Chinese Journal of Oceanology and Limnology   2017, Vol. 35 issue(3): 668-680     PDF       
http://dx.doi.org/10.1007/s00343-017-5329-9
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

QI Yarong(漆亚瑢), WANG Xin(王欣), CHENG Jay Jiayang(成家杨)
Preparation and characteristics of biosilica derived from marine diatom biomass of Nitzschia closterium and Thalassiosira
Chinese Journal of Oceanology and Limnology, 35(3): 668-680
http://dx.doi.org/10.1007/s00343-017-5329-9

Article History

Received Nov. 19, 2015
accepted in principle Jan. 25, 2016
Preparation and characteristics of biosilica derived from marine diatom biomass of Nitzschia closterium and Thalassiosira
QI Yarong(漆亚瑢)1, WANG Xin(王欣)1, CHENG Jay Jiayang(成家杨)1,2        
1 School of Environment and Energy, Shenzhen Graduate School of Peking University, Shenzhen, 518055, China;
2 Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC, USA
ABSTRACT: In this study, biosilica of high purity was successfully prepared from marine diatom (Nitzschia closterium and Thalassiosira) biomass using an optimized novel method with acid washing treatment followed by thermal treatment of the biomass. The optimal condition of the method was 2% diluted HCl washing and baking at 600℃. The SiO2 contents of N. closterium biosilica and Thalassiosira biosilica were 92.23% and 91.52%, respectively, which were both higher than that of diatomite biosilica. The SiO2 morphologies of both biosilica are typical amorphous silica. Besides, N. closterium biosilica possessed micropores and fibers with a surface area of 59.81 m2/g. And Thalassiosira biosilica possessed a mesoporous hierarchical skeleton with a surface area of 9.91 m2/g. The results suggest that the biosilica samples obtained in this study present highly porous structures. The prepared porous biosilica material possesses great potential to be used as drug delivery carrier, biosensor, biocatalyst as well as adsorbent in the future.
Key words: biosilica     preparation     diatom     Nitzschia closterium     Thalassiosira    
1 INTRODUCTION

Living microorganisms can generate nanostructured biomaterials through precise control of biomineralization (Mohammed, 2015). Typically, diatom which is a kind of eukaryotic unicellular and photosynthetic microalgae can absorb silicate and convert it into silica skeleton called as frustule. The three dimension nanostructured porous frustule plays an important role in mechanical protection and nutrient and waste exchange for diatom cell (Dolatabadi and de la Guardia, 2011). The diatom biosilica is a kind of hierarchically structured nanomaterials (Meyers et al., 2008). And the nanostructure morphology varies with diatom species. Moreover, diatom contributes greatly to silicon cycle, as well as to the majority of carbon fixation in the ocean (Mohammed, 2015). Diatom captures atmospheric CO2 and dissolved carbonate as carbon source to grow, which directly alleviates the emission of the problematic greenhouse gases (CO2) (Moll et al., 2014). Therefore, diatom culture provides a promising green system and sustainable platform for nanostructured porous material synthesis.

The diatom biosilica possesses superior properties of chemical and thermal stability, non-toxicity, tunable porosity, and compatibility compared to other materials. It has been proposed in various areas such as biotechnological and biomedical applications as natural made nano-devices (Gordon et al., 2009). The outstanding textural properties of diatom with high surface area, light weight and strong mechanical strength may be owed to the tiny pores that vary from one species to another (Lu et al., 2015). These advantages make diatom (no matter fossil or living diatom) as an ideal source of biosilica (Meyers et al., 2008; Dolatabadi and de la Guardia, 2011). Fossilized diatom, known as diatomite or diatomaceous earth (DE), was usually used for porous biosilica preparation at present (Sheng et al., 2009). These porous silica materials possess excellent properties and have been widely used as drug delivery carriers, biosensors, biocatalysts as well as adsorbents (Dolatabadi and de la Guardia, 2011). Still, there are some disadvantages during production process of it. First, diatomite is a mixture of diatom biosilica from different types of species with impurities, making it very difficult to get pure biosilica with varied particular structures for varied applications (Sheng et al., 2009). Second, natural diatomite contains large amount of impurities (non-silica) including clay, volcanic glass, terrigenous particles, organic matter or inorganic oxides that depend on the local environment conditions. Thus, it is difficult to remove impurities from diatomite and obtain pure isolated silica by present treatment. Usually, purification procedures of raw diatomite include crushing of diatomite rocks, removal of large aggregates and removal of oxide impurities, followed by particle size separation and sedimentation processes. The purification operations are complex and usually cause secondary pollution, and diatomite mining would disrupt and deteriorate local environment (Meyers et al., 2008). Besides, although porous silica with different size, morphology or other certain properties for various applications can be obtained by chemical synthesis (De Stefano et al., 2009a), materials with complex multi-property is really rare. Therefore, there is a need to explore alternative approaches for porous silica material production.

Natural biological synthesis for porous silica material by diatom silicification provides an alternative approach (Meyers et al., 2008). Diatom frustule have arrays of unique pores that vary greatly with diatom species, and the pore size of it range from 50 nm to several microns. And it is chemically composed of amorphous hydrated silica (SiO2•2H2O) with organic macromolecules (Wu et al., 2012). This superior physicochemical property makes it much easier to extract pure biosilica from diatom biomass (De Stefano et al., 2009b). However, there is few detailed preparation methods of biosilica from marine diatom biomass (Jeffryes et al., 2015). In some methods, detergent was used to remove large amount of organic mass, which was proved ineffective (Hildebrand et al., 2009). And some used strong acids with high concentration (70% nitric acid, 97% sulfate acid) to treat diatom, which would not only destroy the unique surface structure but also cause discharge of waste acid of high concentration (De Stefano et al., 2009a). Therefore, to explore an effective preparation method with consideration of maintaining the structure of biosilica and optimizing the use of chemical reagents is of great significance.

The aim of this work is to prepare nanostructured biosilica from marine diatom biomass (N. closterium and Thalassiosira). In this study, firstly, the gross marine diatom biomass was used as object to explore the biosilica preparation method. And the parameters of method exploration (acid type, concentration of acid solution, baking temperature and treatment time) were optimized. Then, the explored method was applied to two pure diatom strains. The characteristics (morphology, BET surface area, surface chemical groups) of two types of pure biosilica and gross biosilica materials were also investigated.

2 MATERIAL AND METHOD 2.1 Preparation of diatom samples

The dry biomass sample of gross marine diatom used in this study was collected from outdoor microalgae cultivation site of Key Engineering Laboratory for Algal Biofuels near Dapeng bay of Shenzhen City. Two pure marine diatom strains, N. closterium and Thalassiosira were separated from Dapeng Bay of Shenzhen City, China. The cultivations of pure marine diatom were carried out in f/2 media (Guillard, 1975) under aeration condition and harvested at the end of stationary phase. The harvested diatom solution was centrifuged at 4 000 r/min for 10 min (Hettich UNIVERSAL 5810R, Germany). The collected pellets were re-suspended in deionized water and centrifuged under the same condition for three times. Then the diatom pellets were freezing dried in refrigerated compressed air dryer (Labconco freezone 2.5, USA). The obtained dry biomass samples were maintained in drying basin for the preparation experiments.

2.2 Method exploration of biosilica preparation 2.2.1 Chemical treatment

The preliminary organic mass removal of diatom was carried out by acid solution washing treatment (Alyosef et al., 2014). And HCl was selected as common washing solvent (Mazumder et al., 2010). The effect of acid concentration on the impurities removal rate of diatom biomass was investigated. 2.50 g raw diatom biomass was added into a 250 mL flask. The varied acid solution concentrations contained 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15% and 20%. The flasks were shaken at 180 r/min at room temperature (approximately 25℃) for 0.5 h. The reaction products were centrifuged (Hettich UNIVERSAL 5810R, Germany) at 4 000 r/min for 6 min. Then the deposited sediments were washed with deionized water and centrifuged for 3 times to remove traces of acid. After washing process, the samples were kept in an Electric Blast Drying Oven (Shanghai Yiheng DHG-9055A, China) at 60℃ for 48 h to remove retained moisture.

2.2.2 Thermal treatment

After the acid washing treatment, the dried washed diatom samples were treated in a Digital Muffle furnace (Shanghai Yangguang SZX-4-1, China) for 0.5 h. The baking temperature was optimized. The baking temperature was set to be 200℃, 400℃, 600℃ and 800℃, respectively.

2.3 Biosilica preparation

Two pure marine diatom strains, N. closterium and Thalassiosira were used for pure biosilica preparation according to the explored method in this study.

2.4 Characterization of biosilica

The elemental compositions of the biosilica samples were analyzed by Energy Dispersive Spectrometer (EDS, EDAX GENESIS Xm2 SYSTEM 60x, USA). The XRD analyses were performed with an X-ray powder diffractometer (Rigaku D/MAX-2500/PC, Japan). The surface chemical groups of the biosilica samples were measured by Fourier transform infrared (FTIR) spectroscopy using SHIMADZU IR Prestige-21 FTIR Spectrometer with KBr pellets in the range 400-4 000/cm (SHIMADZU, Japan). The surface morphologies of samples was examined by SEM (Hitachi, S4800, Japan). The Brunauer-Emmett-Teller (BET) surface areas and the pore size distributions of the biosilica samples were determined using a TriStarⅡ 3020 Series Surface Area and Porosity Analyzer (Micromeritics Instrument Corporation, USA). SiO2 contents of biosilica extracted from three types of marine diatoms were tested using thermogravimetric analysis based on ISO 2598-1-1992.

3 RESULT AND DISCUSSION 3.1 Method exploration of biosilica preparation 3.1.1 Acid solution washing treatment

The raw diatom biomass was treated by a normal inorganic acid, HCl, to remove the acid-soluble organic and inorganic impurities. As shown in Fig. 1a, the mass removal rate of raw diatom biomass increased gradually until the acid concentration up to 2%, where the mass removal rate reached about 66%. So the optimal HCl concentration for raw diatom biomass washing is 2%, which is much lower than that reported in other methods (Mejía et al., 2013).

Figure 1 HCl washing of raw diatom biomass for 0.5 h (a) and baking of 2% HCl washed diatom for 0.5 h (b)
3.1.2 Baking treatment

After acid washing treatment, the color of residue was dark green, suggesting there may be other organic impurities remaining in the washed diatom biomass. The washed diatom biomass needs to be further treated to remove the residual organic impurities. Some solvents were reported effective in organic matter removal (De Stefano et al., 2009a; Wu et al., 2012). So, the dark green diatom samples were later treated by several solvents, including ethanol (99.9%), acetone (99.9%), or processed by hydrogen peroxide solution (50%) or 1 mol/L KNO3 solution combined with ultrasonic vibration. However, the diatom residue samples still showed dark green and no obvious mass decrease of diatom samples could be observed. These results suggested that the above chemical methods were ineffective on removing acid insoluble impurities in diatom biomass. While baking is known to be an effective way for organic matter removal (De Stefano et al., 2009b; Wang et al., 2012; Mejía et al., 2013), so it was chosen as further treatment for diatom biosilica purification. And Fig. 1b shows the mass removal rate of 2% HCl washed diatom biomass after being baked under different temperatures. It is clear that the mass removal rate of 2% HCl washed diatom biomass at 600℃ was up to 88.31%, which is near to that under 800℃ baked treatment. The result indicates that baking treatment at 600℃ for 0.5 h is enough for decomposing the residual impurities of 2% HCl washed diatom biomass.

3.1.3 Method confirmation

The sample color of raw diatom biomass, 2% HCl washed diatom biomass, and diatom biosilica of gross diatom can be seen in Fig. 2a-c. Before treatment, raw gross diatom biomass sample was in green. Gradient changes in the color of samples after washing and baking treatment can be observed. And the final white product was marine diatom biosilica. Since further experiments showed that 2% HCl washed diatom biomass baked at 500℃ was still in gray color, temperature at 600℃ would be more efficient for baking treatment. The result indicates that acid washing pre-treatment is necessary for biosilica preparation from marine diatom biomass. Besides, the biosilica preparation rate of gross diatom biomass was 11.69% as showed in Table 1. Also, the impurities removal rate of gross diatom biomass by 2% HCl solution washing was 67.01%. And the accumulated impurities removal rate of gross diatom biomass after baking treatment was 88.31%. Finally, an optimal method of biosilica preparation from marine diatom biomass was obtained.

Figure 2 Samples of (a) gross diatom powder, (b) 2% HCl washed gross diatom, (c) gross diatom biosilica, (d) diatom powder of N. closterium, (e) 2% HCl washed diatom of N. closterium, (f) diatom biosilica of N. closterium, (g) diatom powder of Thalassiosira, (h) 2% HCl washed diatom of Thalassiosira, (i) diatom biosilica of Thalassiosira
Table 1 Biosilica preparation rates and impurities removal rates for diatoms

The SEM images of raw diatom, 2% HCl washed diatom, 20% HCl washed diatom, biosilica of diatom baked at 600℃, and biosilica of diatom baked at 800℃ were showed in Fig. 3a-e, respectively. Obviously, the surface of raw gross diatom was clogged with impurities. After being washed by 2% HCl, the surface impurity was clearly less than raw diatom. While after 20% HCl treatment, the surface structure of gross diatom was eroded. With further treatment of baking at 600℃, the micro-pores can be seen clearly on its surface, confirming the decomposition of the surface impurities. While after baking treatment at 800℃, the skeleton structure was destroyed. The result indicated that baking treatment at 600℃ can remove the impurities effectively as well as keep its skeleton structure complete. Thus, the method of acid washing treatment followed by 600℃ baking treatment is more favorable for marine diatom biosilica preparation.

Figure 3 SEM images of (a) raw gross diatom, (b) 2% HCl washed gross diatom, (c) 20% HCl washed gross diatom, (d) biosilica of gross diatom baked at 600℃, (e) biosilica of gross diatom baked 800℃

Energy dispersion X-ray spectroscopy (EDS) was used to analyze the chemical composition of gross diatom biosilica. Powder samples were fixed on a copper tape rather than carbon tape to avoid the disturbance of carbon element. Atomic percentage change of Si and C during the gross diatom biosilica preparation process can be seen in Fig. 4a. The atomic content of C element dropped dramatically after each treatment, while Si atomic content increased, suggesting impurities were successfully removed. But the atomic content of Si element decreased as the baking temperature increased to 800℃. Result shows that baking temperature at 600℃ is more efficient for biosilica preparation, where the Si atomic content increased from 8.45% to 24.94%, while C atomic content decreased from 41.98% to 8.05%.

Figure 4 (a) Si and C atomic content, (b) FT-IR spectra of gross diatom before and after treatments, (c) XRD pattern of gross diatom biosilica, (d) nitrogen adsorption isotherms of gross diatom before and after treatments, (e) pore size distributions of gross diatom before and after treatments

Figure 4b shows the main characteristic peaks of biosilica from gross diatom and raw gross diatom. The chemical groups on the surface of raw diatom includes Si-O-Si bending at 470/cm, Si-O-Si stretching at 1 040-1 095/cm, and O-H stretching of hydroxyl groups at 3 435/cm, -COOH at 1 790/cm, -COO-at 1 400-1 651/cm and-OH from-COOH at 2 523/cm, -SO-at 1 231/cm, -R3C-OH at 1 072/cm, and-CH2-, -CH3-, -CH4-at 2 850-2 960/cm (De Stefano et al., 2009b). After biosilica preparation, groups of-COOH, -COO-, -OH from-COOH, -SO-, or-R3C-OH were removed. Functional groups of gross diatom biosilica are Si-O-Si bending at 470/cm, Si-O-Si bending at 797-800/cm, Si-O stretching of Si-OH groups at 950/cm, Si-O-Si stretching at 1 040-1 095/cm, and O-H stretching of hydroxyl groups at 3 435/cm, which contains adsorbed water and Si-O-H stretching. The peaks of 1 630-1 640/cm and 3 740-3 780/cm stand for the Si-OH bonds. In comparison with raw diatom, most organic chemical groups disappeared, indicating the successful removal of impurities from raw gross diatom through acid washing and baking treatments.

The X-ray powder diffraction pattern of gross diatom biosilica was shown in Fig. 4c to characterize components of the gross diatom biosilica product. As shown in the figure, a broad reflection centered at 2θ=20°-25° appeared, suggesting the amporphous phase of SiO2 in the biosilica. This result confirms the extracted biosilica coming from diatom biomass. Meanwhile, several sharp peaks centered at 21° and 26° were also observed, indicating that there was SiO2 in well-crystallized quartz form existing in the prepared biosilica sample. This is because the gross diatom biomass sample in this study was collected from a large-scale outdoor cultivation site. And it was cultured using seawater without 0.45 μm membrane filtration process, making the culture solution includes natural impurities such as sand etc. which are usually in quartz phase. Therefore, the well-crystalized quartz of SiO2 contained in the biosilica was attributed to the introduced natural SiO2 impurities such as sand from sea water. Besides, a little amount of Al2O3 and Fe2O3 were also observed in the biosilica. In this study, the SiO2 content of the extracted biosilica from gross marine diatom was measured to be 94.76% (Table 2), which is much higher than that of purified diatomite in other studies (65%-82.95%) (Sheng et al., 2009; Danil de Namor et al., 2012). The results suggest that the gross marine diatom biosilica extracted using the explored method is of high purity.

Table 2 SiO2 contents of marine diatom biosilica prepared from three types of diatoms

Nitrogen adsorption isotherms of raw gross diatom and its biosilica were shown in Fig. 4d. It can be seen that quantity adsorbed by gross diatom biosilica was higher than raw gross diatom in the given pressure range, showing the biosilica has a better adsorption ability. The BET surface area of gross diatom and its biosilica was shown in Table 3. The BET surface area of the gross diatom biosilica was 142.47 m2/g, which was much bigger than that of raw gross diatom of 5.72 m2/g, and also higher than that of biosilica from diatomite with a surface area range of 18.8-30.92 m2/g (Sheng et al., 2009; Aw et al., 2012). The pore size distributions were shown in Fig. 4e. The average pore size of biosilica extracted from gross diatom was calculated to be 22.53 nm. From pore-size distribution figure, it is clear that incremental pore volume of raw diatom increased greatly after preparation treatments. As shown in figure, the highest incremental pore volume of gross diatom biosilica appeared in 21.0-54.8 nm interval. The result indicates that gross diatom biosilica obtained in this study mainly possesses mesopores.

Table 3 BET test results of raw diatoms and biosilica from gross diatom, N. closterium and Thalassiosira
3.2 Preparation of N. closterium biosilica and Thalassiosira biosilica

The marine diatom biosilica preparation method explored in this study was further used for biosilica preparation from pure diatom strains. Two kinds of separated marine diatom strains, N. closterium and Thalassiosira, were selected for biosilica preparation. Biomass of N. closterium and Thalassiosira were first washed by 2% HCl solution for 0.5 h, and then were baked at 600℃ for another 0.5 h. Finally, white products of N. closterium biosilica and Thalassiosira biosilica were obtained.

3.2.1 Biosilica preparation efficiency

Sample pictures of N. closterium and Thalassiosira biomass before and after treatments were shown in Fig. 3. As shown in Fig. 3d-f, for N. closterium, the raw diatom color was green, and the sample color after 2% HCl washing treatment was dark green, while the final sample after baking treatment was white. As for Thalassiosira, as shown in Fig. 3g-i, the raw diatom was yellow green, the 2% HCl washed diatom biomass sample was grey green, after further baking treatment, the final product was also white. Besides, biosilica preparation rates and impurities removal rates during the biosilica preparation process were shown in Table 1. The biosilica preparation rates of N. closterium and Thalassiosira were calculated to be 13.15% and 22.98% respectively, which were higher than that of gross diatom (11.69%).

3.3 Characterization of N. closterium biosilica 3.3.1 Morphology

The SEM images of raw diatom and diatom biosilica of N. closterium were shown in Fig. 5. Similar to raw gross diatom sample, the skeleton and micropores and fibers of raw N. closterium were clogged with macromolecule impurities before preparation treatments. In comparison, after being treated, the hierarchy skeleton and the biosilica surface with micropores and fibers can be seen clearly. The N. closterium biosilica skeleton was mainly composed of nano-channels and nano-fibers, with a few of micropores. This result confirms the impurities removal from raw N. closterium biomass. The biosilica product prepared from N. closterium biomass possesses complete skeleton and cleaner surface compared with that from diatomite (Alyosef et al., 2014).

Figure 5 SEM images of (a), (b) raw diatom of N. closterium; (c), (d) diatom biosilica of N. closterium
3.3.2 XRD analysis

The XRD pattern of N. closterium biosilica was shown in Fig. 6a. The diffraction spectrum of N. closterium biosilica sample is typical for amorphous silica (a broad reflection centered at 2θ=20°-25°) (Jin et al., 2014) without other obvious peaks. The result suggests that the N. closterium biosilica mainly consisted of SiO2. The amorphous band described in Fig. 6a may be attributed to the glass formation of SiO2 peaks at 21°, 26° (Aw et al., 2012).

Figure 6 XRD pattern of N. closterium biosilica (a), FT-IR spectra of raw N. closterium and its biosilica (b), nitrogen adsorption isotherms of raw N. closterium and its biosilica (c), pore size distributions of raw N. closterium and its biosilica (d)
3.3.3 Chemical composition

Si and C atomic contents of N. closterium before and after biosilica preparation were shown in Table 4. The result showed the Si atomic percentage increased severely, which was 13.28 times of its raw diatom. On the contrary, the C atomic percentage of N. closterium decreased by 89.05%. And the elements of N, S, Cl and Na and Ca in N. closterium disappeared after extraction process, suggesting organic impurities were successfully removed. Meanwhile, different to N. closterium biomass sample, the elements of Mg, Fe and Al in N. closterium biosilica sample were detected, which may exist in the biosilica in their oxidized forms. The result indicates the high efficiency of explored method for diatom biosilica preparation in this study. As shown in Table 2, the SiO2 content of N. closterium biosilica was 92.23%, which was higher than that of gross diatom biosilica (91.27%) or of modified diatomite (82.95%). The result confirms that the prepared N. closterium biosilica is of high purity.

Table 4 Atomic percentages before and after biosilica preparation of N. closterium and Thalassiosira
3.3.4 Chemical groups

As shown in Fig. 6b, the surface chemical groups of raw N. closterium including-COOH, -COO-, -OH, may mainly came from organic impurities. Whereas, after acid washing and baking treatment, the main groups of biosilica product were the groups that contain Si element, including Si-O-Si bending at 470/cm and 797-800/cm, Si-O stretching at 950/cm, Si-O-Si stretching at 1 040-1 095/cm, and Si-OH bonds at 1 630-1 640/cm and 3 740-3 780/cm. The result indicates the successful removal of organic impurities from raw N. closterium biomass.

3.3.5 BET surface area

Nitrogen adsorption isotherms of raw diatom and biosilica derived from N. closterium were shown in Fig. 7c. The adsorbed quantity of N. closterium biosilica was higher than its raw diatom. As shown in Table 3, the BET surface area of N. closterium biosilica was 59.81 m2/g. In comparison, the BET surface area of raw N. closterium was 4.61 m2/g.

Figure 7 SEM images of (a), (b) raw diatom of Thalassiosira; (c), (d) diatom biosilica of Thalassiosira
3.3.6 Pore size distribution

The biosilica pore size distribution was presented in Fig. 6d. The average pore size of the biosilica prepared from N. closterium was calculated to be 18.45 nm. As shown in Fig. 6d, N. closterium biosilica pore size were mainly among 21.4-54.8 nm interval, indicating its surface pores were mainly mesopores. The pore size range of the biosilica became wider comparing with raw biomass, since micropores within the 1.7-10.3 nm interval appeared after treatment. Moreover, micropores within each interval increased dramatically after treatment. These results confirm the obtained biosilica of hierarchical mesoporous surface.

3.4 Characterization of Thalassiosira biosilica 3.4.1 Morphology

The SEM images of raw diatom and diatom biosilica of Thalassiosira were shown in Fig. 7. As shown in Fig. 7a-b, the micropores of Thalassiosira were clogged with impurities before treatment. But after being treated, the hierarchy structure with mesopores on biosilica surface can be seen clearly. And Thalassiosira biosilica hierarchical structure was mainly consisted of mesopores on upper layers and nanopores on the lower layers. The result provides evidence for the impurities removal from raw Thalassiosira. The biosilica product from Thalassiosira also possesses a better skeleton structure and cleaner pores than that from diatomite (Alyosef et al., 2014).

3.4.2 XRD analysis

The XRD pattern of Thalassiosira biosilica was shown in Fig. 8a. The diffraction spectrum of Thalassiosira sample was also typical for amorphous silica without other obvious peaks (Jin et al., 2014). The result suggests the Thalassiosira biosilica mainly consisted of SiO2. And the amorphous band described in Fig. 8a may be due to the glass formation of SiO2 peaks at 21°, 26° (Aw et al., 2012).

Figure 8 XRD pattern of Thalassiosira biosilica (a), FT-IR spectra of raw Thalassiosira and its biosilica (b), nitrogen adsorption isotherms of raw Thalassiosira and its biosilica (c), pore size distributions of raw Thalassiosira and its biosilica (d)
3.4.3 Chemical composition

Si and C atomic percentages of Thalassiosira before and after biosilica preparation were shown in Table 4. It is clear that the Si atomic percentage increased greatly after being treated. And Si atomic percentage of Thalassiosira was 7.34 times of raw Thalassiosira. On the contrary, the C atomic percentage of Thalassiosira decreased by 91.47%. Similar to N. closterium, the elements of N, S, Cl and Na in Thalassiosira disappeared after extraction process, suggesting organic impurities were successfully removed. And the elements of Ca, Mg, Fe and Al were detected in Thalassiosira biosilica. Besides, the SiO2 content of Thalassiosira biosilica was 91.52% (Table 2), which was also higher than that of gross diatom biosilica and modified diatomite. These results indicate the high efficiency of explored method for diatom biosilica preparation.

3.4.4 Chemical groups

As shown in Fig. 8b, the chemical groups on the surface of raw Thalassiosira including-COOH, -COO-, -OH and-CH mainly came from organic impurities. And all of them disappeared after being treated. After treatments, the main chemical groups of final biosilica product were the groups containing Si, including Si-O-Si bending at 470/cm and 797-800/ cm, Si-O stretching at 950/cm, Si-O-Si stretching at 1 040-1 095/cm, and Si-OH bonds at 1 630-1 640/cm and 3 740-3 780/cm. The result shows that main chemical groups in Thalassiosira biosilica resembles those in gross diatom biosilica and N. closterium biosilica, with the main groups containing Si. These results indicate the successful preparation of Thalassiosira biosilica from Thalassiosira biomass.

3.4.5 BET surface area

Nitrogen adsorption isotherms of raw diatom and its biosilica of Thalassiosira were shown in Fig. 8c. The adsorbed quantity value of Thalassiosira biosilica was higher than that of raw Thalassiosira, which also suggests a better adsorption ability of diatom biosilica. As shown in Table 3, the BET surface area of biosilica from Thalassiosira was 9.91 m2/g. In comparison, the BET surface area of raw Thalassiosira was 0.000 5 m2/g.

3.4.6 Pore size distribution

The biosilica pore size distribution was shown in Fig. 8d. The average pore size of Thalassiosira biosilica was calculated to be 65.17 nm. The Thalassiosira biosilica pores were macropores within the range of 20.0-172.9 nm. In comparison, the pore size of raw Thalassiosira biomass was nearly zero. This result indicates that the obtained biosilica in this study possesses a highly porous surface structure.

4 CONCLUSION

In this study, highly pure biosilica samples were successfully prepared from marine diatom biomass, N. closterium and Thalassiosira, using the method of acid solution washing followed by thermal treatment. The optimal condition of the biosilica preparation method for marine diatom biomass is 2% HCl washing for 0.5 h followed by baking at 600℃ for another 0.5 h. After biosilica preparation treatment, the Si element percentage content increased dramatically. And the chemical groups of final biosilica products were the groups mainly containing Si. The SiO2 contents of N. closterium biosilica and Thalassiosira biosilica are significantly higher than that of diatomite biosilica. The SiO2 morphologies of both N. closterium biosilica and Thalassiosira biosilica are typical amorphous silica. Besides, the N. closterium biosilica has micropores and fibers with average pore size of 18.45 nm and the BET surface area of 59.81 m2/g. Meanwhile, the Thalassiosira biosilica has a hierarchical skeleton with mesopores with average pore size of 65.17 nm and the BET surface area of 9.91 m2/g. The result shows that the biosilica obtained in this study possess highly porous surface structure.

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