Biofilm Polymer for Biosorption of Pollutant Ions

Procedia Environmental Sciences 17 ( 2013 ) 179 – 187

1878-0296 © 2013 The Authors. Published by Elsevier B.V.Selection and peer-review under responsibility of SUSTAIN conference’s committee and supported by Kyoto University; (OPIR), (GCOE-ES), (GCOE-HSE), (CSEAS), (RISH), (GCOE-ARS) and (GSS) as co-hosts.doi: 10.1016/j.proenv.2013.02.027

Available online at www.sciencedirect.com

The 3rd International Conference on Sustainable Future for Human Security SUSTAIN 2012

Biofilm polymer for biosorption of pollutant ions

Andi Kurniawanab* and Tatsuya Yamamotob aFaculty of Fisheries and Marine Science, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia

bGraduate School of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu Shiga 525-8577, Japan

Abstract

The utilization of biofilm as an adsorbent of pollutant ions is one of the promising technologies for water contamination treatment. An understanding of biofilm adsorption property is therefore essential to predict how biofilm contributes to mobility and bioavailability of pollutant ions in the environments. The aim of this study is investigating adsorption properties of biofilm, i.e., existing in the biofilm polymer and clarifying the ability of biofilm as an adsorbent in the biosorption of pollutant ions. The biofilms applied in this study are natural biofilms formed on the surface of stones collected from Lake Biwa. The characterization of the biofilm polymer is revealed by electrophoretic mobility measurements and acid-base titrations. To clarify the ability of biofilm for biosorption of pollutant ions, we study the adsorption kinetics of lithium ion to biofilms formed in winter and spring. The followings are observed as results of this study: 1) both negative and positive charged sites exist on biofilm polymer; 2) the ion adsorption by biofilm polymer is a physicochemical process where the electrostatic interaction between ion and the charged sites in biofilm polymers is a main driving force; 3) the adsorbing ion promotes ions desorption from biofilms through ion exchange mechanism; 4) the biofilms components change seasonally and appear to affect the ability of biofilms to adsorb ions. From all of similar studies, this study is the first one investigating utilization of natural biofilms formed in different seasons for biosorption of heavy metal ion i.e., lithium ion. This study also reveals that biofilm may become a promising adsorbent for pollutant ions. © 2012 The Authors. Published by Elsevier B.V. Selection and/or peer-review under responsibility of SUSTAIN conferences committee and supported by Kyoto University; (OPIR), (GCOE-ES), (GCOE-HSE), (CSEAS), (RISH), (GCOE-ARS) and (GSS) as co-hosts. Keywords: biofilm; biofilm polymer; biosorption; pollutant ions; lithium.

1. Introduction

Biofilm is formed when bacteria and other microorganisms attach to a surface and then replicate with producing extracellular polymers [1]. Biofilm has high sorption capacities and low production cost. It

* Corresponding author. Tel.: +62-341-553512. E-mail address: [email protected].

Available online at www.sciencedirect.com

© 2013 The Authors. Published by Elsevier B.V.Selection and peer-review under responsibility of SUSTAIN conference’s committee and supported by Kyoto University; (OPIR), (GCOE-ES), (GCOE-HSE), (CSEAS), (RISH), (GCOE-ARS) and (GSS) as co-hosts.

180 Andi Kurniawan and Tatsuya Yamamoto / Procedia Environmental Sciences 17 ( 2013 ) 179 – 187

attracts attention to specialist in the field of water treatment [2, 3, 4]. However, the study concerning utilization of biofilm formed in natural environment for biosorption of pollutant ion is rarely conducted. Most of the recent works has focused on the adsorption of heavy metal to bacteria and/or single bacterial strain biofilm that intentionally grown in the laboratory [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17].

One of the main parts of biofilm is biofilm polymer [18]. Biofilm polymer is enriched in functional groups acting as sorption sites for variety of aqueous chemical substances, including contaminants [19, 20]. To develop biofilm as an adsorbent for pollutant ions in the real environment, this study focus to polymer of biofilm formed in natural environment, especially correlation between adsorption properties of biofilm polymer and adsorption behaviour of biofilm, in essential.

Pollutant ions such as heavy metal ions are one of the biggest water contamination problems [2, 16, 17]. One of heavy metals that recently become a pollutant is lithium ion. Lithium is utilized as an important compound of various things such as high-performance grease, heat-resistant ceramics and flux for welding, batteries and pharmaceuticals [21, 22]. It is also used in nuclear fusion furnace [23, 24]. Thus, the recovery of lithium is an important subject [25].

In this study, the polymer characteristics of biofilms which are collected from the surface of stone in Lake Biwa (as one of the environmental biofilm) are clarified. The utilization of natural biofilm for adsorption of heavy metal ion (i.e., lithium) is also investigated. Moreover, the comparison of lithium adsorption capacity of biofilms formed in different seasons is also conducted. We recognize that this is the first stud characterizing polymer of natural biofilms in order to utilize natural biofilms for the biosorption of lithium ion.

2. Materials and Methods

2.1. Sampling Site and Sample Preparation

Lake Biwa is the largest lake in Japan. This lake is located in central part of Japanese Archipelago ×1010

m3. The samples used in this study were collected from the shore of the southern basin (Akanoiwan) of Lake Biwa. Biofilm samples were collected in winter (November and December 2008) and spring (March 2009) as the previous study founded that biofilm thickness reach maximum amount in these seasons [26]. Stones were taken from the depth of 50 70 cm and brought back to the laboratory in a plastic container filled with nearby lake water; the container was maintained at 4°C.

Biofilms on the surfaces of the stones (ca. 100 stones in each sampling) were removed using a toothbrush and suspended in sterilized distilled water. Biofilm pellets were prepared by centrifuging (8,000×g at 4°C for 10 min) the biofilm suspensions. Biofilm collected in November 2008 was applied as sample in electric charge measurements of biofilm polymer and acid-base titration. Biofilms collected in December 2008 and March 2009 were applied as samples in kinetics adsorption analysis of lithium ion.

2.2. Dry Weight of Biofilm

Biofilm pellet (±1 wet-g) were taken and then dehydrated for 3 days until the weight was stable to be dry weight.


2.3. Electric Charge of Biofilm

The biofilm was washed three times with 10 mM NaCl aqueous solution by centrifugation (8,000×g at 4°C for 10 min) and the supernatant was discarded. The obtained biofilm pellet (ca. 0.03 g) was suspended in 1 mL of 10 mM NaCl aqueous solution. The suspension was mixed vigorously with vortex for 5 min, then sonicated (2510J-MT, Yamato Scientific, Tokyo, Japan; 42 kHz, 125 W) for 10 min, followed by vortex for 10 s. The obtained suspension was mixed with 10 mM of PBS at a ratio of 1:19 and applied to analyze electric charge of BF polymer. The electrophoretic mobility (EPM) of biofilm was measured on ZETASIZER Nano-Z (Malvern Instruments, Ltd., Worcestershire, England) in PBS varying in pH values from 2.0 to 9.0. Buffer pH was adjusted with 20 mM of HCl or NaOH. PBS pHs before and after addition of biofilm and after measurement of EPM were recorded.

2.4. Acid-base Titration

The biofilms were washed six times with distilled water by centrifugation. One gram of the pellet of biofilm was placed in a plastic cup and distilled water was added to give a total weight of 40 g. Then, 10 mM of HCl or 10 mM of NaOH aqueous solution was titrated onto the samples using automatic titration machine (DL50, Mettler, Toledo, OH, USA). The pH changes were recorded and analyzed. Distilled water (40 gram) was also subjected to titration. From the results, the adsorbed amount of H+ or OH- by biofilm unit weight was calculated as follows:

)/()1010()( )()( wvHC bfpHdwpH (1)

Where, C(H+) is the uptake capacity of H+ by biofilm unit weight ( mol/wet-g), pH(dw) and pH(bf) is the pH value of distilled water and biofilm suspension when HCl solution was added, respectively. Moreover, v is the volume of sample solution (L) and w is the wet-weight of biofilm (gram). When NaOH solution was added, the uptake capacity of OH- ion, C(OH-), by biofilm unit weight ( mol/wet-g), was given by the similar equation as follows:

)/()1010()( )()( wvOHC dwpHbfpH (2)

The uptake capacity of H+ and OH- ions by biofilm was calculated by applying above equations.

2.5. Adsorption of Lithium

Biofilms were washed six times with 5 mM phosphate-buffered saline (PBS) of pH 7 by centrifugation. Biofilm pellets were stored at -40°C until ion adsorptions analyses were conducted. 1 wet-g of biofilm pellet was re-suspended in 50 mL of 5 mM PBS of pH 7. The suspension was mixed vigorously with vortex for 5 min then sonicated for 10 min, followed by the vortex for 30 s. Next, 5.0 mL of 20 mM of solution of reagent grade LiCl, prepared by diluting chemical compound (Wako Pure Chemical Industries, Osaka, Japan) in 5 mM PBS of pH 7, was added to the suspension. The suspension temperature was maintained at 25°C using a water thermostat, and mixed well using magnetic stirrer.


Suspension aliquots were taken after 1 min 300 min, and then centrifuged (15,000×g at 4°C for 1 min) to separate supernatant and pellet. Ion concentration in the solution was measured using capillary electrophoresis method (CAPI-3300, Otsuka electronics, Osaka, Japan). The adsorbed amount of ion to biofilm was calculated from the difference between ion concentration in the supernatant and in the control (only PBS and ion). The desorbed ion (different species from added ion) from biofilm due to addition of ion was also investigated. As a control of investigation of desorbed ion, same amount of biofilm was re-suspended in 50 ml of 5 mM PBS of pH 7, and then treated with similar treatment described above but without ion addition. The adsorption experiment was also conducted using one dry-g of strong acidic resin (SP-650M; Toyopearl, Tokyo, Japan) and weak acidic resin (CM-650M; Toyopearl).

3. Results and Discussion

3.1. Electric Charge of Biofilm

A biofilm suspension is put in an electric field, and then electrophoretic velocity is measured under various pH conditions. The electrophoretic mobility is normalized by dividing it with electric field strength to obtain electrophoretic mobility (EPM). The EPM of biofilm is shown as a function of pH (Fig. 1).

Fig. 1. EPM of biofilm measured in various pHs.

At pH 7, biofilm shows negative EPM values, whereas these values decrease at smaller pH values, especially with greater extent around pH 4. It is due to ionization depression of functional groups, such as carboxylic group which pKa is around pH 4, at the acidic condition.

At pH 2, biofilm shows positive EPM value. This indicates the existence of positively charged functional group (s), which is supposed to be amino group (to be discussed later) in biofilm polymer. pH values of buffer solution shift to greater value from its original when biofilm samples are added to buffer solution; e.g., the original pH values of 4.0 and 5.0 shift to 5.7 and 6.0, respectively after adding biofilm samples. It indicates decrease in proton concentration by adding the biofilm sample. It appears that proton is applied to de-ionize acidic functional group in the biofilm. Carboxylic acid is the most probable to be functional group, as discussed above and further confirmed by buffer action as shown in acid-base titration section.

-2.5

-1.5

-0.5

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0 1 2 3 4 5 6 7 8 9

EPM

(x10

-8 m

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s-1)

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3.2. Acid-base Titration

Acid base titrations are applied to biofilm and distilled water (Fig. 2). The acid-base titration curve of biofilm is different from that of distilled water (as background solution). This clearly reveals the presence of ionizeable functional groups associated with biofilm polymer.

Fig. 2. (a) Acid titration to biofilm and distilled water; (b) Base titration to biofilm and distilled water.

From the result of acid-base titration (Fig. 2), H+ and OH- uptake capacities by biofilm are calculated (Fig. 3). Maximum difference of pHs in acid-base titration curve between biofilm and distilled water is around pH 4 for HCl addition and around pH 11 for NaOH addition. Similarly, maximum uptake capacity of biofilm appears at pH 3 - 4 for proton adsorption and pH 10 - 11 for hydroxyl ion adsorption (Fig. 3). It appears that around pH 4 and pH 11 there are some functional groups in biofilm polymer having ability to adsorb proton and hydroxide ions respectively. The peak at pH 4 may indicate existence of carboxyl groups (pKa = ca. 4), and that at pH 11, existence of amino groups (pKa = ca.11).

0

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Fig. 3. The adsorbed amount of H+ (black column) or OH- (white column) per unit weight of the biofilm.

The existences of the functional groups explain well the EPM change along with pH as shown in Fig. 1. In case of greater amount of negative charge than positive charge in the biofilm, the biofilm would have net negative charge. The negative EPM of biofilm at pH 7 corresponds to this case. Thus, the biofilm polymer carries both positive and negative charge in the environment of lake water (pH value ca. 7).

3.3. Adsorption of Lithium

3.3.1. Kinetics of Adsorption The time course of lithium ion adsorption by biofilm is shown in Fig. 4. The adsorption process is very

fast as the adsorption amount reached, within 1 min, to as much value as observed in later stage. This indicates that lithium adsorption by biofilm is a physicochemical process where electrostatic interaction between ions and the charged sites in biofilm polymers is a driving force to adsorb ions to get inside biofilm.

Fig. 4. Time course of lithium accumulation to winter biofilm. The experiments are repeated 3 times, independently (average values are shown). Bars represent standard error.

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In order to evaluate biofilm potential as an adsorbent in biosorption of heavy metal ion (i.e., lithium ion), adsorbed amount of ions to biofilms is compared with strongly and weakly cation exchange resins as one of common adsorbents in adsorption of heavy metal ions such lithium (Table 1). Adsorbed amount of ions is greater to biofilm than those to resins for a dry gram of these substrates. This indicates more charged sites in biofilm or greater attractive force by biofilm polymers compared with ion exchange resins. This result suggests that biofilm may become promising adsorbent for the biosorption of pollutant ions such as heavy metal ions.

Table 1. Adsorbed of Lithium to Biofilm and Cation Exchange Resins

Adsorbent Absorbed Li+ (mmol/dry-g)

Biofilm (winter) 0.081

Strongly cation exchange resin 0.0303

Weakly cation exchange resin 0.024

3.3.2. Desorption of Ions Various kinds of ions are desorbed from biofilms or cation exchange resins by adsorbing ions (Table

2). It seems that adsorbing lithium replace previous adsorbed ions bound to negative charged site of the biofilms. The adsorbing ion seems to promote desorption of ion from biofilms or cation exchange resins through ion exchange mechanisms.

Table 2. Adsorbed and Desorbed Amount of Ions to and from Biofilm

Absorbed amount of Li+ Desorbed ions

(mmol/dry-g) Ion Amount (mmol/dry-g)

0.081

K+ 0.00030

Ca2+ 0.010

Mg2+ 0.0030

Na+ 0.032

3.3.3. Comparison with Biofilm Collected in Spring The time course of adsorption of lithium to biofilm sampled in spring is also studied (Fig. 5). The

result is compared with adsorption to biofilm sampled in winter. The adsorbed amount of ions to biofilms sampled in winter (0.081 mol/dry-g) is greater, compared with those sampled in spring (0.0080

mol/dry-g). This indicates that more adsorption sites in biofilm sampled in winter compared with biofilms sampled in spring.

If this result is correlated with microscopic observation result, it reveals that biofilms sampled in winter contained more polymer like substances (data not shown). It appears that adsorption sites of cation (negatively charge sites) mostly exist in the polymer like substances. Thus, the biofilms sampled in winter (that have more polymers like substances) can adsorb greater amount of ions than biofilms sampled in spring. However, further investigation is needed to confirm this assumption.

The difference of composition of biofilms indicates that the biofilms components change seasonally along with the change in the environmental factors, such as temperature and light intensity. This should be considered in biofilm utilization in the biosorption of pollutant ions.


Fig.5. Time course of lithium accumulation to spring biofilm. The experiments are repeated 3 times, independently (average values are shown). Bars represent the standard error.

4. Conclusion

In this study, it can be concluded that: 1) both negative and positive charged sites exist on biofilm polymer; 2) the ion adsorption by biofilm polymer is a physicochemical process where electrostatic interaction between ion and charged sites in biofilm polymers is a main driving force; 3) the adsorbing ion promote the desorption of ions from biofilms through ion exchange mechanism; 4) the biofilms components change seasonally and seems to affect the ability of biofilm to adsorb ions. From all of similar studies, this study is the first one investigating utilization of natural biofilms formed in different seasons for biosorption of heavy metal ion i.e., lithium ion. Based on the conclution, biofilm may become a promising adsorbent for pollutant ions such heavy metal ions.

Acknowledgements

This study is part of research project supervised by Prof. Hisao Morisaki from Graduate School of Science and Engineering, Ritsumeikan University. The authors are grateful to Dr. Yuki Tsuchiya from Faculty of Life Science, Ritsumeikan University for data analysis and discussion.

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Biofilm Polymer for Biosorption of Pollutant Ions

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