Copper (II) removal by heat inactivated Streptomyces fradiae biomass

ISSN: 1314-6246

Kirova et al.

J. BioSci. Biotech. 2012, SE/ONLINE: 77-82.

RESEARCH ARTICLE Gergana Kirova 1 Zdravka Velkova 1 Velizar Gochev 2

Copper (II) removal by heat inactivated Streptomyces fradiae biomass: Surface chemistry characterization of the biosorbent ABSTRACT

Authors’ addresses: 1 Department of Chemistry and Biochemistry Medical University Plovdiv, Plovdiv, Bulgaria. 2 Department of Biochemistry and Microbiology, Plovdiv University "Paisii Hilendarski", Plovdiv, Bulgaria.

Correspondence: Gergana Kirova Department of Chemistry and Biochemistry Medical University Plovdiv, 15-A Vassil Aprilov Blvd., 4000 Plovdiv, Bulgaria Tel.: +359 88 4881679 e-mail: [email protected]

The effect of chemical pretreatment on copper (II) uptake capacity of heat inactivated Streptomyces fradiae biomass and the role of the surface chemistry of the biomass on biosorption behaviour were investigated. Treatment of the biosorbent with alkali chemicals, salts, acids and organic solvents showed varying effects on the removal efficiency of copper (II). Pretreatment of the biomass with sodium hydroxide and disodium salt of ethylenediaminetetraacetic acid significantly increased biosorption of copper (II) in comparison with the heat inactivated Streptomyces fradiae biomass. The maximum copper (II) uptake of pretreated Streptomyces fradiae biomass with sodium hydroxide was 44,17 mg/g at initial pH = 5,0. All treatments with acids decreased biosorption capacity of the biosorbent. The surface properties of the biomass were investigated by potentiometric titrations and Fourier transform infrared spectroscopy. The obtained results indicated that important functional groups presented on the biomass were carboxyl, amino and hydroxyl groups. Only carboxyl and hydroxyl groups were found to play an important role in copper (II) biosorption. Key words: Streptomyces pretreatment, biosorption

Introduction The intensive development of industry has a significant impact on the environment. Modern technologies are leading to the formation of significant amount of waste products contaminated with a variety of organic and inorganic substances. One of the most serious waste water problems is contamination with heavy metals. They have toxic and carcinogenic effect not only on humans but on all living organisms. Copper is an essential trace nutrient that is required in small amounts (5-20 micrograms per gram) by humans, other mammals, fish and shellfish for carbohydrate metabolism and the functioning of more than 30 enzymes. It is also needed for the formation of haemoglobin and haemocyanin, the oxygen-transporting pigments in the blood of vertebrates and shellfish respectively. However, copper concentrations that exceed 20 μg/g can be toxic, as explained by Bradl (2005) and Wright and Welbourn (2002). Copper is one of the most toxic metals to aquatic organisms and ecosystems. The most

fradiae,

potentiometric

titration,

chemical

bioavailable and therefore most toxic form of copper is the cupric ion Cu (II) (Wright & Welbourn, 2002). The standard physical and chemical methods for removal of metal ions from aqueous solutions, in particular Cu (II), have a number of shortcomings, making the pollution of the aquatic ecosystems with heavy metals a major environmental problems of contemporary society. Biosorption is a costeffective alternative for purification of waste water, especially when it comes to low concentrations or trace metal impurities (Veglio & Beolchini, 1997; Vijayaraghavan & Yun, 2008). It is based on the ability of different biomaterials such as bacteria, fungi, yeasts, algae, etc. to keep on their surface or concentrate metal ions from aqueous solutions. In recent decades studies on the potential application of biosorption as an alternative to chemical technologies are particularly intense and focused on the following areas: screening of effective biosorbents; increasing the biosorption efficiency by pretreatment of the biomass; optimization of the process parameters; study the mechanisms of biosorption. In order to increase the economic efficiency of the

SPECIAL EDITION / ONLINE Section “Ecology and biodiversity” National Youth Conference “Biological sciences for a better future”, Plovdiv, October 19-20, 2012

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ISSN: 1314-6246

Kirova et al.


RESEARCH ARTICLE process, the attention is focused on the opportunities from utilization of waste microbial biomass from fermentation and food industries, as biosorbents (Bailey et al., 1999). Because of their easy accessibility, low cost and numerous technological features, waste biomasses from the production of antibiotics were selected and tested for biosorbents of variety of heavy metals (Golab et al., 1994; Bal et al., 2003; Chergui et al., 2007; Sahmoune et al., 2008; Simeonova et al., 2008). The aim of this work was to study the effect of chemical pretreatment on copper (II) biosorption capacity by heat inactivated waste Streptomyces fradiae biomass from tylosin production, obtained from Biovet Peshtera, Bulgaria and the role of the surface structure of the biomass on the sorption behaviour. Assessment of change in biosorption efficiency was made for chemically pretreatment of the biomass by acids, alkali and salts. The functional groups involved in Cu(II) biosorption were identified by potentiometric titration, modification of functional groups by chemical treatment and FTIR analysis.

Materials and Methods Biosorbent preparation The waste Streptomyces fradiae biomass from tylosin production was provided by “Biovet” AD, Peshtera, Bulgaria. The raw biomass was powdered by mortar and pastel, filtered under vacuum, washed several times with distilled water until pH 6 was obtained for the filtrate and oven-dried at 80°C for 12 h. The dried biomass was stored at 4°C until further use. Chemical pretreatment of the biomass The heat inactivated Streptomyces fradiae biomass was pretreated in the presence of HCl (0.1 M), H 3PO4 (1.76 M), H2C2O4 (0.1 M), NaOH (0.1 M) and C10H14N2Na2O8 (0.025 M) (Kapoor & Viraraghavan, 1998; Goksungur et al., 2003). Five grams of biomass was pretreated as described below: - stirred at 300 rpm for 6 h in 250 ml of 0.1 M HCl - boiled at 110°C for 20 min in 100 ml of 1.76 M H3PO4 - stirred at 300 rpm for 2 h in 50 ml of 0.1 M H2C2O4 - boiled at 110°C for 15 min in 100 ml 1 M NaOH - stirred at 300 rpm for 60 min in 50 ml 0.025 M C10H14N2Na2O8. For 100 g of the raw biomass total lipid extraction was made with chloroform/methanol using the Folch method (Folch et al., 1957).

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All the pretreated samples were filtered under vacuum, washed with distilled water and oven- dried at 800C for 12 h. Surface chemistry characterization of the biomass Surface chemistry of the biomass was characterized by potentiometric titrations, modification of functional groups by chemical treatment and FTIR analysis. Potentiometric titrations of the biomass For the potentiometric titrations 0.05g biomass was soaked in 50 ml and 0.1 mol/l NaNO3 was used to stabilize the system at a fixed ionic strength. The suspension was stirred magnetically. Afterward 0.0484 mol/l HCl was added and the equilibrium pH value of the suspension became lower than 3. The biomass was converted to hydrogen form. The back titration process was conducted with 0.0507 mol/l NaOH until the pH of the suspension was raised to about 11. Corresponding blank titration was carried out on a solution of the background electrolyte (NaNO3 solution) with the same procedure as the sample except for the presence of the biomass phase (Pan et al., 2007). After each 0.25 ml of titrant added, the system was allowed to equilibrate until a stabile pH was observed. The potentiometric titration curve was constructed, plotting the volume of titrant against the recorded pH. The pH measurements were recorded using a WTW inoLab pH 720 pH meter with a SenTix® 41 pH electrode. The pH electrode was calibrated with buffers pH 4.01, pH 7.00 prior to use. Modification of functional groups of the biomass Methylation of amino groups Five grams of raw biomass were suspended in 100 mL of formaldehyde (HCHO) and 170 ml of formic acid (HCOOH). The reaction mixture was shaken for 5 h at 170 rpm and subsequently filtered under vacuum, washed with distilled water and oven- dried at 80°C for 8 h. (Kapoor & Viraraghavan, 1997). Esterification of carboxyl groups Five grams of the heat inactivated biomass were mixed with 500 mL of methanol (CH3OH) and 5 ml of concentrated hydrochloric acid. The reaction mixture was agitated (170 rpm) for 6 h. (Drake et al., 1996).The modified biomass was filtered under vacuum, washed with distilled water and dried at 800C for 8h. Esterification of hydroxyl groups Hydroxyl groups of the biosorbent were modified using mixture of pyridine and acetic anhydride. 0.2 g of raw biomass was suspended in 10 ml mixture of pyridine and


ISSN: 1314-6246

Kirova et al.


RESEARCH ARTICLE

Fourier transform infrared spectroscopy To complete the study of functional groups, an IR analysis was performed with a FTIR spectrometer (Thermo Nicolet Avatar 330 FTIR). Five mg of examined biomass was mixed and ground with 200 mg of KBr. The tablet was immediately analyzed. FTIR analysis was applied to the treated biomass before and after biosorption. Batch biosorption studies The stock solution of cooper (II) (1000 mg/l) was prepared by dissolving required quantity of CuSO 4.5H2O (Merck, p.a.) in distilled water. Fresh dilutions were made for the sorption experiments. The biosorption experiments were carried out in 250 ml Erlenmeyer flasks containing 0.1g of the biomass and 100 ml of 50 mg/l cooper (II) solution, adjusted to pH 5 (biosorption conditions were previously determined but not published). Samples were stirred on a magnetic stirrer at 300 rpm for the period of contact time (2h). Copper (II) ions left in solution, after biosorption, at the end of each experiment were separated from the biomass by centrifugation at 3000 rpm for 20 min and the supernatant was analyzed for cooper (II) concentration. The Cu (II) uptake was calculated using the equation (1): (1) V (c  c )

q

0

t

Results Effect of chemical treatment of Streptomyces fradiae cells on the biosorption process The cell wall plays an important role in the metal biosorption by non-viable cells, metal biosorption may be increased by physical or chemical pre-treatment. To investigate the effect of pre-treatment the heat inactivated waste biomass of Streptomyces fradiae was treated with acids (HCl, H3PO4, H2C2O4), sodium hydroxide, ethylenediaminetetraacetic acid disodium salt and organic solvents (chloroform/methanol). The metal uptake obtained by the chemically treated cells has been presented in Figure 1. As demonstrated in Figure 1 the highest metal uptake 44.17 mg/g was observed when caustic treated cells were used, and in the case of disodium salt of ethylenediaminetetraacetic acid and chloroform/methanol pretreatments the copper (II) uptake was found 34, 53 and 24.60 mg/g respectively. The control (heat treated cells) showed 19. 97 mg/g copper (II). 50

40

30

q (mg/g)

acetic anhydride (12 to 18 volume ratio) and heated for 2 h at 60°C (Tsekova et al., 2006). After reaction time the biomass was filtered under vacuum, washed with distilled water and dried at 80°C for 8h.

20

W

where: q – metal uptake, mg/g; c0 and ct – initial and final concentrations of Cu(II) ions, mg/l; V – volume of Cu(II) solution, l; W – quantity of biomass, g. Removal efficiency was calculated using equation (2): (2)

 c  ct R   0  c0

  x100,% 

Determination of the concentration of Cu (II) solutions The concentration of cooper (II) ions was determined by direct titration method with ethylenediaminetetraacetic acid disodium salt and Fast Sulphon Black F as an indicator (Jeffery et al., 1989).

10

0 a

b

c

d

e

f

g

Type of chemical treatment

Figure 1. Effect of different pre-treatments on the Cu(II) uptake of waste S. fraidae biomass; a - control; b - oxalic acid pretreated biomass; c - phosphoric acid pretreated biomass; d - hydrochloric acid pretreated biomass; e ethylenediaminetetraacetic acid disodium salt pretreated biomass; f - sodium hydroxide pretreated biomass; g chloroform/methanol pretreated biomass. The reason for high value of copper (II) uptake from caustic treated cells might be that this pretreatment could


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RESEARCH ARTICLE had more negative effect on copper biosorption than the esterification of carboxyl groups. These results alluded that carboxyl groups also take part in Cu (II) biosorption but less than the hydroxyl groups. Kapoor and Viraraghavan (Kapoor & Viraraghavan, 1997) reported that Aspergillus niger, one of the fungi, was capable of removing heavy metals such as lead, cadmium and copper from aqueous solutions. The experimental results suggested that carboxylate and amine groups be important in metal ion biosorption on A. niger biomass.

20

15

q (mg/g)

remove the polysaccharides and change the structure of the cell wall of the biomass. At the same time, sodium hydroxide could dissolve the inclusions in the cell and expose more active binding sites that improve the biosorption capacity. Furthermore sodium hydroxide dissociated H+ from the cell wall, resulting in the increase of negative functional groups. Similar conclusions were made by previous studies (Goksungur et al., 2003; Chergui et al., 2007). Extraction of the lipid fraction of the biomass increased the biosorption uptake with 23,18% compared to heat inactivated biomass (control). Extraction with organic solvents removes the protein and lipid fractions of the biomass surface (Ashkenazy et al., 1997). Thus, this treatment might expose more metal binding sites and improve the adsorptive property of the biomass (Park et al., 2005). All treatments with acids result in decreased Cu (II) uptake compared to heat inactivated S. fradiae biomass which is in agreement with previous studies (Huang & Huang, 1996; Javaid et al., 2011). It could possibly be explained in terms of hydrogen ions binding to the biomass after the acid treatment being responsible for the reduction in biosorption of heavy metals. This indicated that the acids destroyed the sorbing groups and their positive ions (H+) may covalently bonded to the absorbing surfaces. The order of metal uptake capacities was found: H2C2O4 < H3PO4 ≤ HCl < control.

10

5

0 a

b

c

d

Type of chemically modified Streptomyces fradiae biomass

Chemical modification of functional groups of the biomass The S. fradiae heat inactivated biomass was applied to various chemical modifications to reveal the role of the modified groups in copper (II) biosorption. As shown in Figure 2, the treatment of the biomass with formaldehyde and formic acid (methylation of amino groups) did not influenced significantly the Cu (II) uptake compared to the heat inactivated biomass (control), a reduction with 11.96% was found. Biomass with esterification of the carboxyl groups showed decreased copper (II) uptake with 58.98 % compared to the control. Treatment of the biomass with mixture of pyridine and acetic anhydride decreased the copper uptake with 65.69% vs. heat inactivated biomass. The decreased of the Cu (II) uptake by the biomass with esterification of carboxyl groups and esterification of hydroxyl groups in comparison to the control suggested the involvement of these groups in the biosorption process. According to Bal et al., (2003) amino, hydroxyl and carboxyl groups are likely to ensure potential interaction and offer particularly abundant metal-binding capacity. The esterification of hydroxyl groups

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Figure 2. Cu(II) uptake capacities (mg/g) of control and chemically modified functional groups of Streptomyces fradiae biomass; a - control; b - methylation of amino groups; c - esterification of carboxyl groups; d esterification of hydroxyl groups. Potentiometric titration Figure 3 shows S. fradiae biomass potentiometric titration curve compared to the blank sample. The groups presented on the biomass and their corresponding pKa values were obtained by inflection points of the titration curve. The titration curve of the biomass showed two flexion points corresponding to pKa of acidic binding groups at pH 3.53 and 3.71. Flexion points corresponding to alkaline functional groups were at pH 8.99, 9.88, and 10.32. These values are comparable to the values reported for carboxylic group (pKa 1.7-4.7). Alkaline functional groups were amines (pKa 8.0-11.0), sulfhydryl (thiol) (pKa 8.0–10.0) and hydroxyl (pKa 9.5–13) (Volesky, 2007; Loukidou et al., 2004; Ramrakhiani et al., 2011).


ISSN: 1314-6246

Kirova et al.


RESEARCH ARTICLE Fourier transform infrared spectroscopy For better understanding the nature of the functional groups involved in the copper (II) adsorption FT-IR analysis of the NaOH pretreated biomass before and after Cu (II) biosorption was examined and presented at Figure 4 and Figure 5. 12

1411cm-1 (stretching of C=O groups OH (primary and secondary)) decreased. Slight decreased in the intensity was observed for the peak at 3415cm-1 (–OH stretching vibration). The FTIR analysis confirmed the presence of amino, carboxyl and hydroxyl groups on the surface of the pretreated biosorbent. The results also suggested that carboxyl groups and hydroxyl groups are involved in Cu (II) biosorption. Similar result was obtained by Bal et al. (2003).

1 2

Sample 1.JDX: *Mon May 14 11:06:54 2012 (GMT+01:00) 100

10

721.2822

1234.2796 1461.8500 1411.7073

1041.4235

1868.7765

1652.7776 1646.9919 1562.1352

0

4

2921.7713 2850.4145

3415.4832

6

3745.2673

Transmittance

pH

50

3855.1953

8

2 0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

-50

-1

mL NaOH(0.05 mol.L )

4000

3000

2000

1000

Wavenumbers

Sample 3.JDX: *Mon May 14 11:22:21 2012 (GMT+01:00)

4000

3000

2000

721.2822

1226.5654 1033.7092

1733.7772 1648.9205 1558.2781 1465.7071 1375.0647

2917.9142 2850.4145

0

3446.3402

50

3853.2667

1868.7765

100

3745.2673

The FTIR spectra’s of metal loaded and unloaded biomass were taken in the range of 400–4000cm−1. The spectra of unloaded biomass displayed a number of peaks that indicated the complex nature of the biosorbent. Broad and distinctive band at 3415 cm-1 was indicative for –OH stretching vibration. Peak at 2921cm-1 indicated C-H asymmetric stretch vibration of aliphatic chains. Band at 1652 cm-1 was indicative for -C=O stretching vibration or NH2 groups. The bands at 1646 cm-1 and 1562cm-1 indicated stretching of C=O groups. The bands at 1411cm-1 and 1041 cm-1 are due to stretching of COOH, OH (primary and secondary). The band at 1234 cm-1 is indicative for the presence of OH (primary and secondary), COOH stretching or amide III stretching. Peak at 721cm-1 and corresponding band at 1465 cm-1 was indicative for long-chain polymer. FTIR spectra of Cu (II) loaded biomass compared to unloaded biomass to confirm the deferens and groups responsible for Cu(II) biosorption. Changes in the intensity of the bands after biosorption are observed. Substantial increase and shifting in the intensity of -COOH band from 1041 cm-1 to 1033 cm-1 while peaks around 1646 cm-1, 1562cm-1 and

Figure 4. FTIR spectrum of S. fradiae biomass pretreated with NaOH.

Transmittance

Figure 3. Potentiometric titration curves of heat inactivated S. fradiae biomass (2) and blank sample (1).

1000

Wavenumbers

Figure 5. FTIR spectrum of S. fradiae biomass pretreated with NaOH and adsorbed Cu (II).

Conclusions Based on the results obtained the following conclusions were made:


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RESEARCH ARTICLE Chemical treatment of the biomass with 1M NaOH, Ethylene Diamine Tetraacetic Acid Disodium salt (0.25 M) and lipid extraction with chloroform/methanol enhanced the metal uptake. 1M NaOH treated biomass had the highest metal uptake 44.17 mg/g. Chemical modification of the functional groups showed that important groups taking part in the biosorption are carboxyl and hydroxyl groups. Functional groups presented on the biomass and suggested by potentiometric titration was carboxyl, hydroxyl and amino may be and thiol groups, but thiol groups was rejected from the FTIR analysis. The FTIR analysis confirmed the presence of the carboxyl, amino and hydroxyl groups. The spectrum of Cu (II) loaded biomass compared to the unloaded biomass provided further evidence for the role of hydroxyl and carboxyl groups in Cu (II) biosorption.

References Ashkenazy R, Gottlieb L, Yannai S. 1997. Characterization of acetone-washed functional groups involved in lead biosorption. Biotechnology Bioeng., 55(1): 1-10. Bailey E, Bricka T, Adrian M. 1999. A review of potentially lowcost sorbents for heavy metals. Water Res., 33(11): 2469–2479. Bal Y, Bal KE, Lallam A. 2003. Removal of Bi (III) and Zn (II) by nonliving Streptomyces rimosus biomass from nitric solutions. The European Journal of Mineral Processing and Environmental Protection, 3(1): 42-48. Bradl H. 2005. Heavy Metals in the Environment: Origin, Interaction and Remediation. Elsevier/Academic Press, London. Chergui A, Bakhti MZ, Chahboub A, Haddouma S, Selatnia A, Junter GA. 2007. Simultaneous biosorption of Cu2+, Zn2+ and Cr6+ from aqueous solution by Streptomyces rimosus biomass. Desalination, 206: 179–184. Drake L, Lin S, Rayson G, Jackson P. 1996. Chemical modification and metal binding studies of Datura innoxia. Environmental Science and Technology, 30(1): 110-114. Folch J, Lees M, Sloane-Stanley GH. 1957. A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues. J. Biol. Chem., 226: 497–509. Goksungur Y, Uren S, Guvench U. 2003. Biosorption of Copper Ions by Caustic Treated Waste Bakers Yeast Biomass. Turk. J. Biol., 27: 23-29.

82

Golab Z, Breitenbach M, Jezierski A. 1994. Sites of copper binding in Streptomyces pilosus. Water, Air, & Soil Pollution, 82(3-4): 713-721. Huang CP, Huang C. 1996. Application of Aspergillus oryzae and Rhizopus oryzae. Water Research, 30: 1985-1990. Javaid A, Bajwa R, Manzoor T. 2011. Biosorption of heavy metals by pretreated biomass of Aspergillus niger, Pak. J. Bot., 43(1): 419-425. Jeffery GH, Basset J, Mendham J, Denney RC. 1989. Vogel’s Textbook of Quantative Chemical Analysis, 5thEdition. Longman Scientific & Technical, UK Ltd., ELBS, England, UK, 326. Kapoor A, Viraraghavan T. 1998. Biosorption of heavy metal on Aspergillus niger: effect of pretreatment. Bioresour. Technol., 63: 109-113. Kapoor A, Viraraghavan T. 1997. Heavy metal biosorption sites in Aspergillus niger. Bioresource Technol., 61: 221-227. Loukidou M, Zouboulis A, Karapantsios T, Matis K. 2004. Equilibrium and kinetic modeling of chromium (VI) biosorption by Aeromonas caviae. Colloid Surf. A., 242: 93-104. Pan J, Liu R, Tang H. 2007. Surface reaction of Bacillus cereus biomass and its biosorption for lead and copper ions. Journal of Environmental Sciences, 19: 403-408. Park D , Yun YS , Park JM. 2005. Studies on hexavalent chromium biosorption by chemically-treated biomass of Ecklonia sp. Chemosphere, 60: 1356-1364 Ramrakhiani L, Majumder R, Khowala S. 2011. Removal of hexavalent chromium by heat inactivated fungal biomass of Termitomyces clypeatus: Surface characterization and mechanism of biosorption. Chemical Engineering Journal, 171: 1060-1068. Sahmoune MN, Louhab K, Addad J. 2008. Chromium Biosorption by Waste Biomass of Streptomyces Rimosus Generated from the Antibiotic Industry. Journal of Applied Sciences Research, 4(9): 1076-1082. Simeonova A, Godjevargova T, Ivanova D. 2008. Biosorption of heavy metals by dead Streptomyces fradiae. Environmental Engineering Science, 25(5): 627–633. Tsekova K, Christova D, Ianis M. 2006. Heavy metal biosorption sites in Penicillium cyclopium. J. Appl. Sci. Environ. Mgt., 10(3): 117- 121. Veglio F. Beolchini F. 1997. Removal of metals by biosorption: a review. Hydrometallurgy, 44: 301–16. Vijayaraghavan K, Yeoung-Sang Y. 2008. Bacterial biosorbents and biosorption. Biotechnology Advances, 26: 266-291. Volesky B. 2007. Biosorption and me. Water Res., 41: 4017-4029 Wright D, Welbourn P. 2002. Environmental Toxicology. Cambridge University Press, Cambridge, U.K.
