te ria and their role in plant growth pro mo tion. Bio tech nol ogy Ad -vances, 17: 319–339.
Rosas, S. B., Andres, J. A., Rovera, M. & Cor rea, N. S., 2006.
Phos phate-solubilizing Pseu do mo nas putida can in flu ence the rhizobia–le gume sym bi o sis. Soil Bi ol ogy and Bio chem is try, 38: 3502–3505.
Ruby, M. V., Da vis, A. & Nichol son, A.,1994. In situ for ma tion of lead phos phates in soil as a method to im mo bi lize lead. En vi -ron men tal Sci ence and Tech nol ogy, 28: 646–654.
Rugierro, Ch. E., Boukhalfa, H., Forsythe, J. H., Lack, J. G., Hersman, L. E. & Neu, M. P., 2005. Ac ti nide and metal tox ic -ity to pro spec tive bioremediation bac te ria. En vi ron men tal Mi crobi ol ogy, 7: 88–97.
Sayer, J. A., Kierans, M. & Geoffrey, M. G., 1997. Solubilization of some nat u rally oc cur ring metal-bear ing min er als lime scale and lead phos phate by Asperigillus niger. FEMS Mi cro -bi ol ogy Let ters, 154: 29–35.
Scheckel, K. G. & Ryan, J. A., 2002. Ef fect of ag ing and pH on dis so lu tion ki net ics and sta bil ity of chloropyromorphite. En -vi ron men tal Sci ence and Tech nol ogy, 36: 2198–2204. Shen, Y., Storm, L., John son, J. & Ty ler, G., 1996. Low-mo lec u lar
or ganic ac ids in the rhizosphere soil so lu tion of beech for est cambisols de ter mined by chro ma tog ra phy us ing sup ported liq uid mem brane en rich ment tech nique. Soil Bi ol ogy and Bio -chem is try, 28: 1163–1169.
Strobel, B. W., 2001. In flu ence of veg e ta tion on low-mo lec u -lar-weight carboxylic ac ids in soil so lu tion – a re view. Geo-derma, 99: 169–198.
Topolska, J., Latowski, D., Kaschabek, S., Manecki, M., Mer kel, B. & Rakovan, J., 2014. Pb remobilization by bac te ri ally me -di ated -dis so lu tion of pyromorphite Pb5(PO4)3Cl in pres ence
of phos phatesolubilizing Pseu do mo nas putida. En vi ron men -tal Sci ence and Pol lu tion Re search, 21: 1079–1089. Traina, S. J. & Laperche, V., 1999. Con tam i nant bioavailability in
soils, sed i ments and aquatic en vi ron ments. In: Smith, J. V. (ed.), Col lo quium on Ge ol ogy, Min er al ogy, and Hu man Wel -fare. Pro ceed ings of the Na tional Acad emy of Sci ences, 96: 3365–3371.
United States En vi ron men tal Pro tec tion Agency, 2005. Best man -age ment prac tices for lead at out door shoot ing ranges. EPA-902-B-01-00, re vised June 2005. Di vi sion of En force ment and Com pli ance As sis tance, RCRA Com pli ance Branch, New York, New York 10007-1866, 103 pp. http://www2.epa. gov/lead/bestman age mentprac ticesleadout doorshoot -ing-ranges-epa-902-b-01-001-re vised-june-2005 [05.02.2011]
Vicedomini, M., 1983. Potentiometric in ves ti ga tion of the cop per(II)gluconate sys tem in acid so lu tion. Jour nal of Co or di -na tion Chem is try, 12: 307–312.
Vyas, P. & Gulati, A., 2009. Or ganic acid pro duc tion in vi tro and plant growth pro mo tion in maize un der con trolled en vi ron -ment by phos phate-solubilizing flu o res cent Pseu do mo nas. BMC Microbiolology, 9: 174–189.
Welch, S. A. & Ullman, W. J., 1996. Feld spar dis so lu tion in acidic and or ganic so lu tions: Compositional and pH de pend ence of dis so lu tion rates. Geochimica et Cosmochimica Acta, 60: 2939–2948.
Xie, L. & Giammar, D., 2007. Equi lib rium sol u bil ity and dis so lu tion rate of the lead phos phate chloropyromorphite. En vi ron -men tal Sci ence and Tech nol ogy, 41: 8050–8055.
