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Article HttP://DX.DOi.OrG/10.5504/BBeQ.2013.0054 A&eB

Biotechnol. & Biotechnol. eq. 27/2013/4

Biotechnol. & Biotechnol. eq. 2013, 27(4), 3918-3922Keywords: carbon fiber, surface modification, microorganism immobilization, adhesion, wastewater treatment

IntroductionWater pollution is one of the most pressing environmental problems in the last two decades. As a promising technology for wastewater treatment, biofilm treatments have been successfully used for higher effluent quality (22). Biofilm reactors function with a higher rate of removal of chemical oxygen demand (COD) and lower excess sludge production, and allow high concentrations of mixed liquor suspended solids (26, 28), which are difficult to be achieve effectively by conventional activated sludge processes. In the biofilm treatment technology, the choice of suitable biofilm carrier is one of the most important steps, and many organic and inorganic materials are used as conventional biofilm carriers in biofilm reactors (such as Ca-alginate, polyvinyl chloride, polythene, etc.). It is well known that carbon has good biocompatibility (23), so carbon-based materials may have very broad development prospects as biofilm carriers. Researchers have already found that carbon fibers (CF) could make a large number of microorganisms adhere to them in a short time, much better than other carriers (10, 17). That means that, as a biofilm carrier, CF will create more favorable conditions for water treatment.

Microorganism adhesion is the foundation of biofilm formation, which is significant in biofilm water treatment. Many references (4, 12, 14, 16) show that material surface characteristics, such as hydrophilicity, chemistry, charge,

roughness and rigidity, can greatly affect the adhesion capacity of different types of cells on material surfaces. And some efforts (7, 13, 15) show that the adhesion capacity of bacterial or eukaryotic cells could be improved if the material surface is modified to a better hydrophilicity state by producing various oxygen-based functional groups, such as hydroxyl group, carboxylic acid, carboxylic ester, ether, ketone, aldehyde, etc. However, a very hydrophilic surface can also hinder the microorganism adhesion because the oxygen-based functional groups give the carrier surface a strong negative charge, which repels the negatively charged cell surface with ionized amino acids (6). Our previous study (2) showed that the metal ions adsorbed on CF surfaces may improve the immobilization ability of microorganisms by connecting both electronegative oxygen groups and electronegative extracellular polymeric substances (EPS).

A lot of researchers (5, 11, 20) have studied the adhesion behavior of bacterial or eukaryotic cells on treated carbon-based samples, and strong acid is a conventional and useful surface oxidation treatment to modify various types of carbon materials (such as carbon fiber, activated carbon, carbon nanotube, etc.) by producing oxygen-based functional groups (9, 19, 21). However, there is little research about the immobilization behavior of microorganisms on modified CF biofilm-carriers. Previously, maleic anhydride and urea were grafted on strong acid oxidized CF surface, and these complex surface modifications could greatly improve the adhesion capacity of microorganisms (1, 3). In the present work, the effects of different surface nature on the immobilization of microorganisms, including the content of various oxygen-based functional groups, were synthetically investigated and

TIME-GRADIENT NITRIC ACID MODIFICATION OF CF BIOFILM-CARRIER AND SURFACE NATURE EFFECTS ON MICROORGANISM IMMOBILIZATION BEHAVIOR IN WASTEWATER

Yanling Bao and Guangze DaiSouthwest Jiaotong University, School of Materials Science and Engineering, Chengdu, P.R. ChinaCorrespondence to: Guangze DaiE-mail: g.dai@163.com

ABSTRACTPAN-based carbon fiber (CF) biofilm-carrier was modified by nitric acid for a different oxidation time. The CF surface morphology and nature were characterized by laser confocal microscopy (LCM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and degree of moisture (DM). In addition, the biocompatibility of CF was discussed based on the immobilization behavior of microorganisms. The results indicated that the HNO3 oxidation treatment could be regarded as a favorable surface treatment for the CF carrier. The surface oxygen content and hydrophilicity were shown to affect the immobilization ability of microorganisms and influence biofilm formation on the CF surface. Among these, the carboxyl content on the CF surface contributed greatly to microorganism immobilization. The configurations and adhesion forms of cells were considerably affected by the properties of the CF surface. The results from this study could be helpful in the study of microorganism adhesion mechanisms and could be regarded as promising in view of preparation of high biocompatibility bio-carriers in the field of water treatment.

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Fig. 1. LCM photographs of CF samples: CF-0 (a), CF-1 (b), CF-2 (c), CF-3 (d), and CF-4 (e).

Fig. 2. LCM images of cells attached onto CF surfaces after 2 h of incubation: CF-0 (a), CF-1 (b), CF-2 (c), CF-3 (d), and CF-4 (e).

analyzed in order to study the modification mechanism of microorganisms. The effects of CF surface morphology, nature and microorganism adhesion ability were characterized by laser confocal microscopy (LCM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), the degree of moisture (DM), and the immobilization ratio of microorganisms (IRM), respectively.

Materials and MethodsSurface modificationPAN-CF (Commercial T300) was chosen as a carrier material. Prior to usage, CF was immersed in acetone for 24 h to remove the sizing agent from the CF surface, then washed with distilled water and dried at 120 °C for 4 h (denoted as CF-0). The modification was as follows: CF-0 was immersed into concentrated HNO3 and shaken slowly at 100 °C for 1 h, 2 h, 3 h and 4 h, then washed with distilled water to pH 7 and dried at 120 °C for 4 h (denoted as CF-1, CF-2, CF-3 and CF-4, respectively). The oxidation was aimed at increasing the oxygen-based functional groups and improving the wettability of the bio-carrier samples.

Characterizations and equipmentThe morphology of the specimens was observed by laser confocal microscopy (VK-9710, Keyence Corporation, Japan). The chemical properties of the CF surfaces were assessed by FTIR and XPS. The FTIR spectra (KBr dispersed pellets) in the range of 400 cm-1 to 4000 cm-1 were recorded on a fully computerized Nicolet 5700 spectrometer at a resolution of 4 cm-1. The X-ray photoelectron spectroscopy (XSAM800, Kratos Analytical Ltd, England) measurement of CF surface was performed with an Al X-ray source (1486.6 eV); the base pressure in the sample chamber was about 2×10-7 Pa. The hydrophilicity of the CF surface was represented by the degree of moisture (DM) defined as the weight (g) of water adsorbed per gram CF after the CF surface has been kept hanging in a closed container with a saturated solution of ammonium sulfate in the bottom for 24 h (85 % relative humidity). If the

specimen has better hydrophilicity and wettability, the DM value will be higher.

Microorganisms and growth conditionsActivated sludge from the Sanwayao Wastewater Treatment Plant (Chengdu, China) was chosen for study. The microorganisms in the activated sludge were cultured and enriched at 25 °C in a thermostatic container with an aeration rate of 0.2 m3/h by feeding beef extract peptone medium (3 g/L beef extract, 10 g/L peptone and 5 g/L NaCl). The concentration of mixed liquor suspended solids (MLSS) in the activated sludge suspension was adjusted to 4500 mg/L (8), and the pH was adjusted to about 7.8 with a 1 mol/L NaOH solution.

Microorganism immobilization and definitionCF specimens were immersed in the activated sludge suspension solution, where aeration was provided throughout the process, and microorganisms began to attach and adhere to the CF surface. Specimens were taken out from the solution after different duration of immersion, lightly rinsed in sterile distilled water and dried in a desiccator overnight before LCM examination. The immobilization ability of microorganisms was expressed as the immobilization ratio of microorganisms (IRM), i.e. the specific dry weight (g) of microorganisms immobilized on 1 g of CF after the CF surface has been kept immersed in the medium for 24 h.

Results and DiscussionFig. 1 shows the LCM photographs of CF-0, CF-1, CF-2, CF-3 and CF-4 samples. As expected, CF-0 exhibited a relatively smooth surface with shallow grooves (Fig. 1a). the micrographs (Fig. 1b–e) of acid-treated CF show that, with enhancement of the oxidation degree by HNO3 treatment, the ridges of grooves became thinner and black etch pits were created. This indicates that the surface roughness of CF increased, implying great corrosiveness of HNO3 on the CF surface. CF-4 displayed the roughest surface (Fig. 1e) for long duration of acid oxidation.

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Fig. 2 illustrates the images of microorganism cells attached on different CF samples after the CF surfaces had been kept hanging for 2 h in the sludge suspension medium containing microorganisms (with aeration), and indicates that the configurations and the adhesion forms of cells were considerably affected by the properties of the CF surface. On the CF-0 surface, the preferred manner of attachment of cells was along the grooves at the beginning of adhesion (Fig. 2a). However, besides a little number of cells adhering along the channels, most of the microorganism cells adhered cross the grooves on the CF-3 surface (Fig. 2d). As a whole, there were more cells adhering onto the treated CF surfaces than onto CF-0.

Clearly, only a few cells had attached onto the CF-0 and the CF-3 surface. The CF-1 and the CF-4 surface were just partly covered by microorganisms, and a biofilm had not developed fully. However, the cell distribution on CF-1 was more uniform than that on CF-4, and the quantity of cells on CF-1 was lager. The microorganisms on CF-2 had already covered the whole CF surface and a developed biofilm could be observed, indicating that CF-2 has the best biocompatibility and potential for immobilization of microorganisms.

According to Fig. 1 and Fig. 2, CF-4 has the roughest surface, but not the best cell adhesion ability, which indicates that the surface roughness of the CF carrier does not have a considerable effect on microorganism adhesion.

X-ray photoelectron spectroscopy is a unique and valid technique for obtaining detailed information about the atomic structures of elements and molecules from carbon fiber surfaces (27). Table 1 presents the element content information for O and C from XPS spectra. The data obtained from five different kinds of CF samples showed an obvious change of element content on the CF surfaces. The original surfaces usually have an oxygen-containing surface because of the oxygen groups in the sizing agent, and part of the sizing agent can be removed by washing in acetone (24). The oxygen content was found to increase from CF-0 to CF-4 with the extension of oxidation time, which demonstrated that oxygen groups were successfully produced on CF surfaces after acid treatment.

TABLE 1 Atomic content of different samples obtained from XPS spectra

Samples C1s [Area] O1s [Area] O/C [%]CF-0 2895.6 738.6 25.5CF-1 2662.6 784.7 29.4CF-2 2234.8 828.3 37.1CF-3 2597.3 1028.0 39.6CF-4 2863.8 1673.1 58.4

The FTIR spectra of CF samples are drawn in Fig. 3. For CF-0 and CF-acid (CF after HNO3 oxidation), the wide peak between 3600 cm-1 and 3100 cm-1 reflects OH stretching vibrations (18). A number of overlapping bands appeared between 1400 cm-1 and 1000 cm-1, which suggest that there are C–O–C, C–O–N or >C–N groups (23). Peaks appearing

at 1610 cm-1 and 1240 cm-1 are ascribed to available oxygen functionalities (highly conjugated C=O stretching, C–O stretching in carboxylic groups, and carboxylate moieties) (9). in Fig. 3, the peaks of CF-acid are stronger than the ones of CF-0, which indicates that the oxidation treatment gave rise to an increase of oxygen-containing groups. These results are consistent with the XPS results in Table 1.

Fig. 3. FTIR spectrum of CF-0 and CF-acid (CF after HNO3 oxidation).

Fig. 4. Core region spectra C1s of different CF samples.

TABLE 2 Component peak positions used in C1s curve fitting

Functionality Graphitic carbon c–o C=O O–C=O

Binding energy [eV] 284.8 285.9 288.0 289.4

The core region spectra of C1s from XPS for different fiber samples are presented in Fig. 4. The main C1s peak is at 284.8 eV, which corresponds to the graphitic linkage of CF. In addition, there are some peaks at higher binding energy positions than the main C1s peak and they correspond to different oxygen-containing groups. Table 2 gives the different oxygen-containing carbon structural components possibly existing on CF samples, and the particular range of binding energies for each functional group. According to Table 2, the concentrations of oxygen groups in different samples from

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computing the percentage area of the peaks in C1s spectra (Fig. 4) are shown in Table 3. The data for the degree of moisture (DM) and immobilization ratio of microorganisms (IRM) are also given.

As shown in Table 3, the main oxygen-containing groups on the CF surface are C–O, C=O, and O–C=O. When CF samples were modified by oxidation, the degree of moisture of CF increased gradually from 1.20 % to 12.7 % with increasing the total content of oxygen groups on the CF surface. This indicates that the hydrophilicity and wettability of CF are positively correlated with the oxygen content on the CF surface.

TABLE 3 Fitted data of C1s curve of different CF samples

SamplesConcentration of oxygen groups [%] DM

[%]IRM [%]C–O C=O O–C=O Total

CF-0 11.39 2.45 2.90 16.74 1.20 117.1CF-1 13.87 3.68 3.30 20.85 5.18 153.9CF-2 17.26 3.19 3.68 24.13 6.13 227.0CF-3 23.57 4.21 2.60 30.38 6.71 131.6CF-4 24.66 3.17 2.90 30.73 12.7 142.1

However, as the oxygen content and DM value on the surface increased, the immobilization ratio of microorganisms rose from 117.1 to 227.0 at first but then decreased to 142.1. This means that hydrophilicity is not the only factor that affects the adhesion ability of microorganisms.

In order to show intuitive relationships among the degree of oxidation, hydrophilicity and immobilization capacity of CF, Fig. 5a and Fig. 5b were drawn according to Table 3. The graphs clearly indicate that the adhesion capacity of microorganisms improved if the material surface was modified to a better hydrophilicity state by producing oxygen-based functional groups, but a very hydrophilic surface can hinder microorganism immobilization as well. In other words, the microorganisms showed a preference for adhering onto carrier surfaces with moderate hydrophilicity and oxygen content. This is consistent with the LCM results in Fig. 2.

Taking into account the content of different oxygen functional groups given in Table 3, the relationship between the O–C=O content and microorganism immobilization is additionally shown in Fig. 5c. With increasing the hno3treatment time, the O–C=O content increased at first as a result of oxidation, but then decreased because of corrosion, which converted O–C=O to CO2. As shown in Fig. 5c, the variation trend of the O–C=O content is positively correlated with the immobilization ratio of microorganisms, which indicates that the O–C=O groups on the CF surface have a great effect on the adhesion of microorganisms. A possible explanation could be that hydrogen bonds caused by carboxyl groups influence the adhesion capacity (25).

Fig. 5. Effects of oxidation time on the total oxygenic functional group content and microorganism immobilization (a), on the degree of moisture and microorganism immobilization (b), and on O–C=O content and microorganisms immobilization (c).

Fig. 6. Photos of microorganism immobilization state on different CF samples following 24 h of incubation: sample before microorganism immobilization (a), CF-0 (b), CF-2 (c).

The IRM data in Table 3 also show that 2 h HNO3 oxidation for CF-2 is a favorable surface modification for the CF carrier, which demonstrated a notably promoted potential for immobilization of microorganisms on samples and an obviously improved biocompatibility of CF. Macrography photos of the microorganism immobilization state on the CF-0 and the CF-2 surface after the CF had been kept hanging for 24 h in the sludge suspension medium containing microorganisms were also taken (Fig. 6), to display the superiority of CF-2 for immobilization.

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ConclusionsThe results from this study showed that HNO3 oxidation treatment could be considered as a favorable surface treatment for CF carriers, and that the degree of oxidation has a great influence on modification. The configurations and the adhesion forms of cells were notably affected by the properties of the CF surface. The immobilization ratio of microorganisms (IRM) increased from 117.1 to 227.0 when the CF surface was modified to better hydrophilicity by producing oxygen-based functional groups, but a very hydrophilic surface can also hinder microorganism immobilization, since the IRM was reduced back to 142.1. In other words, the microorganisms showed preference for adhering onto carrier surfaces with moderate hydrophilicity and oxygen content with a DM of 6.13 %. The O–C=O groups on the CF surface have a great effect on the adhesion of microorganisms, and the variation trend of the O–C=O content was positively correlated with IRM. The obtained results could be helpful in the study of microorganism adhesion mechanisms and could be regarded as promising in view of preparation of high biocompatibility bio-carriers in the field of water treatment.

AcknowledgementsThe present work was supported partly by Key Projects in the National Science & Technology Pillar Program during the 11th Five-Year Plan Period (No. 2009BAG12A07), the Fundamental Research Funds for the Central Universities (No. SWJTU12CX011, No. SWJTU12CX012, and No. SWJTU11CX051), the Special Founds for 2012 Central University to Improve the Basic Conditions Running, the SWJTU Special Founds for 2012 Key Specialty Construction.

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