**5. Abiotic U(VI) reduction**

=

Microcentrifuge (Eppendorf, Hamburg,

**3. Determination of U(VI)**

190 Applied Bioremediation - Active and Passive Approaches

spectrometry (ICP-MS).

soil samples.

U(VI) reductase activity was determined by measuring the decrease in U(VI) in the solution using UV/vis spectrophotometer (WPA, Light Wave II, and Labotech, South Africa). Arsenazo III (Sigma-Aldrich, St. Louis, Missouri, USA) (1, 8-dihydroxynaphthalene-3, 6 disulphonic acid-2, 7-bis [(azo-2)-phenylarsonic acid]), a non-specific chromogenic reagent, was selected as the complexing agent for facilitating U(VI) detection. The accuracy and the precision of the method on the UV/vis spectrophotometer was determined by measuring the concentration of standard U(VI) solution in the range of (0-80 mg/L). A linearized U(VI) standard curve was generated by plotting the absorbance at 651 nm versus the known U(VI) standard concentra‐ tion. Standard curve for U(VI) measurement demonstrated high degree of accuracy with R2

Measurement of U(VI) was carried out by withdrawing 2 mL of homogenous solution from a 100 mL serum bottle using a disposable syringe. The sample was then centrifuged for about

Germany). The sample (0.5-1 mL) was then diluted with 0.4 mL of 2.5% diethylene-triamine‐ penta acetic acid (DTPA) and then diluted up to mark with basal mineral medium (BMM) (1:10) dilution. The homogenous solution was then mixed with 2 mL of complexing reagent (Arsenazo III ) and then allowed to stand for full development of the pink-violet color prior analyses for U(VI) at a wavelength of 651 nm (10 mm light path) against a reagent blank. Total uranium in the unfiltered sample was measured using inductively-coupled plasma mass

In order to achieve *ex situ* and *in situ* biological treatment of water and soil contaminated by U(VI) and transuranic elements, it is necessary to search for microorganisms capable of reducing U(VI) under natural conditions. In a current investigation at the University of Pretoria, cultures isolated from a uranium mine dump were tested for uranium (VI) reduction under anaerobic and micro-aerobic conditions. The following section presents results from detailed batch experiments conducted with non-purified and purified cultures isolated from

Microorganisms were isolated from the soil samples collected from tailings dumps of an abandoned uranium mine. Background uranium concentration in the original sample was detected at levels as high as 29 mg/kg, much higher than values observed in natural soils (0.3-11.7 mg/kg). To select U(VI) tolerant species, microorganisms from soil were cultured overnight into a 100 mL of sterile basal mineral medium (BMM) amended with glucose as sole carbon source and a dose of U(VI) (75 mg/L uranium (VI) as uranyl nitrate). The inoculum was grown under anaerobic conditions for 24 hours at 30±2°C in 100 mL serum bottles purged with pure (nitrogen) N2 gas (99.9% pure grade) for about 5-10 minutes to expel residual oxygen before sealing the bottle with rubber stoppers and aluminium seal. After 24 hours enriched

99.7% and was used to estimate unknown U(VI) concentration.

**4. Uranium (VI) reduction capability in pure cultures**

10 minutes at 6000 rpm (2820 *g*) using a Minispin®

Heat-killed cultures and sodium azide exposed cultures were used to determine the extent of abiotic U(VI) reduction in batch experiments. For U(VI) reduction experiments cultures were grown over night in a sterile nutrient or Luria-Bettani (LB) broth under anaerobic conditions. Overnight grown cells were heat killed by autoclaving at 121°C for 20 minutes and another set of overnight grown cells were incubated with 0.1% of sodium azide (NaN3 ). Cells were then harvested by centrifuging at 6000 rpm (2820 *g*) for 10 minutes. The supernatant was then decanted and the remaining pellet was washed three times with sterile 0.85% NaCl solution. The washed pellet was then re-suspended in 100 mL serum bottle containing U(VI) stock solution and sterile basal mineral medium (BMM) amended with D-glucose as sole carbon source. The serum bottles were then purged with 99.9% (N2 ) for about 5-10 minutes to expel residual oxygen before sealing the bottle with rubber stoppers and aluminium seal. The experiments were all conducted at 30±2°C with continuous shaking on lateral shaker (Labotec, Gauteng, South Africa). The experimental conditions in the abiotic controls and the live cells experiments were kept the same (100 mL serum bottles containing BMM amended with Dglucose, 100 mg/L U(VI) solution, and incubated at 30±2°C under anaerobic conditions). A sample was withdrawn at regular time interval using a disposable syringe for U(VI) reduction analysis as described above.

The results showed insignificant difference U(VI) reduction between live and heat-killed cells (Figure 3). The instantaneous U(VI) reduction in heat-killed cells may be due to the presence of the cells that escaped destruction by heat. The reduction of U(VI) observed during the first 2 hours in all treatment containing biomass presented an anomaly. It was clear from these results that another mechanism rather than the direct metabolic process was involved in the U(VI) removal from solution. On the other hand abiotic control (without bacteria) showed that U(VI) reduction process is biologically mediated.

Reverting back to the biosorption studies, it is suggested that functional groups on the cell wall surfaces (-OH, -NH2 , and –COOH) may serve as ligands for U(VI)-U(IV) complexation with the cell surface. U(VI) reduction may serve as a step towards this complexation step. To evaluate these effects we conducted experiments where the pH was varied and the oxidation reduction potential (ORP) was measured with time. Results presented in Figure 4 show that that the rate of U(VI) reduction was pH dependent (Figure 4a). Electronegative conditions under anaerobic conditions created a strongly reducing environment as expected, after which the ORP increased to electropositive values (Figure 4b). As a result insignificant change over time in ORP indicated poor oxidation-reduction, while significant change in ORP over time indicated that the oxidation-reduction process approaches completeness.

**Figure 3.** Evaluation of abiotic U(VI) reduction in heat-killed and azide exposed cells

U(VI) at 400 mg/L

300

400

500 **Figure 3.** Evaluation of abiotic U(VI) reduction in heat-killed and azide inhibited cells

400

#### **6. U(VI) Reduction by non-purified cultures (Consortium)**

U concentration, mg/L0 100 200 300 Total U Protein concentration, mg/L0 100 200 Protein concentration Preliminary experiments were conducted over a wide range of U(VI) concentration (30-400 mg/L) under similar experimental conditions (100 mL serum bottles containing BMM amended with D-glucose, U(VI) solution, and then incubated at 30±2°C under anaerobic conditions) using a reconstituted consortium culture of several identified U(VI) tolerant species. Results showed complete U(VI) reduction in batch cultures at initial U(VI) concentration up to 300 mg/L within 24 hours. In all batch studies with U(VI) concentra‐ tion up to 400 mg/L (80-100%) U(VI) removal was achieved within the first 5 hours of incubation. However, inhibition of the reduction process was observed at the initial U(VI) concentration of 400 mg/L over time (Figure 5a).

Time, h **Figure 7.** Evaluation of U(VI) reduction, protein concentration and total uranium under an initial concentration of 400 mg/L. U(VI) reduction trends in batches using purified cultures with single species per batch showed similar trends of U(VI) reduction. Figure 5b shows the summary of results from the best performing cultures labeled Y1, Y5, and Y6. The species characterisation results for these pure cultures are presented later in the chapter. The results in Figure 5b show that the microorgan‐ isms existing as a community possess significant stability and metabolic capabilities which can be linked to the effectiveness of synergistic interactions among members of bacterial com‐ munities [64].

0 10 20 30 40 50

2

conditions.

indicated poor oxidation-reduction, while significant change in ORP over time indicated that the oxidation-reduction process

Preliminary experiments were conducted over a wide range of U(VI) concentration (30-400 mg/L) under similar experimental conditions (100 mL serum bottles containing BMM amended with D-glucose, U(VI) solution, and then incubated at 30±2ºC under anaerobic conditions) using a reconstituted consortium culture of several identified U(VI) tolerant species. Results showed complete U(VI) reduction in batch cultures at initial U(VI) concentration up to 300 mg/L within 24 hours. In all batch studies with U(VI) concentration up to 400 mg/L (80-96%) U(VI) removal was achieved within the first 5 hours of incubation. However,

U(VI) reduction trends in batches using purified cultures with single species per batch showed similar trends of U(VI) reduction. Figure 5b shows the summary of results from the best performing cultures labeled Y1, Y5, and Y6. The species characterisation results for these pure cultures are presented later in the chapter. The results in Figure 5b show that the microorganisms existing as a community possess significant stability and metabolic capabilities which can be linked to the effectiveness of synergistic

Bioremediation of Radiotoxic Elements under Natural Environmental Conditions

Figure 4. (a) U(VI) reduction at different pH values – U(VI) reduction rate increased with increasing pH, and (b) data showing loss of U(VI)

Figure 5. (a) U(VI) reduction in reconstituted consortium culture from mine soil under the initial concentration of 300 and 400 mg/L, and (b) comparative performance of three pure isolates against the reconstituted consortium culture. The reconstituted consortium culture shows the best

 Time, h 0 20 40 60 80

Proportional distribution of uranium precipitates in the medium and cells can be used to determine the location of U(VI) reductase activity in the culture system. This is because most the precipitates are formed from reduced uranium species. Transmission electron microscopy (TEM) was used to establish the distribution and localization of uranium deposits in the cells. The energy dispersive X-ray (EDX) spectrometer coupled to the TEM was also used for elemental characterization of the metal deposits in the medium. TEM result images show crystal structures in the medium and very little crystallisation inside the cells (Figure 6a). EDX analysis of the crystals deposited on the cell surfaces confirmed the accumulation of uranium elements in the crystal matrix. Extracellular depositions of uranium also indicate that bacteria are excellent nucleation sites for mineral formations. EDX spectra derived from the uranium deposits show that they are composed of the following elements U>Cu>P>Os>Ca>Co>Fe (according to their descending order of their weight %). The higher copper (Cu) peak results from the specimen to support grid used. Phosphorous observed in the spectrum could either be from the added phosphorous or could microbially produced. On the other

Figure 6. (a) TEM Scan of bacterial cells indicating deposition of uranium species on cell surface and (b) EDX spectrum of precipitate.

Proteins make up a large fraction of the biomass of actively grown microbes. To determine microbial activity over time, protein concentration was determined using a UV/Vis Spectrophotometer (WPA, Light Wave II, and Labotech, South Africa) at the wavelength of 595 nm using Coomassie Dye as a complexing agent to facilitate protein detection. Samples required pre-treatment to reduce interferences during the spectrophotometric analyses. Cell lysis was achieved by ultrasonification of acid treated cells. Results showed that microbial activity decreased with increasing U(VI) reduction (Figure 7). These results served as a confirmation

The phylogenetic characterization of cells from the mine dump soil was conducted after sub-culturing the cells in nutrient or Luria-Bettani broth. Individual colonies from a serially diluted preparation were carefully examined for colony morphology and cell morphology by Gram-staining. This process, we recognize, could eliminate a wide range of potential U(VI) reducers especially anaerobic species in the samples. But at this stage, we were targeting the species that can survive under facultative anaerobic

The purified colonies were streaked on nutrient agar followed by incubating at 30°C for 18 hours in preparation for 16S rRNA gene sequence analysis. Microbial pure cultures were grown from loop-fulls from individual colonies, transferred to fresh media containing low amounts (30-75 mg/L) of uranyl nitrate. The process was repeated at least three times for each colony type to

Time, h 0 5 10 15 20 25 30

U(VI) at 100 mg/L

http://dx.doi.org/10.5772/56909

Y1 Y5 Y6 mixed culture

ORP

ORP, mV

193


(a) (b)

**Figure 4.** (a) U(VI) reduction at different pH values – U(VI) reduction rate increased with increasing pH, and (b) data

(a) (b)

**Figure 5.** (a) U(VI) reduction in reconstituted consortium culture from mine soil under the initial concentration of 300 and 400 mg/L, and (b) comparative performance of three pure isolates against the reconstituted consortium culture. The reconstituted consortium culture shows the best performance possibly due to symbiotic interactions within the

Proportional distribution of uranium precipitates in the medium and cells can be used to determine the location of U(VI) reductase activity in the culture system. This is because most the precipitates are formed from reduced uranium species. Transmission electron microscopy (TEM) was used to establish the distribution and localization of uranium deposits in the cells. The energy dispersive X-ray (EDX) spectrometer coupled to the TEM was also used for elemental characterization of the metal deposits in the medium. TEM result images show crystal structures in the medium and very little crystallisation inside the cells (Figure 6a). EDX analysis of the crystals deposited on the cell surfaces confirmed the accumulation of uranium elements in the crystal matrix. Extracellular depositions of uranium also indicate that bacteria are excellent nucleation sites for mineral formations. EDX spectra derived from the uranium

U(VI) concentration, mg/L

0

20

40

60

80

100

U(VI) concentration, mg/L

inhibition of the reduction process was observed at the initial U(VI) concentration of 400 mg/L over time (Figure 5a).

approaches completeness.

U(VI) concentration, mg/L

U(VI) concentration, mg/L

U(VI) concentration, mg/L

culture system.

0

100

200

300

400

Figure 3. Evaluation of abiotic U(VI) reduction in heat-killed and azide inhibited cells

Time, h 0 10 20 30 40 50

interactions among members of bacterial communities [64].

U(VI) at PH 6.5 U(VI) at PH 2

reduction capacity as ORP increases.

showing loss of U(VI) reduction capacity as ORP increases.

Time, h 0 10 20 30 40 50

performance possibly due to symbiotic interactions within the culture system.

U(VI) at 300 mg/L U(VI) at 400 mg/L

hand no uranium was observed in the metal free biomass (Results not shown).

**8.1. Correlation of U(VI) reduction to enzyme activity** 

**7. Fate of reduced Uranium species in a cell** 

**7. Fate of reduced uranium species in a cell**

Time, h 0 10 20 30 40 50

**8. Microbial characterisation and activity** 

of enzymatic activity as responsible agent for U(VI) reduction.

achieve close to a pure culture of each identified species.

**8.2. Culture composition analysis** 

**6. U(VI) Reduction by non-purified cultures (Consortium)** 

Sodium Azide Heat killed cell free control Live-cells

Preliminary experiments were conducted over a wide range of U(VI) concentration (30-400 mg/L) under similar experimental conditions (100 mL serum bottles containing BMM amended with D-glucose, U(VI) solution, and then incubated at 30±2ºC under anaerobic conditions) using a reconstituted consortium culture of several identified U(VI) tolerant species. Results showed complete U(VI) reduction in batch cultures at initial U(VI) concentration up to 300 mg/L within 24 hours. In all batch studies with U(VI) concentration up to 400 mg/L (80-96%) U(VI) removal was achieved within the first 5 hours of incubation. However,

U(VI) reduction trends in batches using purified cultures with single species per batch showed similar trends of U(VI) reduction.

inhibition of the reduction process was observed at the initial U(VI) concentration of 400 mg/L over time (Figure 5a).

indicated poor oxidation-reduction, while significant change in ORP over time indicated that the oxidation-reduction process

approaches completeness.

U(VI) concentration, mg/L

Figure 3. Evaluation of abiotic U(VI) reduction in heat-killed and azide inhibited cells

Time, h 0 10 20 30 40 50

interactions among members of bacterial communities [64].

**6. U(VI) Reduction by non-purified cultures (Consortium)** 

Sodium Azide Heat killed cell free control Live-cells

Figure 4. (a) U(VI) reduction at different pH values – U(VI) reduction rate increased with increasing pH, and (b) data showing loss of U(VI) reduction capacity as ORP increases. **Figure 4.** (a) U(VI) reduction at different pH values – U(VI) reduction rate increased with increasing pH, and (b) data showing loss of U(VI) reduction capacity as ORP increases.

Figure 5. (a) U(VI) reduction in reconstituted consortium culture from mine soil under the initial concentration of 300 and 400 mg/L, and (b) comparative performance of three pure isolates against the reconstituted consortium culture. The reconstituted consortium culture shows the best performance possibly due to symbiotic interactions within the culture system. **7. Fate of reduced Uranium species in a cell Figure 5.** (a) U(VI) reduction in reconstituted consortium culture from mine soil under the initial concentration of 300 and 400 mg/L, and (b) comparative performance of three pure isolates against the reconstituted consortium culture. The reconstituted consortium culture shows the best performance possibly due to symbiotic interactions within the culture system.

Proportional distribution of uranium precipitates in the medium and cells can be used to determine the location of U(VI) reductase

#### activity in the culture system. This is because most the precipitates are formed from reduced uranium species. Transmission electron microscopy (TEM) was used to establish the distribution and localization of uranium deposits in the cells. The energy **7. Fate of reduced uranium species in a cell**

**8. Microbial characterisation and activity** 

of enzymatic activity as responsible agent for U(VI) reduction.

achieve close to a pure culture of each identified species.

**8.2. Culture composition analysis** 

**8.1. Correlation of U(VI) reduction to enzyme activity** 

2

conditions.

Time, h 0 10 20 30 40 50

Protein concentration

Preliminary experiments were conducted over a wide range of U(VI) concentration (30-400 mg/L) under similar experimental conditions (100 mL serum bottles containing BMM amended with D-glucose, U(VI) solution, and then incubated at 30±2°C under anaerobic conditions) using a reconstituted consortium culture of several identified U(VI) tolerant species. Results showed complete U(VI) reduction in batch cultures at initial U(VI) concentration up to 300 mg/L within 24 hours. In all batch studies with U(VI) concentra‐ tion up to 400 mg/L (80-100%) U(VI) removal was achieved within the first 5 hours of incubation. However, inhibition of the reduction process was observed at the initial U(VI)

0 10 20 30 40 50

Time, h 

U(VI) reduction trends in batches using purified cultures with single species per batch showed similar trends of U(VI) reduction. Figure 5b shows the summary of results from the best performing cultures labeled Y1, Y5, and Y6. The species characterisation results for these pure cultures are presented later in the chapter. The results in Figure 5b show that the microorgan‐ isms existing as a community possess significant stability and metabolic capabilities which can be linked to the effectiveness of synergistic interactions among members of bacterial com‐

**Figure 3.** Evaluation of abiotic U(VI) reduction in heat-killed and azide exposed cells

cell-free control Sodium Azide Heat-killed cells Live-cells

> U(VI) at 400 mg/L Total U

Protein concentration, mg/L

0

100

200

300

400

U(VI) concentration, mg/L

0

U concentration, mg/L

0

concentration of 400 mg/L over time (Figure 5a).

400 mg/L.

munities [64].

100

200

300

400

500

**Figure 3.** Evaluation of abiotic U(VI) reduction in heat-killed and azide inhibited cells

**6. U(VI) Reduction by non-purified cultures (Consortium)**

20

40

60

80

100

120

192 Applied Bioremediation - Active and Passive Approaches

**Figure 7.** Evaluation of U(VI) reduction, protein concentration and total uranium under an initial concentration of dispersive X-ray (EDX) spectrometer coupled to the TEM was also used for elemental characterization of the metal deposits in the medium. TEM result images show crystal structures in the medium and very little crystallisation inside the cells (Figure 6a). EDX analysis of the crystals deposited on the cell surfaces confirmed the accumulation of uranium elements in the crystal matrix. Extracellular depositions of uranium also indicate that bacteria are excellent nucleation sites for mineral formations. EDX spectra derived from the uranium deposits show that they are composed of the following elements U>Cu>P>Os>Ca>Co>Fe (according to their descending order of their weight %). The higher copper (Cu) peak results from the specimen to support grid used. Phosphorous observed in the spectrum could either be from the added phosphorous or could microbially produced. On the other hand no uranium was observed in the metal free biomass (Results not shown). Proportional distribution of uranium precipitates in the medium and cells can be used to determine the location of U(VI) reductase activity in the culture system. This is because most the precipitates are formed from reduced uranium species. Transmission electron microscopy (TEM) was used to establish the distribution and localization of uranium deposits in the cells. The energy dispersive X-ray (EDX) spectrometer coupled to the TEM was also used for elemental characterization of the metal deposits in the medium. TEM result images show crystal structures in the medium and very little crystallisation inside the cells (Figure 6a). EDX analysis of the crystals deposited on the cell surfaces confirmed the accumulation of uranium elements in the crystal matrix. Extracellular depositions of uranium also indicate that bacteria are excellent nucleation sites for mineral formations. EDX spectra derived from the uranium

Figure 6. (a) TEM Scan of bacterial cells indicating deposition of uranium species on cell surface and (b) EDX spectrum of precipitate.

Proteins make up a large fraction of the biomass of actively grown microbes. To determine microbial activity over time, protein concentration was determined using a UV/Vis Spectrophotometer (WPA, Light Wave II, and Labotech, South Africa) at the wavelength of 595 nm using Coomassie Dye as a complexing agent to facilitate protein detection. Samples required pre-treatment to reduce interferences during the spectrophotometric analyses. Cell lysis was achieved by ultrasonification of acid treated cells. Results showed that microbial activity decreased with increasing U(VI) reduction (Figure 7). These results served as a confirmation

The phylogenetic characterization of cells from the mine dump soil was conducted after sub-culturing the cells in nutrient or Luria-Bettani broth. Individual colonies from a serially diluted preparation were carefully examined for colony morphology and cell morphology by Gram-staining. This process, we recognize, could eliminate a wide range of potential U(VI) reducers especially anaerobic species in the samples. But at this stage, we were targeting the species that can survive under facultative anaerobic

The purified colonies were streaked on nutrient agar followed by incubating at 30°C for 18 hours in preparation for 16S rRNA gene sequence analysis. Microbial pure cultures were grown from loop-fulls from individual colonies, transferred to fresh media containing low amounts (30-75 mg/L) of uranyl nitrate. The process was repeated at least three times for each colony type to deposits show that they are composed of the following elements U>Cu>P>Os>Ca>Co>Fe (according to their descending order of their weight %). The higher copper (Cu) peak results from the specimen to support grid used. Phosphorous observed in the spectrum could either be from the added phosphorous or could microbially produced. On the other hand no uranium was observed in the metal free biomass (Results not shown).

grown from loop-fulls from individual colonies, transferred to fresh media containing low amounts (30-75 mg/L) of uranyl nitrate. The process was repeated at least three times for each

Genomic DNA was extracted from purified colonies according to the protocol described for the Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI, USA). 16S rRNA genes were amplified by a reverse transcriptase-polymerase chain reaction (RT-PCR) using primers pA and pH1 (Primer pA corresponds to position 8-27; Primer pH to position 1541-1522 of the 16S gene under the following reaction conditions: 1 min at 94°C, 30 cycles of 30s at 94°C, 1 min at 50°C and 2 min at 72°C, and a final extension step of 10 min at 72°C). PCR fragments were then cloned into pGEM-T-easy (Promega) [Promega Wizard® Genomic DNA Purifica‐ tion Kit (Version 12/2010)]. The 16S rRNA gene sequences of the strains were aligned with reference sequences from *Desulfovibrio sp.*, *Geobacter sp.*, *Acinetobacter sp.*, *Anthrobacter sp.*, and *Shewanella putrefaciens* using Ribosomal Database Project II programs. Sequence alignment was verified manually using the program BIOEDIT. Pairwise evolutionary distances based on an unambiguous stretch of 1274 bp were computed by using the Jukes and Cantor method [65].

U(VI) reducing colonies were identified from the genera *Bacilli*, *Acinobacter*, *Actinomycetes* and *Chrysebactreium*. Sections of phylogenetic tree diagrams with closet associations to know species are shown in Figures 8d. The associations shown Figure 8 have been reported among U(VI) reducing groups in literature. Fowle et al. [66] has shown that *Bacillus* species are effective biosorbents for uranium. Additionally, the capability of *Anthrobacter* species isolated from a uranium-contaminated site in accumulating uranium intracellularly as uranium

cell-free control Sodium Azide Heat-killed cells Live-cells

Bioremediation of Radiotoxic Elements under Natural Environmental Conditions

http://dx.doi.org/10.5772/56909

195

U(VI) at 400 mg/L Total U

**Figure 7.** Evaluation of U(VI) reduction, protein concentration and total uranium under an initial concentration of

Protein concentration, mg/L

0

100

200

300

400

2

precipitates closely associate with polyphosphate granules was also reported [21].

Protein concentration

Time, h 0 10 20 30 40 50

0 10 20 30 40 50

Time, h

400 mg/L. **Figure 7.** Evaluation of U(VI) reduction, protein concentration and total uranium under an initial concentration of 400

**Figure 3.** Evaluation of abiotic U(VI) reduction in heat-killed and azide exposed cells

colony type to achieve close to a pure culture of each identified species.

U(VI) concentration, mg/L

0

U concentration, mg/L

0

mg/L.

100

200

300

400

500

20

40

60

80

100

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**Figure 6.** (a) TEM Scan of bacterial cells indicating deposition of uranium species on cell surface and (b) EDX spectrum of precipitate.
