Sequenced Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion – a New Way of Resource Reclaiming

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Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion

Sequenced Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion – A New Way of Resource Reclaiming –

Wolfgang Dott, Maxime Dossin and Petra Schacht

1. Bioleaching...... 739

2. Biological phosphate enrichment...... 741

3. Combination of bioleaching and biologically induced phosphate recovery...... 743

4. Abstract...... 749

5. Bibliography...... 749

Reutilization of heavy metal contaminated solids, incineration ash in particular, is being increasingly problematic, since the use of ash in agriculture or construction industry is often not possible, due to its potential toxicity. Methods of bioleaching, known from ore extraction, may be used as an alternative remediation concept for heavy metal depletion in contaminated solids. The bioleaching bacteria of Acidithiobacillus species oxidize metal sulfides into soluble metal ions, which are then brought in solution. At the same time, sulfur compounds are oxidized by these bacteria in sulfuric acid: acid-soluble heavy metals are thereof brought in solution. In this work, contaminated solids are treated with the bioleaching process: focus is made on depletion of heavy metals in ash and their subsequent fixation, to prevent any pollution of groundwater. In addition, a selective recovery of phosphorus is achieved. Phosphorus is an absolute essential element for life, without any possible substitution through other element. This irreplaceable compound presents limited reserve, which are unequally spread in the world. It is found in marine-sedimentary deposits (approximately 90 % of inventories) and in igneous rocks (10 %) [1]. The marine-sedimentary deposits are concentrated in North Africa (Tunisia and Morocco), China and in the southeastern United States. The magmatic deposits are located mainly in Russia and Brazil. Most of the phosphate is used in agriculture in the form of phosphatic fertilizer. With the increasing world population, demand for fertilizers will continue to increase. A responsible use of phosphate would go through a recovery of this element, where it is lost up to now, namely in sewage sludge or sewage sludge ash.

1. Bioleaching The bioleaching has experienced a drastic development during the last 20 years: from un- controlled leaching of copper out of piles, to a developed biotechnological process branch [2]. With a part of up to 25 % for the mining of copper in Chile, Canada and the USA, bioleaching is now a strong economic field. The technical application of the process is the

739 Wolfgang Dott, Maxime Dossin, Petra Schacht conversion of insoluble copper, zinc and uranium ores in water-soluble metal sulfates, which are recovered after drainage, precipitation and evaporation. The development of commercial bioleaching process has made great steps in recent years. The advantages of bioleaching over conventional metal extraction are followings: • economical leaching of low concentrated or unpurified ores, • leaching proceeds at low temperatures and atmospheric pressure, • the addition of expensive chemicals is eliminated by the biogenic production of sulfuric acid, • processing is easy,

• no emission of CO2 (low energy input, microorganisms fix CO2)

3+ Fe3+ Fe

+ MO O2 H MO O2 MS MS

Figure 1: 2+ Fe2+ Fe

2+ Reaction A: oxidation of metal 2+ 2- M + H2 S (H 22S ) M + S2 O3 sulfides (MS) by microorganisms (MO), release of heavy 3+ 3+ metals and thiosulfate, oxidation MO Fe , O MO Fe , O2 2 of thiosulfate to sulfuric acid. Reaction B: proton attack by sul- 2- S O , S H2 S n furic acid on the metal sulfides, n86 release of heavy metals, oxidati- 3+ 3+ MO Fe , O2 MO Fe , O2 on of reduced sulfur compounds to sulfuric acid.

Source: Hollender, J.; Dreyer, U.; Kron- H24 SO 2- + SO4 + H berger, L.; Kämpfer, P.; Dott, W.: Selective enrichment and characterization of a Reaction A: thiosulfate Reaction B: polysulfide phosphorus-removing bacterial con- mechanism mechanism sortium from activated sludge. Applied Microbiology and Biotechnology, 2002, 58, 106-111

The leaching potential of Acidithiobacillus is based on two reactions, represented in Figure 1. Both reactions lead to a release of an important part of the heavy metals contained in the ash. The basis of bioleaching is the utilization by sulfur-oxidizing bacteria of inorganic electron donors, mainly compounds with reduced form of sulfur or elementar sulfur, which are used for energy production [4]. For bacteria, it concerns Acidithiobacillus species for example, a group of aerobic, gram-negative, chemolithotrophic bacteria that are capable of producing sulfuric acid through oxidation of reduced metal sulfides. Metals are then brought in solution. Through the biogenic production of sulfuric acid, most of phosphorus is also brought in solution into the form of phosphate anion. Table 1 summarizes the metal content of various contaminated solids (mainly incineration ash) implemanted before (left) and after (right) the leaching process. Values exceeding the German limits are highlighted. A massive decontamination of heavy metals is obtained.

740 Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion

Table 1: Metal content before (left) and after (right) in bioleaching mg/kg

Metal content EOS WS RA ZA EA AS BS EOS WS RA ZA EA AS BS of ash As 17.4 14.5 25.2 10.0 36.4 19.6 4.4 8.2 8.5 11.7 3.8 3.0 0.3 4.4 Cd – – 0.1 13.7 93.5 – 0.3 – – – 1.6 3.2 – 0.3 Cr 4,723 288 119 35 47 92 281 2,858 219 66 19 47 55 136 Cu 121 135 120 95 305 291 37 102 44 39 23 122 36 29 Pb 4.5 201.6 1.3 29.3 431.0 171.7 2.4 5.2 100.1 1.3 19.0 431.0 43.8 2.2 Ti 0.05 0.15 0.01 1.30 17.90 0.12 – – 0.04 0.01 0.70 17.59 0.03 – V 935.4 717.6 31.2 29.1 25.1 35.3 – 910.3 21.0 – – – 25.3 – Zn 198 5,647 38 876 13,434 1,420 180 209 784 13 153 270 146 – fat: Exceeding the limits LAGA ZO/Z1.1

EOS: electric furnace slag, WS: rolling mud, RA: bottom ash, ZA: cyclone fly ash, EA: electrostatic fly ash, AS: digested sludge, BS: Sittard´s soil

Source: Dott, W.: Metalllaugung von Verbrennungsaschen, 2010

2. Biological phosphate enrichment Phosphate removal in sewage treatment plants takes place mainly through chemical precipitation with iron and aluminum salts. This leads to an increase of the salinity of treated water and an increase of metal loading in waste sludge. To reduce these effects, biological phosphate removal is now used in an increasing extent. With this process, phosphate is eliminated from wastewater without any addition of coagulant. Phosphate is fixed in the biomass, which form flakes that settle in the sewage sludge. Essential mechanism of the biological removal is the ability of certain bacteria to save phosphate under the form of polyphosphate. The biological phosphate elimination is known as EBPR [6] (Enhanced Biological Phosphorus Removal) and is applied in wastewater treatment since 2000.

anaerob aerob

PHF

Acetat PHF

3- Poly-P PO4 P EPS EPS

EPS: extracellular polymeric substance PHF: Poly(hydroxy fatty acid)

Figure 2: Mechanism of storage of carbon and polyphosphate in EBPR under aerobic and anaerobic conditions

741 Wolfgang Dott, Maxime Dossin, Petra Schacht

Figure 2 presents the nechanism underlying the biological phosphate elimination. Under anaerobic conditions (left), microorganisms use acetate as carbon source, and convert it to acetyl coenzyme A (Acetyl-CoA). During this process, the consumed adenosine tri- phosphate (ATP) is restored through a transfer of phosphate molecule from intracellular polyphosphate. During the hydrolysis of intracellular polyphosphate chains, inorganic phosphate is also released out of the cell into the surrounding solution. The Acetyl-CoA molecules are condensed and stored under the form of poly(hydroxy fatty acids). When switching to aerobic conditions (right), poly(hydroxy fatty acids) are then used as a source of energy. Phosphate in the surrounding solution is then incorporated by the bacteria and stored as intracellular polyphosphate. Bacteria take more phosphate than they would need, a phenomenon called luxury uptake [7, 8, 9, 10]. Identity of the phosphate-storing microorganisms as well as the underlying mechanism, circumstances and reasons for the luxury uptake are discussed again and again. Figure 3 shows an example of the phosphate incorporation by a special culture named RSAS. The concentration of inorganic phosphate is measured during sequential aerobic and anaerobic phases. These results are a clear representation of the phosphate incorporation under aerobic condition and the phosphate release under anaerobic conditions.

phosphate acetate mg/L mg/L 80 3.000 aerob anaerob aerob

70 2.500

60 2.000 50

40 1.500

30 1.000

20 500 10

0 0 0 10 20 30 40 50 60 70 80 time h addition of acetate

Figure 3: Concentrations of inorganic phosphate and acetate in solution with the special culture RSAS. 72 h of fumigation with change between aerobic and anaerobic conditions every 24 h.

Source: Schacht, Petra: Mikrobiologische Gewinnung von langkettigen Polyphosphaten, 2011

Figure 4 gives an overview of the protein content of the solution, it means the quantity of biomass. A continuous growth of the biomass is observed.

742 Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion

protein content mg/L 800 aerob anaerob aerob 700

600

500

400

300

200

100

0 0 10 20 30 40 50 60 70 80

time h addition of acetate

Figure 4: Time course of protein content of the special culture RSAS during sequential change between aerobic and anaerobic phases

Source: Schacht, Petra: Mikrobiologische Gewinnung von langkettigen Polyphosphaten, 2011

Figure 5 gives a representation of yield and composition of polyphosphate chain lengths obtain with for three different special crops, which were cultivated under similar conditions. Extraction and determination of the distribution of the polyphosphate chains were done following the method from Clark et al.[12].

3. Combination of bioleaching and biologically induced phosphate recovery Goal of the work is to find a biotechnological process that would perform a selective recovery of phosphate after a bioleaching of heavy metals contaminated solids. In presented process, the dissolution of phosphorus from the ash, and subsequent removal of dissolved phosphate is performed in one step. A schematic representation of the mechanism is given on Figure 6. The AEDS culture (Acidithiobacillus enriched digested sludge) is used in this work. The bacterial population consists of several bacterial species/genera, whose distribution of population is represented on Figure 7. 16S rRNA gene sequence analysis and FTIR spectroscopy were used for the identification of microorganisms in the AEDS solution, and to study their activity.

743 Wolfgang Dott, Maxime Dossin, Petra Schacht

polyphosphate mg 6

147,6 mg acetate (1 x acetate) 442,73 mg acetate (3 x acetate) 885,45 mg acetate (6 x acetate)

polyphosphate polyphosphate polyphosphate mg mg mg 6 6 6

5 5 5

4 4 4

3 3 3

2 2 2

1 1 1

0 0 0 RSAS BSKN BPSB Phosphate residues: Long-chain (250-750) Medium-chain (20-250) Short-chain (< 20)

Figure 5: Composition of polyphosphates for different chain lengths (short chains: chain length <20 phosphate residues, medium: chain length of 20-250 phosphate residues, long chain: chain length of 250-750 phosphate residues) in the special crops RSAS, BSKN and BPSB with one-, three-time and six-time acetate addition

Source: Schacht, Petra: Mikrobiologische Gewinnung von langkettigen Polyphosphaten, 2011

744 Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion

EPS: extracellular polymeric substances; Me2+: metal ion

Figure 6: Mechanism of microbial phosphate storage (left) and heavy metals from solution (right)

Source: Dott, W.: Metalllaugung von Verbrennungsaschen, 2010

Iron oxidizing bacteria Polyphosphate 6 % accumulating bacteria e.g. Acinetobacter Iwoffii, New genus Rhodanobater Pseudomomas spp. 8 % 14 %

New genus Fulvimonas Acidithiobacillus ferrooxidans 19 % 52 %

Figure 7: New synthrophe bacterial population for the acid recovery of phosphorus from sewage sludge ash

745 Wolfgang Dott, Maxime Dossin, Petra Schacht

The process of selective phosphorus recovery can be described in three points: • Bioleaching by Acidithiobacillus ferrooxidans of phosphorus and heavy metals from sewage sludge ash and release of these elements in solution. • Phosphorus accumulation by polyphosphate-storing micro-organisms of the class Ac- tinobacteria. • Microbial induced Fe(III)-phosphate precipitation by Acidithiobacillus ferrooxidans. The resulting product is a phosphate-rich solid, which consists of Fe(III)-phosphate and polyphosphate. The experiments were first conducted in a laboratory-scale bioleaching reactor, schematizes on Figure 8. The bacterial suspension is sprinkled on the ash in a lysimeter: most heavy metals and phosphate dissolve. The bioleaching solution is then collected through a glass frit. The process is repeated several times in order to obtain sufficient phosphate and metal concentrations in the bacterial solution. During the continuous sprinkling of the solution, a biofilm forms on the ash.

Pump Aeration

Mixed culture Bioleaching medium: Bioleaching of percolator leaching pH 1.9 bacteria With sewage • Disolved sludge ash on pH: 2.0-1.5 metals glas frit • Disolved phosphate

Probes for analytics

Figure 8: Schematic representation of bioleaching reactors

Figure 9 represents concentrations of phosphate and some heavy-metals in the bacterial solution during the whole process. The first part of curves shows that phosphate and heavy metals are leached from the sewage sludge ash and released in the bacterial solution. The second part of curves show that phosphate is then incorporated in the biomass and removed from the solution: its concentration declines rapidly. During the incorporation of phosphate, heavy metals remain in solution: concentrations stay quasi constant und don’t decline like for phosphate. The phosphate is then removed from the solution, and is found in the biomass. This process combines succesfully a simultaneous release of phosphate and heavy-metals from sewage sludge ash, following by a selective fixation of phosphate in the biomass. This solid biomass, poor in heavy metal, can be easily separated by sedimentation and filtration.

746 Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion

Leaching % 80

0 1325days 4 Mn Al Cu Zn PO4

Figure 9: Phosphate and heavy metal concentration during the process of selective recovery of phosphate from sewage sludge ash. The results shown were obtained for 6 experiments.

Source: Dott, W.; Zimmermann, J.: Recovery of phosphorus from sewage sludge incineration ash by fractionated bioleaching and fate of heavy metals, 12th international symposium of microbial ecology, ISME 12, Cairns, Australia, August 17-22, 2008

Figure 10 gives a schematization of the process in a technical scale. Same kind of results were obtained in technical scale during the leaching of several kilograms of ash.

1. AEDS-production 2. Phosphate and 3. Phosphate recovery and heavy metal release heavy metal separation

Phosphate industry P-enriched product

agriculture

Figure 10: Schematisation of the presented P-recovery process in technical scale

747 Wolfgang Dott, Maxime Dossin, Petra Schacht

Figure 11 presents the ratio of phosphorus and metals in the combustion ash (left) and in the P-enriched biomass (right). Proportion of Metals is drastic reduced in the P-enriched sludge in comparison to raw ash.

Ratio of phosphorus and metals % 100

0 Figure 11: Incineration ash AEDS P-product Metals* Phosphorus Ratio of phosphorus and metals * sum of the metals Al, Ca, Mg, Pb, Cu, Cr, Zn in the incineration ash (left) and P-enriched biomass (right)

mg/l 350

300 Organic phosphate accumulation 30 % 250

200

150 Iron- phosphate 100 Precipitation 70 % 50

0 0 12 345678 9 10 11 12 days Fe P

Figure 12: Evolution of the concentration of iron and phosphate in the bioleaching medium

748 Bioleaching and Bioaccumulation of Phosphorus from Sludge Combustion

Iron plays an important role in the metabolism of Acidithiobacillus species: micororganisms win energy through the oxidation of Fe2+ in Fe3+. Therefore, it is presumed that iron plays an important role in the fixation of phosphate, beside the formation of polyphosphates, as can be seen on figure 12. The evolution of the concentrations of iron and phosphate in AEDS solution are represented. In the first days, evolutions of iron and phosphate concentrations show that phosphate is incorporated in the biomass. Then, a precipitation of iron-phosphate is observed. The ratio between polyphosphate and iron-phosphate is simplified on Figure 13.

Organic P (polyphosphate) 26 %

Figure 13:

FePO4 74 % Ratio of polyphosphates and iron-phosphate in the bioleaching medium

4. Abstract The recovery of phosphorus from sewage sludge incineration ash as well as the separation of heavy metals from ash was investigated by using the biotechnological process of bioleaching and bioaccumulation of released phosphorus by newly developed syntrophic population of bioleaching bacteria, Acidithiobacillus spec. strains, and polyphosphate (poly-P) accumulating bacteria, the AEDS-population (Acidithiobacillus spec. enriched digested sludge). The biologically performed solubilization of phosphorus from sewage sludge incineration ash is accompanied by the release of toxic metals. Therefore a combined process to sepa- rate phosphorus from heavy metals by achieving a plant available phosphorus-enriched product and a metal depleted ash was designed. Leaching experiments were conducted in leaching reactor containing a bacterial stock culture of Acidithiobacillus spec. Following step was the enhancement of P-recovery in combining bioleaching with simultaneous bio- P-accumulation by AEDS-population. The uptake of phosphorus in biomass reaches up to 66 % of the mobilized phosphorus by bioleaching. The combined biologically performed technology of phosphorus leaching and separation from toxic metals by simultaneous bioaccumulation developed in this work is a promising economical and ecological process for the recovery of phosphorus from waste solids.

5. Bibliography [1] Wellmer, F. W.; Becker-Platten, J. D.: Mit der Erde leben, Beiträge Geologischer Dienste zur Daseinsvorsorge und nachhaltigen Entwicklung. Berlin Heidelberg: Springer-Verlag, 1999, S. 129-13 [2] Olson, G.J.; Brierley, J.A.; Brierley, C.L.: Bioleaching review part B- Progress in bioleaching: Applications of microbial processes by the mineral industries. Applied Microbiology and Bio- technology, 2003, 63: 249-257

749 Wolfgang Dott, Maxime Dossin, Petra Schacht

[3] Hollender, J.; Dreyer, U.; Kronberger, L.; Kämpfer, P.; Dott, W.: Selective enrichment and characterization of a phosphorus-removing bacterial consortium from activated sludge. Applied Microbiology and Biotechnology, 2002, 58, 106-111 [4] Silverman, M.; Lundgren, D.: Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans – I. An improved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology, 1959, 77: 642-647 [5] Dott, W.: Metalllaugung von Verbrennungsaschen, 2010 [6] Oehmen, A.; Lemos, P.C.; Carvalho, G.; Yuan, Z.; Keller, J.; Blackall, L.L.; Reis, M.A.M: Advances in enhanced biological phophorus removal: From micro to maccro scale. Water Research, 2007, 41: 2271-2300 [7] Bark, K.; Kämpfer, P.; Sponner, A.; Dott, W.:Polyphosphate-dependent enzymes in some cory- neform bacteria isolated from sewage sludge. FEMS (Federation of European Microbiological Societies) Microbiology Letters, 1993, 107: 133-138 [8] Bark, K.: Enzyme des Polyphosphatstoffwechsels unterschiedlicher Bakterien im Zusammen- hang mit der biologischen Phosphateliminierung aus Abwasser. Dott, W.; Rüden, H. (Hrsg.): Veröffentlichungen aus dem Fachgebiet Hygiene der Technischen Universität Berlin und dem Institut für Hygiene der Freien Universität Berlin 10. Berlin, 1992 [9] Hoffmeister, D.; Weltin, D.; Dott, W.: Untersuchungen zur bakteriellen Phsophateliminierung. GWF Wasser Abwasser. 1990, 131(5): 270-277 [10] Hoffmeister D., Dynamik der extra- und intrazellulären Phosphorverbindungen bei der biologischen Phosphatelimination. Dott, W.; Rüden, H. (Hrsg.): Veröffentlichungen aus dem Fachgebiet Hygiene der Technischen Universität Berlin und dem Institut für Hygiene der Freien Universität Berlin 16. Berlin, 1993 [11] Schacht, Petra: Mikrobiologische Gewinnung von langkettigen Polyphosphaten, 2011 [12] Clark et al., 1986 [13] Dott, W.; Zimmermann, J.: Recovery of phosphorus from sewage sludge incineration ash by fractionated bioleaching and fate of heavy metals, 12th international symposium of microbial ecology, ISME 12, Cairns, Australia, August 17-22, 2008

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