Enrichment and isolation of phosphorus solubilizing bacteria Outi K. Priha, Tuija H. Sarlin, Mona E. Arnold, Päivi H.-M. Kinnunen*

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1 Advanced Materials Research Online: ISSN: , Vol. 825, pp doi: / Trans Tech Publications, Switzerland Enrichment and isolation of phosphorus solubilizing bacteria Outi K. Priha, Tuija H. Sarlin, Mona E. Arnold, Päivi H.-M. Kinnunen* VTT Technical Research Centre of Finland, P.O. Box 1000, FI VTT Espoo, Finland Keywords: bioleaching, phosphorus, Burkholderia, iron ores Abstract. The aim of this study was to enrich phosphorus solubilizing microorganisms from highphosphorus iron ores, apatite ores and phosphogypsum waste. Phosphorus solubilizing microorganisms can be utilized in dephosphorization of high-phosphorus iron ores and in phosphorus leaching from fluorapatite ores. Low grade fluorapatite ore (3.6% P, ph 6.8), fluorapatite concentrate (13% P, ph 8.3), phosphogypsum waste (0.7% P, ph 2.3), iron ore 1 (0.19% P, ph 7.6) and iron ore 2 (0.18% P, ph 7.6) were used as potential sources of phosphorus solubilizing microorganisms. The samples were cultured in NBRIP media at ph 5 and 8 with either glucose or sucrose as a carbon source, and in modified 9K media at ph 1.5 and 2.5 for 3 weeks. Phosphorus solubilizing bacteria were enriched only from the fluorapatite concentrate at the ph of 8. The four obtained heterotrophic isolates were identified by 16S rrna gene sequencing, and were shown to be closest related to Burkholderia fungorum. These results indicate that the diversity of culturable phosphorus solubilizing bacteria present in apatite and iron ores is relatively low. The isolated Burkholderia strain showed phosphorus solubilizing potential. Introduction The maximally acceptable phosphorus content in iron ore for the use of steel producers is 0.075% [1]. Currently, the industry is blending high-phosphorus ore with low-phosphorus ore to meet the given specifications. However, low-phosphorus ore deposits are depleting creating a need to find strategies to decrease the phosphorus content to the desired level. Physical separation can remove some phosphorus from the ore, but generally results in iron losses. Again, the limitations of strong acid leaching are iron loss, additional processing requirements, and the cost of acid reagents. There is an increased interest in developing more cost-efficient ways to separate phosphorus from iron ore, and biological dephosphorization could be one economical process alternative. Biosolubilization of phosphorus from phosphate rock has been researched for fertilizer production purposes [2, 3]. In order to be economically feasible in regards to traditional chemical wet process, the phosphate ores need to contain over 30% P 2 O 5 with a CaO/P 2 O 5 ratio below 1.6, MgO less than 1%, and Fe 2 O 3 and Al 2 O 3 content less than 2.5%. This requirement is generally obtained by screening, scrubbing, heavy media separation, washing, roasting, calcinations, leaching and flotation. [4] Biosolubilization has the potential for selective leaching and for utilization of resources unsuitable for conventional physico-chemical processes. The aim of this study was to enrich phosphorus solubilizing microbes from high-phosphorus iron ores, apatite ores and phosphogypsum waste to be utilized in various phosphorus leaching applications such as bioleaching of apatite and biological dephosphorization of high-phosphorus iron ores. The use of indigenous microorganisms would be advantageous in these kinds of bioprocesses, since the microorganisms have adapted to specific materials and can generally outcompete exogenous microorganisms. Research on the microorganisms naturally associated with phosphate containing ores and wastes, and evaluation of their potential for phosphate solubilisation is necessary in assessing suitable biobeneficiation processes for these materials. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-05/03/16,06:57:19)

2 Advanced Materials Research Vol Materials and methods Low grade fluorapatite ore (3.6% P, ph 6.8), fluorapatite concentrate (13% P, ph 8.3), phosphogypsum waste (0.7% P, ph 2.3), iron ore 1 (0.19% P, ph 7.6) and iron ore 2 (0.18% P, ph 7.6) were used as potential sources of phosphorus solubilizing microorganisms (PSM). For isolation of heterotrophic PSM, the materials (5 g) were incubated in 100 ml NBRIP broth (10 g glucose, 5 g Ca 3 (PO 4 ) 2, 5 g MgCl 2 x 6 H 2 O, 0.25 g MgSO 4 x 7 H 2 O, 0.2 g KCl, 0.1 g (NH 4 ) 2 SO 4 per litre [5] for 3 weeks at 30 C, 140 rpm. At the end of the incubation the samples from the enrichment broth were cultured on NBRIP agar plates (NBRIP + 20 g agar l -1 ) in order to isolate PSM. Material samples were also directly cultured on NBRIP agar with either glucose or sucrose as a carbon source, and ph adjusted to 5 or 8. Ten grams of materials were shaken (140 rpm) in 90 ml of Ringer's solution for 24 h, and cultured on NBRIP plates as a dilution series and incubated at 30 C for 12 d. PSM were identified based on the formation of visible halo/zone on agar plates. For enrichment of autotrophic PSM, the materials (5 g) were incubated in modified 9K medium (3 g (NH 4 ) 2 SO 4, 0.5 g MgSO 4 x 7 H 2 O, 0.5 g K 2 HPO 4, 0.1 g KCl, 0.01 g Ca(NO 3 ) 2, 44.2 g FeSO 4 x 7 H 2 O and 10 g S 0 l -1 ; [2,3] for 3 weeks at 30 C, 140 rpm. The ph of the medium was adjusted to 1.8 with 0.5 M H 2 SO 4. The enrichment cultures were followed by ph and Redox measurements and by microscopy. The isolates with visible halo/zone on NBRIP agar plates were purified and identified by partial sequencing of their 16S rrna gene. An approximately 500-bp fragment from the 5' end of the 16S rrna gene was amplified with universal primers BSF8/20 (5 -AGAGTTTGATCCTGGCTCAG- 3 ) and BSR534/18 (5 -ATTACCGCGGCTGCTGGC-3 ). DNA extraction, PCR amplification and sequencing reactions have been described in detail in [6]. The obtained sequences were compared against GenBank sequences with BLASTN search [7]. The phosphorus bioleaching potential of the obtained isolate was studied with hydroxyapatite Ca 5 (PO 4 ) 3 OH (Sigma 21223), FeSO 4 (Sigma ), low grade fluorapatite ore, and fluorapatite concentrate. The materials were sterilized by keeping them at 105 C over-night. The bioleaching experiments were made with 180 ml of NBRIP medium without added phosphate, 2 g of substrate, and 20 ml of microbial suspension. The flasks were kept for 3 weeks in 30 C, 140 rpm. At the end of the experiment the PO P content of the filtered samples was measured with Hach-Lange LCK349 kit (Hach Lange GMBH, Düsseldorf, Germany). Results and discussion Heterotrophic phosphorus solubilizing bacteria could be enriched only from the fluorapatite concentrate samples cultured either in NBRIP broth or directly on NBRIP agar with glucose, ph 8. All four obtained heterotrophic isolates were identified by 16S rrna gene sequencing, and were shown to be closest related (99%) to Burkholderia fungorum type strain. In the autotrophic enrichment experiments no growth could be detected based on microscopy results or DNA extraction and subsequent PCR of 16S rrna gene. The results from phosphorus leaching experiments with the Burkholderia fungorum isolate are shown in Table 1. Based on the leaching yields after 21 days, B. fungorum solubilized phosphorus most efficiently from hydroxyapatite Ca 5 (OH)(PO 4 ) 3 and low grade fluorapatite ore. Table 1. Results from phosphorus bioleaching experiments with B. fungorum after 21 days. Initial P [%] PO 3-4 -P [mg l -1 ] P solubilized [%] Hydroxyapatite FePO Low grade fluorapatite ore Fluorapatite concentrate

3 64 Integration of Scientific and Industrial Knowledge on Biohydrometallurgy Burkholderia species have previously been reported to have phosphorus solubilizing potential. Lepleux et al. showed in their research on bacteria on forest soils that the most efficient mineral weathering bacteria in the forest soils belonged to the genus Burkholderia [8]. Burkholderia species with phosphate-solubilizing activity have also been isolated from iron ore of region of Minas Gerais in Brazil [9]. The phosphorus solubilizing capability of the B. fungorum isolate from this study is in the same range as in the optimised solubilisation test of the Brazilian Burkholderia isolates [10]. Conclusions As a general rule, indigenous microorganisms compete better in terms of adaptation than exogenous microorganisms [9]. The use of indigenous microorganisms would be advantageous in dephosphorization of high-phosphorus iron ore and in bioleaching of apatite ores. The results however indicated that the diversity of culturable phosphorus solubilizing bacteria present in the selected apatite and iron ores was low. When microbiologically processing these materials, there is likely a need for inoculation of the material with suitable phosphorus solubilizing microorganisms. No growth could be detected in the medium designed for acidophiles (ph 1.8). This emphasizes the fact that the ph of iron ores and apatite ores used in this study were neutral and thus they did not provide appropriate growth conditions for acidophilic microorganisms. The ph of phosphogypsum waste was 2.3, which would be in the optimal ph range for acidophiles, but no culturable acidophiles were detected in the phosphogypsum sample either. The amount of oxidizable sulphur compounds and ferrous iron in the phosphogypsum was probably too low to provide enough energy sources for acidophilic sulphur- and iron oxidizing microorganisms. All four obtained heterotrophic isolates from the fluorapatite concentrate at the ph of 8 were closest related to Burkholderia fungorum. The isolate showed phosphate solubilisation ability from various phosphate sources. The phosphate solubilisation rate obtained within 3 weeks was at a similar level compared to previous studies with Burkholderia sp. Even though the solubilisation rate of heterotrophs is generally lower than that of acidophiles, their optimum ph range is close to the natural ph of several apatite and iron ores. This means that the initial ph does not need to be adjusted in order for the microorganisms to work, and total acid consumption could be decreased. References [1] C.Y. Cheng, V.N. Misra, J. Clough, R. Mun: Dephosphorisation of Western Australian iron ore by hydrometallurgical process. Min Eng 12 (1999) [2] R. Chi, C. Xiao, H. Gao: Bioleaching of phosphorus from rock phosphate containing pyrites by Acidithiobacillus ferrooxidans. Min Eng 19 (2006) [3] C. Xiao, R. Chi, W. Li, Y. Zheng: Biosolubilisation of phosphorus from rock phosphate by moderately thermophilic and mesophilic bacteria. Min Eng 24 (2011) [4] M. Ghabaraghi, M. Irannajad, M. Noaparast: A review of the beneficiation of calcareous phosphate ores using organic acid leaching. Hydrometall 103 (2010) [5] C.S. Nautiyal: An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170 (1999) [6] K. Katina, A. Laitila, R. Juvonen, K.-H.Liukkonen, S. Kariluoto, V. Piironen, R. Landberg, P. Åman, K. Poutanen: Bran fermentation as a means to enhance technological properties and bioactivity of rye. Food Microbiol 24 (2007) [7] C. Camacho, G. Coulouris, V. Avagyan, N. Ma, J. Papadopoulos, K. Bealer, T.L. Madden: BLAST+: architecture and applications. BMC Bioinf 10 (2009) 421.

4 Advanced Materials Research Vol [8] C. Lepleux, M.P. Turpault, P. Oger, P. Frey-Klett, S. Uroz: Correlation of the abundance of betaproteobacteria on mineral surfaces with mineral weathering in forest soils. Appl Env Microbiol 78 (2012) [9] P. Delvasto, A. Valverde, A. Ballester, J. Munoz, F. González, M. Blázquez, J. Igual, C. García-Balboa: Diversity and activity of phosphate bioleaching bacteria from a highphosphorus iron ore. Hydrometall 92 (2008) [10] P. Delvasto, A. Ballester, J. Munoz, F. González, M. Blázquez, J. Igual, A. Valverde, C. García-Balboa: Mobilization of phosphorus from iron ore by the bacterium Burkholderia caribensis FeGL03. Min Eng 22 (2009) 1 9.

5 Integration of Scientific and Industrial Knowledge on Biohydrometallurgy / Enrichment and Isolation of Phosphorus Solubilizing Bacteria /