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Bacterial cellulose of Gluconoacetobacter hansenii as a potential bioadsorption agent for its green environment applications a
a
Bhavna V. Mohite & Satish V. Patil a
School of Life Sciences, North Maharashtra University, P.O. Box. 80, Jalagoan 425001, MS, India Published online: 17 Oct 2014.
To cite this article: Bhavna V. Mohite & Satish V. Patil (2014) Bacterial cellulose of Gluconoacetobacter hansenii as a potential bioadsorption agent for its green environment applications, Journal of Biomaterials Science, Polymer Edition, 25:18, 2053-2065, DOI: 10.1080/09205063.2014.970063 To link to this article: http://dx.doi.org/10.1080/09205063.2014.970063
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 18, 2053–2065, http://dx.doi.org/10.1080/09205063.2014.970063
Bacterial cellulose of Gluconoacetobacter hansenii as a potential bioadsorption agent for its green environment applications
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Bhavna V. Mohite and Satish V. Patil* School of Life Sciences, North Maharashtra University, P.O. Box. 80, Jalagoan 425001, MS, India (Received 8 August 2014; accepted 24 September 2014) Bacterial cellulose (BC) is an interesting biopolymer produced by bacteria having superior properties. BC produced by Gluconoacetobacter hansenii (strain NCIM 2529) under shaking condition and explored for its applications in dye removal and bioadsorption of protein and heavy metals. Purity of BC was confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM) analysis. BC removed azo dye and Aniline blue (400 mg/L) with 80% efficiency within 60 min. The adsorption and elution of Bovine serum albumin (BSA) and heavy metals like lead, cadmium and nickel (Pb2+, Cd2+ and Ni2+) was achieved with BC which confirms the exclusion ability with reusability. The BSA adsorption quantity was increased with increase in protein concentration with more than 90% adsorption and elution ratio. The effect of pH and temperature on BSA adsorption has been investigated. Bioadsorption (82%) and elution ratio (92%) of BC for Pb2+ was more when compared with Cd2+ (41 and 67%) and Ni2+ (33 and 85%), respectively. BC was also explored as soil conditioner to increase the water-holding capacity and porosity of soil. The results elucidated the significance of BC as renewable effective ecofriendly bioadsorption agent. Keywords: bacterial cellulose; bioadsorption; biopolymer; heavy metals
Introduction Bacterial cellulose (BC) is homopolysaccharide having wide industrial applications and it could be a biotechnology’s next high-value product.[1] Nanofibers of BC are about several times thinner compared with plant cellulose making it highly porous with large surface area and high water-holding capacity material. BC is one of the new most abundant, natural biosorbent, renewable, biodegradable and biocompatible polymers.[2] Industrial effluents especially coloured ones from textile, dyes, paper and pulp industries consist of various compounds with complex aromatic structure which make them quite difficult to degrade. Among these, azo dyes account for over 60% of the total dyes produced.[3] The employment of physico-chemical methods for removal of dye from effluent was although effective, but it is cost prohibitive and often leads to extra solid wastes. As a viable alternative, biological processes have received increasing interest owing to their cost-effectiveness.[4] Adsorption has been observed as an effective process for colour removal from such effluents. Adsorption on activated charcoal is an effective method but is prohibitively expensive. Low-cost adsorbents generally *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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have low-adsorption capacities mediating the need for economical and easily available yet highly effective adsorbents.[3] Living and dead cells of bacteria, fungi, algae or plant biomass or a combination there of is generally used for bioremediation.[5] The adaptability of biological methods to varied conditions gives bioremediation an edge over the physical–chemical methods. This is a novel study where we use biopolymer as bioadsorbent for dye removal. The cellulose produced from plant source has importance in bioremediation purpose, but consist of contaminating materials like hemicellulose, lignin along with cellulose which should be separated. Hence, pure cellulose from bacterial source, which is an emerging new industrial product, has an importance favoured by its unique features.[1] Natural products composed primarily of cellulose matrix were tried by number of researchers for removal of heavy metals.[6–8] Soil conditioners reported as efficient tools in increasing water-holding capacity, reducing infiltration rate and cumulative evaporation, and improving water conservation of sandy soils.[9] One of the highly effective methods to enhance and structure is by adding synthetic and natural polymers that improve soil cohesion, porosity, maximum water-holding capacity (MWHC) and various beneficial soil properties. Synthetic polymers cannot be used during organic farming because of their synthetic nature and production from nonrenewable resources. Natural polymers are degraded usually by relatively benign route. Cost-effective and ecofriendly nature of biopolymers makes it an important alternative to synthetic polymers. The hazardous proteins like Tat and Nef or environmental toxins or biotoxins could be removed with polymers like BC. It makes BC an interesting tool for environment cleaning up.[10] In view of this background, the goal was to determine the potential of BC as bioadsorption agent for clean environment applications. Spherical BC produced by shaking method is translucent, loose, porous and has a hydrophilic network structure. Its specific surface area increases with decreasing spherical diameter, so it could be used as a carrier to adsorb or crosslink various kinds of substances (e.g. enzyme, cell, protein, nucleic acid and other compounds).[11] Because of these unique features, there is an increasing interest in new fields of applications.[12] Hence, the present study explores its potential as efficient and ecofriendly adsorbent for the removal of recalcitrant dye, for adsorption and elution of protein and heavy metals. Aniline blue was used as representative azo dye for the dye-removal study. This study is a preliminary attempt to demonstrate the role of BC as polymeric adsorbents for removal of protein with Bovine serum albumin (BSA) as a representative example. Lead, cadmium and nickel were selected for the study owing to the need for removal of these metals due to toxic effects on life. The role of BC as soil conditioner was also confirmed. In our previous study, production and optimization for enhanced BC was achieved,[13,14] which we used in current study for diverse fields of green environment applications that were hardly ever investigated. Methods Micro-organism and cultivation conditions The micro-organism, Gluconoacetobacter hansenii NCIM 2529, used in this study was procured from National Chemical Laboratory (Pune, India). The culture medium for its maintenance contained (%, w/v): mannitol, 2.5; yeast extract, 0.5; peptone, 0.3; agar, 1.5 at pH 5.5. The culture medium used for the production of BC beads was the Hestrin & Schramn (HS) medium consisting of 2.0% (w/v) glucose, 0.5% (w/v) yeast
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extract, 0.5% (w/v) peptone, 0.27% (w/v) Na2HPO4·12H2O, and 0.115% (w/v) citric acid monohydrate.[15] Prior to sterilization at 121 °C, the pH value of the medium was adjusted to 5.0 with 1 N HCl.
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Production and purification of BC The organism was inoculated in a 500 mL flask containing 100 mL of the HS medium and then cultivated at 30 °C and 120 rpm for 120 h. Culture broth containing BC spheres was filtered out. The cellulose beads were purified by treatment with 0.1 N NaOH at 90 °C for 30 min which causes lysis of attached bacterial cells. Thereafter, the solution was filtered to remove dissolved materials. The beads were purified by removal of dead bacterial cells and media components by extensive washing in distilled water at room temperature until the pH of the water became neutral. This obtained purified cellulose was oven dried at 50 °C for 8 h.
BC for green environment applications BC for dye removal The BC was tested for its ability to remove an azo dye. The Aniline blue dye (CAS No. 8004-91-9, Colour Index no. 42755) was used in the present study as an example of azo dyes. Batch adsorption experiment was conducted by shaking 1 g of BC with 50 mL aqueous solution of dye at different concentration (50,100, 200 and 400 ppm). The pH was maintained at 7.0 by using 1.0 N HCl and 1.0 N NaOH. It was kept on shaker at 120 rpm at 28 °C. The adsorbent was removed by centrifugation and decrease in dye concentration in the supernatant liquid was determined spectrophotometrically at 585 nm. Absorption spectra between 400 and 800 nm were also recorded. Adsorption of Aniline blue on BC surface was confirmed by FT-IR analysis. The samples (BC with adsorbed Aniline blue and Aniline blue as control) were well mixed with potassium bromide (KBr) powder and pressed into a small tablet. FT-IR spectra were measured using a Brucker spectrometer (EQUINOX55, Germany) in the transmittance mode with a resolution of 1 cm−1 in the range of 4000–400 cm−1. BC for adsorption and elution of BSA A total of 1 g BC spheres were placed in test tubes with different amounts of the BSA solution (three test tubes for each concentration). Water was added to reach 10 mL and final BSA concentrations were in the range of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL, respectively. Adsorption was carried out at 37 °C for 100 rpm and 2 hours for full adsorption of BSA on BC spheres. Residual concentration of BSA after adsorption was measured by Bradford Method.[16] About 0.1 mol/L NaOH was used as eluent for BC spheres until no BSA was detected in the supernatant. Eluate was collected and the content of BSA was assayed. The amounts of initial, residual and eluated BSA were used for detecting the adsorbed BSA, adsorbed ratio and eluted BSA as follows,[17] Adsorbed BSA ¼ Initial BSA Residual BSA;
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B.V. Mohite and S.V. Patil Adsorbed ratio ð%Þ ¼ The amount of adsorbed BSA=Initial BSA 100%; BSA recovery ratio ð%Þ ¼ Eluted BSA=Adsorbed BSA 100%.
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The effect of pH on BSA adsorption was checked within pH range of 2.0 to 7.0, while the effect of temperature was studied at 28, 37 and at 50 °C. BC for soil augmentation Sandy loam soil samples were collected from 20 cm depth from the vicinity of North Maharashtra University, Jalgaon, India by Latin square fashion method.[9] Soil was analyzed for Water Holding Capacity and Porosity as follows. MWHC of soil MWHC of soil was determined after different wetting cycles using Piper method.[18] Soil samples with and without BC were filled and uniformly packed in keen Roczkowski box and weighed (X). Then boxes (with soil) were kept in tray having water up to 1 cm level and allowed to saturate for 24 h. Boxes were taken out from tray, wiped them to dry from outside and allowed to drain for 30 min. Their weight after saturation (Y) was recorded. The saturated soil was dried in an oven, cooled in dessicator and weighed (Z). Water absorbed by filter paper was determined by weighing filter paper of same size as fitted in keen box. The filter papers were saturated with water, and gently rolled with a glass rod over to squeeze out water uniformly. Weight was taken again and the average amount of water held by one paper (W) was calculated. Each experiment was carried out in triplicate. MWHC of soil was determined by formula, MWHC = (Y − Z − W)/(Z − X) × 100. Porosity of soil Effect of biopolymer amendment on soil porosity was determined by analysing particle and bulk density of amended soil.[19] Soil porosity, or pore space, is the volume percentage of the total soil that is not occupied by solid particles. Porosity was determined by using following formula, Porosity = 1 − Particle density/Bulk density × 100. Adsorption and elution of heavy metals The heavy metal removal ability of BC was checked for lead, cadmium and nickel initially at low concentration (10 mg/l). Adsorption and elution of heavy metals was carried out with BC as follows, 500 mg BC spheres were placed in test tubes with respective heavy metal solution (in triplicate). Adsorption was carried out at 120 rpm for 45 min. Reuse of BC was checked by elution of metal ions with suitable eluents. About 0.1 mol/L Sodium Citrate was used as eluent for lead, 3.0 mol/L nitric acid for nickel and 0.02 mol/L EDTA for cadmium. Elution was carried out at 120 rpm for 30 min. Eluate was collected and the content of metal ion was assayed. Residual concentration of metal ion after adsorption and from eluate was measured by an atomic adsorption spectrophotometer (AA-6800, Shimadzu, Japan). As described above for BSA, the amount of adsorbed metal, adsorbed ratio and recovery ratio was calculated as follows,
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Metal adsorbed ¼ Initial metal Residual metal; Ratio of metal adsorbed ð%Þ ¼ Amount of adsorbed metal=Initial metal 100%; Metal recovery ratio ð%Þ ¼ Eluted metal=Adsorbed metal 100%:
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The adsorption and elution was further carried out for high concentration of metal ion based on the performance at low concentration by the same way as mentioned above. Results and discussion G. hansenii NCIM 2529 produced BC sphere at shaking condition. The BC spheres are white, translucent and have an oval or elliptical shape (Figure 1(a)). The spheres have a loose structure and a diameter between 3 and 5 mm. The scanning electron microscopy (SEM) graph of BC spheres shown in Figure 1(b) proves their loose and porous morphology. It shows a reticulated structure consisting of ultra-fine cellulose fibrils. The microfibrils of BC produced under agitated conditions are curved and entangled resulting in a denser reticulated structure. These unique nano-morphology result in a large surface area that can hold a large amount of water (up to 200 times of its dry mass) and at the same time displays great elasticity, high wet strength and conformability.[20] This result has been supported by previous study.[21] Application of BC for dye removal Aniline blue belongs to triphenylmethane class of dye absorbs maximally at 585 nm and is water-soluble dye. It is used for dyeing wool and cotton directly.[22] Due to its stability, it has long residence time in water. Thus, the studies of colour removal of the dyes are still desirable to keep the environment clean. The dye removal was studied from low to high concentration of dye (50, 100, 200 and 400 ppm). The result for high concentration of dye (400 ppm) was discussed here. The triarylmethane-type dye Aniline blue (400 mg/L) was removed upto 80% by BC in 60 min (Figure 2(a)) measured at 585 nm and further with increase in time, amount of adsorbed BC was remained the same. The dye was adsorbed on the surface of three-dimensional network of BC nanofibers. The UV-visible spectra of Aniline blue and the changes produced
Figure 1.
(a) Morphology of BC spheres; (b) SEM image of porous BC sphere.
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Figure 2. (a) Effect of BC on Aniline blue % removal at 585 nm; (b) changes in absorption spectra (400–800 nm) of Aniline blue (400 mg/L) during bioadsorption by BC.
during dye removal (scans every 10 min.) with BC was shown in Figure 2(b). The relevant decrease in absorption of Aniline blue denotes the removal of dye form the solution. The Figure 3(a) shows the visual removal of Aniline blue by BC along with control, while Figure 3(b) demonstrates the effect of pH on dye removal of Aniline blue. The dye removal was effective for wide pH range from pH 4.0 to 9.0 showing maximum dye removal (about 80%) at pH 7.0. Most of the research for dye removal used microbial cells like bacteria and fungi and their enzymes. D’Souza et al. [22] reported Basidomycete fungus to remove Aniline blue in 4 days, while exoploysacharide (EPS) of the fungus shows 46 and 75% dye removal in 12 and 24 h, respectively. Aspergillus proliferans crude laccase enzyme removes the 40% Aniline blue in 14 h.[23] Vyjayanthi and Suresh [24] reported use of Palladized BC for removal of Drimarene Red dye in a rotating catalyst contact reactor. They claimed that 90% of Drimarene Red dye (100 mg/L) was removed at pH 2 after 25 minutes. In the present study, we found that 90% of Aniline blue dye was removed in 10 min (100 mg/L). In comparison to BC, the dye adsorption rate of wood cellulose modified into polyamide-epichlorohydrin (PAE) polymer cellulose is very slow.
Figure 3. (a) Aniline blue removal by BC compared with control; (b) effect of pH on removal of Aniline blue.
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It required three days at 30 °C to reach equilibrium for Direct Blue 86 with adsorption capacity of only 1 .0 mol/K.[25–27] Application of BC to adsorb metal ions has been reported earlier,[28] but use of BC for dye removal has very few reports. BC fibrils are much thinner than fibers of plant cellulose, much more reactive hydroxyl groups on the surface of BC can be functionalized. The small size of microbial fibrils seems to be a key factor that determines its remarkable performance as an effective adsorbent. This interesting nature of BC could be employed as a biosorbent for the removal of dye from aqueous solution.[29,30] Thus, in present study, BC was applied for one of the azo dye removal, but further it could be used for other important synthetic dyes bioremediation. The adsorption of Aniline blue on BC was confirmed by FT-IR analysis. The characteristic peaks of Aniline blue was observed for stretching vibrations of S = O at 630 cm−1, symmetric stretching at 1174 cm−1 and asymmetric stretching at 1024 cm−1 for C–N.[5,22,26,27,30] The stretching vibrations between C–O showed a band at 977 cm−1. C–N stretching at 1352 cm−1 represented nature of aromatic amine group present in Aniline blue dye, 1492 cm−1 represented the presence of free NH group from dye structure. The stretching between C–H was reported at 2924 cm−1, whereas peak at 1615 cm−1 represented –N=N– stretching of azo group (Figure 4(a)). The presence of characteristic peaks and its intensity indicate its nature as azo dye. The presence of characteristic functional groups of Aniline blue on BC surface confirmed adsorption of Aniline blue on BC surface. The presence of characteristic functional groups present on BC surface included S=O stretching vibrations, symmetric
Figure 4.
FT-IR spectra of (a) Aniline blue and (b) BC with adsorbed Aniline blue.
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and asymmetric stretching, stretching vibrations between C–O and C–N, presence of free NH group, stretching between C–H, stretching of azo group –N=N– (Figure 4(b)).[31]
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Application of BC for adsorption and elution of BSA Adsorption tests were carried out at 100 rpm to avoid the influence of aggregation in BSA solutions. The adsorption quantity had an increasing trend with increasing BSA concentration, while the adsorption ratio had a descending trend; as shown in Figure 5(a), there was a plateau from 1 to 3.0 mg/mL, and then adsorption ratio decreased. When BSA on the surface of BC spheres reached the saturation point, the BC spheres were unable to adsorb continuously even as BSA concentration rises.[32] BSA-adsorbed BC spheres were eluted by 0.1 mol/L NaOH, the recovery ratio was more than 90%, indicating that BC spheres could be reused after elution (Figure 5(a)). Then regenerated BC spheres can be used repeatedly for bioseparation and immobilized reactions expanding their potential applications. According to Ashjaran et al. [30] and Hwang and Chen [26], BC membranes adsorbed approximately 1.7–1.8 mg of protein per mg of BC, while only 0.01 mg of protein adsorbed per mg of polytetrafluroethylene (ePTFE) which is a synthetic polymer as the reference material. BC exhibits great adsorption capacity for cellobiose dehydrogenase, which is a protein showing an affinity to cellulose when compared to wood pulp cellulose.[33] It is well known that the protein adsorption is significantly influenced by surface characteristics, such as hydrophilicity, topography, charge or chemistry.[34] The large surface area of the nanofibrous network of BC allows the binding of a comparatively large quantity of protein.[35] Andrade et al. [36] reported that Plasmsa proteins adhere on BC in relatively high amounts, due to the high surface area of the material and could further be modified into improvement in blood compatibility which seems to be an interesting strategy for the development of BC grafts. The effect of pH and temperature on BSA bioadsorption on BC was studied as shown in Figure 5(b). At low pH of 2.0–3.0, the adsorption capacity was low down which is due to very high positive charge at low pH values resulting into charge repulsion effect.[37] Hence, the maximum surface of protein is available for adsorption at this pH. As the pH increases from 4.0–5.0 acidic residues, the surface of the molecule become protonated and overall protein net charge decreases. If pH is near to 5.0 (pH 4.8 is Isoelectric point of BSA), charge repulsion effect faded and very high-adsorption capacity was achieved. The adsorption capacity was decreased at pH values higher than
Figure 5. (a) Adsorption and elution of BSA by BC; (b) Effect of pH and (c) temperature on bioadsorption of BSA on BC surface.
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5.0. Temperature affects the protein structure, solubility and diffusion at the interface, hence we studied the effect of temperature on BSA bioadsorption, The adsorption capacity for BSA was at 37 °C more than room temperature and further increase to high temperature resulted in decrease in adsorption capacity due to inactivation of protein [38] (Figure 5(c)). The difference in protein adsorption will be due to the change in surface properties of protein. Spherical BC may be applied in bioseparation, immobilized reaction, cell suspension culture, and as an adsorbent for sewage treatment. Application of BC for heavy metal adsorption and elution The methods for heavy metal removal from wastewater streams with heavy metal concentration adsorption gets increasing people’s attention since adsorbents have highspecific surface, strong adsorptive ability, and are suitable for treating a variety of heavy metal ions of low concentration of wastewater.[36,39,40] However, adsorption is confined because adsorbents used for this purpose are too expensive. Therefore, the developing of economic, effective and new adsorbents has become urgent demand for social and economic development. The present study investigates BC for Pb2+, Cd2+ and Ni2+ removal as representatives of heavy metals. Initially, the bioadsorption ability of BC was investigated at low concentration (10 mg/mL) of heavy metals. The bioadsorption and elution ratio was greater for Pb2+ compared with Cd2+ and Ni2+ (Figure 6). Hence to study the effect of BC at higher concentrations of Pb2+ was selected. The adsorbed Pb2+ quantity by BC spheres increased with the (increase) of Pb2+ concentration (Figure 7). Maximum adsorption capacity of Pb2+ was affected by its initial concentration. BC spheres with adsorbed Pb2+ were eluted by 0.1 M/L sodium citrate; the first elution ratio was 90% to 95%, which suggests that recovery of BC spheres is accessible (Figure 7). Thus regenerated BC sphere could be used repeatedly. In contrast to BC, wood cellulose sorbed metal ions in the range of 6.57-75.08% [41] with adsorption capacity of 61.74 mg/g for Pb2+. Various other sorbent materials like coal, activated carbon, microbial biomass, natural polysaccharide gels and functionalized synthetic polymers were used for removal of Pb2+.
Figure 6.
Comparision of adsorption and elution profile of heavy metals by BC treament.
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Figure 7.
B.V. Mohite and S.V. Patil
Adsorption and elution of pb2+ by BC.
In contrast, the adsorption capacity of BC for Pb2+ was reached to 87 mg/g, a value comparable with the values reported for other sorbents (Table 1). It is notably significant than the adsorption capacity of well-known adsorbents for Pb2+ such as activated carbon (30 mg/g),[42] Blast furnace sludge (79 mg/g) [43] and Blast furnace slag (40 mg/g).[44] According to Zahra [45], the equilibrium time of adsorption was attained after 120 min and the maximum removal percentage was achieved at an adsorbent loading weight of 1.5 gram for Pb2+. Application of BC for soil augmentation Addition of BC increased the MWHC of sandy loam soil compared with control, while porosity of soil also increased significantly (Table 2). In dry and semiarid environment, water retention capacity or moisture retention plays a key role in the growth and establishment of crops. The synthetic soil conditioners like polyacrylamide, glycols and hydrogel have major drawbacks like cost, carcinogenicity, sources of origin, etc. Natural polymers like BC may be more suitable as soil conditioners due to its non-toxic and biodegradable nature. The improvement in soil physical properties like porosity accompanied by increase in water-holding capacity correlated with microbial activity, respiration.[47] The amendment in porosity with higher water-holding capacity proves BC as natural soil conditioner. Table 1.
Adsorption capacity of BC for Pb2+ removal compared with different adsorbents.
Adsorbent for Pb2+
Adsorption capacity (mg/g)
Activated carbon Blast furnace slag Blast furnace sludge Plant cellulose BC
30 40 79 61 87
Reference [42] [44] [43] [46] Present study
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Effect of BC on soil properties.
MWHC (%) Porosity (%)
Control soil
Soil with BC
19.5 ± 0.1 65.4 ± 1.2
61% ± 0.23 83.1 ± 0.4
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Conclusions In summary, an effort has been made to show versatile nature of BC produced by G. hansenii NCIM 2529 in diverse green environment application which can provide a platform for its further application as green technology tool. Our preliminary data from the present study demonstrate that BC decolorised 90% of Aniline blue in 10 min at low concentrations of 100 mg/L and further it could be used for other important synthetic dyes bioremediation. Further studies are underway to see the effect of BC at varying higher concentration of Aniline blue and other types of azo dyes. It can be employed as an environmentally friendly adsorbent for the removal of heavy metals like lead, cadmium and nickel from industrial wastewater. BC sphere can be used repeatedly, expanding their potential applications in bioseparation which was proved by adsorption and elution of BSA and heavy metals. The study of adsorption isotherm of BSA and heavy metals could be the future issues to investigate. Acknowledgement The research fellowship to Bhavna Mohite from University Grants Commission, New Delhi under the Research Fellowship in Sciences for meritorious students’ scheme is greatly acknowledged.
References [1] Pansar PS, Chavan YV, Bera MB, Chand O, Kumar H. Evaluation of Acetobacter strain for the production of microbial cellulose. Asian J. Chem. 2009;10:99–102. [2] Richmond PA. Biosynthesis and biodegradation of cellulose, occurrence and functions of native cellulose. In: Haigler CH, Weimer PJ, editors. Biosynthesis and biodegradation of cellulose. New York, NY: Marcel Dekker; 1999. p. 5–23. [3] Fu Y, Viraraghavan T. Fungal decolorization of dye wastewaters: a review. Bioresource Technol. 2001;79:251–262. [4] Banat IM, Nigam P, Singh D, Marchant R. Microbial decolorization of textile-dye containing effluents – a review. Bioresource Technol. 1996;58:217–227. [5] Robinson T, Chandran B, Nigam P. Studies on the production of enzymes by white-rot fungi for the decolorization of textile dyes. Enzyme Microb. Technol. 2001;29:575–579. [6] Bailey SE, Olin TJ, Bricka RM. A review of potentially low-cost adsorbents for heavy metals. Water Res. 1999;33:2469–2479. [7] Chakravarty S, Bhattacharjee S, Gupta KK, Singh M, Chaturvedi HT, Maity S. Adsorption of zinc from aqueous solution using chemically treated newspaper pulp. Bioresource Technol. 2007;98:3136–3141. [8] Rezaee A, Derayat J, Godini H, Pourtaghi G. Adsorption of mercury from synthetic solutions by an Acetobacter xylinum biofilm. Res J. Environ. Sci. 2008;2:401–407. [9] Al-Omran AM, Mustafa MA, Shalaby AA. Intermittent evaporation from soil columns as affected by a gel farming conditioners. Sol. Sci. Soc. Am. J. 1987;51:1593–1599. [10] Cohen M. Environmental toxins and health: the health impact of pesticides. Aust. Fam. Physician. 2007;36:1002–1004.
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[11] Svensson A, Nicklasson E, Harrah T, Panilaitis B, Kaplan DL, Brittberg M, Gatenholm P. Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials. 2005;26:419–431. [12] Klemm D, Schumann D, Udhardt U, Marsch S. Bacterial synthesized cellulose — artificial blood vessels for microsurgery. Prog. Polym. Sci. 2001;26:1561–1603. [13] Mohite B, Kamalja K, Patil S. Statistical optimization of culture conditions for enhanced bacterial cellulose production by Gluconoacetobacter hansenii NCIM 2529. Cellulose. 2012;19:1655–1666. [14] Mohite BV, Salunke BK, Patil SV. Enhanced production of bacterial cellulose by using Gluconacetobacter hansenii NCIM 2529 strain under shaking conditions. Appl. Biochem. Biotechnol. 2013;169:1497–1511. [15] Hestrin S, Schramm M. Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose. Biochem. J. 1954;58:345–350. [16] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 1976;72:248–254. [17] Zhu H, Jia S, Yang H, Jia Y, Yan L, Li J. Preparation and application of bacterial cellulose sphere: A novel biomaterial. Biotechnol. Eq. 2011;25:2233–2236. [18] Piper CS. Soil and plant analysis. New York: Interscience; 1950. [19] Mujumdar SP, Singh RA. Particle size distribution analysis. In: Mujumdar SP, Singh RA, editors. Analysis of soil physical properties. Jodhpur: Agrobios; 2000. p. 51–52. [20] Czaja W, Romanovicz D, Brown RM Jr. Structural investigations of microbial cellulose produced in stationary and agitated culture. Cellulose. 2004;11:403–411. [21] Cheng H, Wang P, Chen J, Wu WT. Cultivation of Acetobacter xylinum for bacterial cellulose production in a modified airlift reactor. Biotechnol. Appl. Biochem. 2002;35:125–132. [22] D’Souza DT, Tiwari R, Sah AK, Raghukumar C. Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes. Enzyme Microbe. Technol. 2006;38:504–511. [23] Eykholt GR, Davenport DT. Dechlorination of the chloroacetanilide pesticides alachlor and metolachlor by iron metal. Environ. Sci. Technol. 1998;32:1482–1487. [24] Vyjayanthi JP, Suresh S. Decolorization of drimarene red dye using palladized bacterial cellulose in a reactor. Water Environ. Res. 2010;82:601–609. [25] Hwang MC, Chen KM. The removal of color from effluents using polyamide-epichlorohydrin-cellulose polymer. I. Preparation and use in direct dye removal. J. Appl. Polym. Sci. 1993a;48:299–311. [26] Hwang MC, Chen KM. Removal of color from effluents using polyamideepichlorohydrincellulose polymer. II. Use in acid dye removal. J. Appl. Polym. Sci. 1993b;49:975–998. [27] Hwang MC, Chen KM. The removal of color from effluents using polyamide-epichlorohydrln-cellulose polymer. III. Use in anionic dye removal in a batch process. J. Appl. Polym. Sci. 1993c;50:735–744. [28] Rezaee A, Drayat J, Mortazavi SB. Removal of mercury from chlor-alkali industry wastewater using Acetobacter xylinum cellulose. Am. J. Environ. Sci. 2005;1:102–105. [29] Zhao YP, Wang JF, Chen FH. Combined removal of arsenic (V) and fluoride from drinking water by Fe (III)-loading ligand exchange cotton adsorbent. Chem. J. Chin. Univ. 2003;24:643–647. [30] Ashjaran A, Yazdanshenas MH, Rashidi A, Rezaee RKA. Biosorption thermodynamic and kinetic of direct dye from aqueous solutions on bacterial cellulose. Afr. J. Microbiol. Res. 2012;6:1270–1278. [31] Nada AMA, El-Wakil NA, Hassan ML. Differential adsorption of heavy metal ions by cotton stalk cation-exchangers containing multiple functional groups. J. Appl. Polym. Sci. 2006;101:4124–4132. [32] Liu MH, Deng Y, Zhan HY. Adsorption and desorption of copper (II) from solutions on a new spherical cellulose adsorbent. J. Appl. Polym. Sci. 2002;84:478–485. [33] Ougiya H, Hioki N, Watanabe K, Morinaga Y, Yoshinaga F, Samejima M. Relationship between the physical properties and surface area of cellulose derived from adsorbents of various molecular sizes. Biosci. Biotechnol. Biochem. 1880;62:1880–1884. [34] Kalme SD, Parshetti GK, Jadhav SU, Govindwar SP. Biodegradation of benzidine based dye direct blue-6 by pseudomonas desmolyticum NCIM 2112. Bioresource Technol. 2006;98:1405–1410.
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[35] Lazaridis NK, Charalambous C. Adsorptive removal of trivalent and hexavalent chromium from binary aqueous solutions by composite alginate-goethite beads. Water Res. 2005;39:4385–4396. [36] Andrade FK, Silva JP, Carvalho M, Castanheira EMS, Soares R, Gama M. Studies on the hemocompatibility of bacterial cellulose. J. Biomed. Mater. Res. A. 2011;98A:554–566. [37] Wrzosek K, Polakovic M. Effect of pH on protein adsorption capacity of strong cation exchangers with grafted layer. J. Chromatogr. A. 2011;1218:6987–6994. [38] Cheng X, Canavan HE, Graham DJ, Castner DGB, Ratner D. Temperature dependent activity and structure of adsorbed proteins on plasma polymerized N-isopropyl acrylamide. Biointerphases. 2006;1:61–72. [39] Hoffman AS. Blood-biomaterials interactions: an overview. In: Cooper SL, Peppas NA, editors. Biomaterials: interfacial phenomena and applications. Vol. 199, Advances in Chemistry Series. Washington: ACS, 1982. p. 3–8. [40] Sagvolden G, Giaever I, Feder J. Characteristic protein adhesion forces on glass and polystyrene substrates by atomic force microscopy. Langmuir. 1998;14:5984–5987. [41] Nwosu FO, Adediran GO, Kazeem SR. Adsorption of wood metal ions of wood cellulose modified with urea and thiourea. IFE J. Sci. 2007;9:145–148. [42] Reed BE, Arunachalam SJJ. Use of granular activated carbon columns for lead removal. Environ. Eng.-ASCE. 1994;120:416–436. [43] López-Delgado A, Pérez C, López SA. Adsorption of heavy metals on blast furnace sludge. Water Res. 1998;32:989–996. [44] Srivastava SK, Gupta VK, Mohan D. Removal of lead and chromium by activated slag – a blast furnace waste. J. Environ. Eng. ASCE. 1997;123:461–468. [45] Zahra N. Lead removal from water by low Cost adsorbents: a review. Pak. J. Anal. Environ. Chem. 2012;13:01–08. [46] O’Connell DW, Birkinshaw C, O’Dwyer TF. Design of a novel cellulose-based adsorbent for use in heavy metal recovery from aqueous waste streams. In: Brebbia CA, Antunes do Carmo JS, editors. Water pollution VIII: modelling, monitoring and management. Vol. 95, WIT transactions on ecology and the environment. Southampton: WIT Press; 2006, p. 489–498. [47] Patil SV, Salunke BK, Patil CD, Salunkhe RB. Studies on amendment of different biopolymers in sandy loam and their effect on germination, seedling growth of Gossypium herbaceum L. Appl. Biochem. Biotechnol. 2011;163:780–791.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Bacterial cellulose of Gluconoacetobacter hansenii as a potential bioadsorption agent for its green environment applications a
a
Bhavna V. Mohite & Satish V. Patil a
School of Life Sciences, North Maharashtra University, P.O. Box. 80, Jalagoan 425001, MS, India Published online: 17 Oct 2014.
To cite this article: Bhavna V. Mohite & Satish V. Patil (2014) Bacterial cellulose of Gluconoacetobacter hansenii as a potential bioadsorption agent for its green environment applications, Journal of Biomaterials Science, Polymer Edition, 25:18, 2053-2065, DOI: 10.1080/09205063.2014.970063 To link to this article: http://dx.doi.org/10.1080/09205063.2014.970063
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 18, 2053–2065, http://dx.doi.org/10.1080/09205063.2014.970063
Bacterial cellulose of Gluconoacetobacter hansenii as a potential bioadsorption agent for its green environment applications
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Bhavna V. Mohite and Satish V. Patil* School of Life Sciences, North Maharashtra University, P.O. Box. 80, Jalagoan 425001, MS, India (Received 8 August 2014; accepted 24 September 2014) Bacterial cellulose (BC) is an interesting biopolymer produced by bacteria having superior properties. BC produced by Gluconoacetobacter hansenii (strain NCIM 2529) under shaking condition and explored for its applications in dye removal and bioadsorption of protein and heavy metals. Purity of BC was confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM) analysis. BC removed azo dye and Aniline blue (400 mg/L) with 80% efficiency within 60 min. The adsorption and elution of Bovine serum albumin (BSA) and heavy metals like lead, cadmium and nickel (Pb2+, Cd2+ and Ni2+) was achieved with BC which confirms the exclusion ability with reusability. The BSA adsorption quantity was increased with increase in protein concentration with more than 90% adsorption and elution ratio. The effect of pH and temperature on BSA adsorption has been investigated. Bioadsorption (82%) and elution ratio (92%) of BC for Pb2+ was more when compared with Cd2+ (41 and 67%) and Ni2+ (33 and 85%), respectively. BC was also explored as soil conditioner to increase the water-holding capacity and porosity of soil. The results elucidated the significance of BC as renewable effective ecofriendly bioadsorption agent. Keywords: bacterial cellulose; bioadsorption; biopolymer; heavy metals
Introduction Bacterial cellulose (BC) is homopolysaccharide having wide industrial applications and it could be a biotechnology’s next high-value product.[1] Nanofibers of BC are about several times thinner compared with plant cellulose making it highly porous with large surface area and high water-holding capacity material. BC is one of the new most abundant, natural biosorbent, renewable, biodegradable and biocompatible polymers.[2] Industrial effluents especially coloured ones from textile, dyes, paper and pulp industries consist of various compounds with complex aromatic structure which make them quite difficult to degrade. Among these, azo dyes account for over 60% of the total dyes produced.[3] The employment of physico-chemical methods for removal of dye from effluent was although effective, but it is cost prohibitive and often leads to extra solid wastes. As a viable alternative, biological processes have received increasing interest owing to their cost-effectiveness.[4] Adsorption has been observed as an effective process for colour removal from such effluents. Adsorption on activated charcoal is an effective method but is prohibitively expensive. Low-cost adsorbents generally *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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have low-adsorption capacities mediating the need for economical and easily available yet highly effective adsorbents.[3] Living and dead cells of bacteria, fungi, algae or plant biomass or a combination there of is generally used for bioremediation.[5] The adaptability of biological methods to varied conditions gives bioremediation an edge over the physical–chemical methods. This is a novel study where we use biopolymer as bioadsorbent for dye removal. The cellulose produced from plant source has importance in bioremediation purpose, but consist of contaminating materials like hemicellulose, lignin along with cellulose which should be separated. Hence, pure cellulose from bacterial source, which is an emerging new industrial product, has an importance favoured by its unique features.[1] Natural products composed primarily of cellulose matrix were tried by number of researchers for removal of heavy metals.[6–8] Soil conditioners reported as efficient tools in increasing water-holding capacity, reducing infiltration rate and cumulative evaporation, and improving water conservation of sandy soils.[9] One of the highly effective methods to enhance and structure is by adding synthetic and natural polymers that improve soil cohesion, porosity, maximum water-holding capacity (MWHC) and various beneficial soil properties. Synthetic polymers cannot be used during organic farming because of their synthetic nature and production from nonrenewable resources. Natural polymers are degraded usually by relatively benign route. Cost-effective and ecofriendly nature of biopolymers makes it an important alternative to synthetic polymers. The hazardous proteins like Tat and Nef or environmental toxins or biotoxins could be removed with polymers like BC. It makes BC an interesting tool for environment cleaning up.[10] In view of this background, the goal was to determine the potential of BC as bioadsorption agent for clean environment applications. Spherical BC produced by shaking method is translucent, loose, porous and has a hydrophilic network structure. Its specific surface area increases with decreasing spherical diameter, so it could be used as a carrier to adsorb or crosslink various kinds of substances (e.g. enzyme, cell, protein, nucleic acid and other compounds).[11] Because of these unique features, there is an increasing interest in new fields of applications.[12] Hence, the present study explores its potential as efficient and ecofriendly adsorbent for the removal of recalcitrant dye, for adsorption and elution of protein and heavy metals. Aniline blue was used as representative azo dye for the dye-removal study. This study is a preliminary attempt to demonstrate the role of BC as polymeric adsorbents for removal of protein with Bovine serum albumin (BSA) as a representative example. Lead, cadmium and nickel were selected for the study owing to the need for removal of these metals due to toxic effects on life. The role of BC as soil conditioner was also confirmed. In our previous study, production and optimization for enhanced BC was achieved,[13,14] which we used in current study for diverse fields of green environment applications that were hardly ever investigated. Methods Micro-organism and cultivation conditions The micro-organism, Gluconoacetobacter hansenii NCIM 2529, used in this study was procured from National Chemical Laboratory (Pune, India). The culture medium for its maintenance contained (%, w/v): mannitol, 2.5; yeast extract, 0.5; peptone, 0.3; agar, 1.5 at pH 5.5. The culture medium used for the production of BC beads was the Hestrin & Schramn (HS) medium consisting of 2.0% (w/v) glucose, 0.5% (w/v) yeast
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extract, 0.5% (w/v) peptone, 0.27% (w/v) Na2HPO4·12H2O, and 0.115% (w/v) citric acid monohydrate.[15] Prior to sterilization at 121 °C, the pH value of the medium was adjusted to 5.0 with 1 N HCl.
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Production and purification of BC The organism was inoculated in a 500 mL flask containing 100 mL of the HS medium and then cultivated at 30 °C and 120 rpm for 120 h. Culture broth containing BC spheres was filtered out. The cellulose beads were purified by treatment with 0.1 N NaOH at 90 °C for 30 min which causes lysis of attached bacterial cells. Thereafter, the solution was filtered to remove dissolved materials. The beads were purified by removal of dead bacterial cells and media components by extensive washing in distilled water at room temperature until the pH of the water became neutral. This obtained purified cellulose was oven dried at 50 °C for 8 h.
BC for green environment applications BC for dye removal The BC was tested for its ability to remove an azo dye. The Aniline blue dye (CAS No. 8004-91-9, Colour Index no. 42755) was used in the present study as an example of azo dyes. Batch adsorption experiment was conducted by shaking 1 g of BC with 50 mL aqueous solution of dye at different concentration (50,100, 200 and 400 ppm). The pH was maintained at 7.0 by using 1.0 N HCl and 1.0 N NaOH. It was kept on shaker at 120 rpm at 28 °C. The adsorbent was removed by centrifugation and decrease in dye concentration in the supernatant liquid was determined spectrophotometrically at 585 nm. Absorption spectra between 400 and 800 nm were also recorded. Adsorption of Aniline blue on BC surface was confirmed by FT-IR analysis. The samples (BC with adsorbed Aniline blue and Aniline blue as control) were well mixed with potassium bromide (KBr) powder and pressed into a small tablet. FT-IR spectra were measured using a Brucker spectrometer (EQUINOX55, Germany) in the transmittance mode with a resolution of 1 cm−1 in the range of 4000–400 cm−1. BC for adsorption and elution of BSA A total of 1 g BC spheres were placed in test tubes with different amounts of the BSA solution (three test tubes for each concentration). Water was added to reach 10 mL and final BSA concentrations were in the range of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL, respectively. Adsorption was carried out at 37 °C for 100 rpm and 2 hours for full adsorption of BSA on BC spheres. Residual concentration of BSA after adsorption was measured by Bradford Method.[16] About 0.1 mol/L NaOH was used as eluent for BC spheres until no BSA was detected in the supernatant. Eluate was collected and the content of BSA was assayed. The amounts of initial, residual and eluated BSA were used for detecting the adsorbed BSA, adsorbed ratio and eluted BSA as follows,[17] Adsorbed BSA ¼ Initial BSA Residual BSA;
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B.V. Mohite and S.V. Patil Adsorbed ratio ð%Þ ¼ The amount of adsorbed BSA=Initial BSA 100%; BSA recovery ratio ð%Þ ¼ Eluted BSA=Adsorbed BSA 100%.
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The effect of pH on BSA adsorption was checked within pH range of 2.0 to 7.0, while the effect of temperature was studied at 28, 37 and at 50 °C. BC for soil augmentation Sandy loam soil samples were collected from 20 cm depth from the vicinity of North Maharashtra University, Jalgaon, India by Latin square fashion method.[9] Soil was analyzed for Water Holding Capacity and Porosity as follows. MWHC of soil MWHC of soil was determined after different wetting cycles using Piper method.[18] Soil samples with and without BC were filled and uniformly packed in keen Roczkowski box and weighed (X). Then boxes (with soil) were kept in tray having water up to 1 cm level and allowed to saturate for 24 h. Boxes were taken out from tray, wiped them to dry from outside and allowed to drain for 30 min. Their weight after saturation (Y) was recorded. The saturated soil was dried in an oven, cooled in dessicator and weighed (Z). Water absorbed by filter paper was determined by weighing filter paper of same size as fitted in keen box. The filter papers were saturated with water, and gently rolled with a glass rod over to squeeze out water uniformly. Weight was taken again and the average amount of water held by one paper (W) was calculated. Each experiment was carried out in triplicate. MWHC of soil was determined by formula, MWHC = (Y − Z − W)/(Z − X) × 100. Porosity of soil Effect of biopolymer amendment on soil porosity was determined by analysing particle and bulk density of amended soil.[19] Soil porosity, or pore space, is the volume percentage of the total soil that is not occupied by solid particles. Porosity was determined by using following formula, Porosity = 1 − Particle density/Bulk density × 100. Adsorption and elution of heavy metals The heavy metal removal ability of BC was checked for lead, cadmium and nickel initially at low concentration (10 mg/l). Adsorption and elution of heavy metals was carried out with BC as follows, 500 mg BC spheres were placed in test tubes with respective heavy metal solution (in triplicate). Adsorption was carried out at 120 rpm for 45 min. Reuse of BC was checked by elution of metal ions with suitable eluents. About 0.1 mol/L Sodium Citrate was used as eluent for lead, 3.0 mol/L nitric acid for nickel and 0.02 mol/L EDTA for cadmium. Elution was carried out at 120 rpm for 30 min. Eluate was collected and the content of metal ion was assayed. Residual concentration of metal ion after adsorption and from eluate was measured by an atomic adsorption spectrophotometer (AA-6800, Shimadzu, Japan). As described above for BSA, the amount of adsorbed metal, adsorbed ratio and recovery ratio was calculated as follows,
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Metal adsorbed ¼ Initial metal Residual metal; Ratio of metal adsorbed ð%Þ ¼ Amount of adsorbed metal=Initial metal 100%; Metal recovery ratio ð%Þ ¼ Eluted metal=Adsorbed metal 100%:
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The adsorption and elution was further carried out for high concentration of metal ion based on the performance at low concentration by the same way as mentioned above. Results and discussion G. hansenii NCIM 2529 produced BC sphere at shaking condition. The BC spheres are white, translucent and have an oval or elliptical shape (Figure 1(a)). The spheres have a loose structure and a diameter between 3 and 5 mm. The scanning electron microscopy (SEM) graph of BC spheres shown in Figure 1(b) proves their loose and porous morphology. It shows a reticulated structure consisting of ultra-fine cellulose fibrils. The microfibrils of BC produced under agitated conditions are curved and entangled resulting in a denser reticulated structure. These unique nano-morphology result in a large surface area that can hold a large amount of water (up to 200 times of its dry mass) and at the same time displays great elasticity, high wet strength and conformability.[20] This result has been supported by previous study.[21] Application of BC for dye removal Aniline blue belongs to triphenylmethane class of dye absorbs maximally at 585 nm and is water-soluble dye. It is used for dyeing wool and cotton directly.[22] Due to its stability, it has long residence time in water. Thus, the studies of colour removal of the dyes are still desirable to keep the environment clean. The dye removal was studied from low to high concentration of dye (50, 100, 200 and 400 ppm). The result for high concentration of dye (400 ppm) was discussed here. The triarylmethane-type dye Aniline blue (400 mg/L) was removed upto 80% by BC in 60 min (Figure 2(a)) measured at 585 nm and further with increase in time, amount of adsorbed BC was remained the same. The dye was adsorbed on the surface of three-dimensional network of BC nanofibers. The UV-visible spectra of Aniline blue and the changes produced
Figure 1.
(a) Morphology of BC spheres; (b) SEM image of porous BC sphere.
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Figure 2. (a) Effect of BC on Aniline blue % removal at 585 nm; (b) changes in absorption spectra (400–800 nm) of Aniline blue (400 mg/L) during bioadsorption by BC.
during dye removal (scans every 10 min.) with BC was shown in Figure 2(b). The relevant decrease in absorption of Aniline blue denotes the removal of dye form the solution. The Figure 3(a) shows the visual removal of Aniline blue by BC along with control, while Figure 3(b) demonstrates the effect of pH on dye removal of Aniline blue. The dye removal was effective for wide pH range from pH 4.0 to 9.0 showing maximum dye removal (about 80%) at pH 7.0. Most of the research for dye removal used microbial cells like bacteria and fungi and their enzymes. D’Souza et al. [22] reported Basidomycete fungus to remove Aniline blue in 4 days, while exoploysacharide (EPS) of the fungus shows 46 and 75% dye removal in 12 and 24 h, respectively. Aspergillus proliferans crude laccase enzyme removes the 40% Aniline blue in 14 h.[23] Vyjayanthi and Suresh [24] reported use of Palladized BC for removal of Drimarene Red dye in a rotating catalyst contact reactor. They claimed that 90% of Drimarene Red dye (100 mg/L) was removed at pH 2 after 25 minutes. In the present study, we found that 90% of Aniline blue dye was removed in 10 min (100 mg/L). In comparison to BC, the dye adsorption rate of wood cellulose modified into polyamide-epichlorohydrin (PAE) polymer cellulose is very slow.
Figure 3. (a) Aniline blue removal by BC compared with control; (b) effect of pH on removal of Aniline blue.
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It required three days at 30 °C to reach equilibrium for Direct Blue 86 with adsorption capacity of only 1 .0 mol/K.[25–27] Application of BC to adsorb metal ions has been reported earlier,[28] but use of BC for dye removal has very few reports. BC fibrils are much thinner than fibers of plant cellulose, much more reactive hydroxyl groups on the surface of BC can be functionalized. The small size of microbial fibrils seems to be a key factor that determines its remarkable performance as an effective adsorbent. This interesting nature of BC could be employed as a biosorbent for the removal of dye from aqueous solution.[29,30] Thus, in present study, BC was applied for one of the azo dye removal, but further it could be used for other important synthetic dyes bioremediation. The adsorption of Aniline blue on BC was confirmed by FT-IR analysis. The characteristic peaks of Aniline blue was observed for stretching vibrations of S = O at 630 cm−1, symmetric stretching at 1174 cm−1 and asymmetric stretching at 1024 cm−1 for C–N.[5,22,26,27,30] The stretching vibrations between C–O showed a band at 977 cm−1. C–N stretching at 1352 cm−1 represented nature of aromatic amine group present in Aniline blue dye, 1492 cm−1 represented the presence of free NH group from dye structure. The stretching between C–H was reported at 2924 cm−1, whereas peak at 1615 cm−1 represented –N=N– stretching of azo group (Figure 4(a)). The presence of characteristic peaks and its intensity indicate its nature as azo dye. The presence of characteristic functional groups of Aniline blue on BC surface confirmed adsorption of Aniline blue on BC surface. The presence of characteristic functional groups present on BC surface included S=O stretching vibrations, symmetric
Figure 4.
FT-IR spectra of (a) Aniline blue and (b) BC with adsorbed Aniline blue.
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and asymmetric stretching, stretching vibrations between C–O and C–N, presence of free NH group, stretching between C–H, stretching of azo group –N=N– (Figure 4(b)).[31]
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Application of BC for adsorption and elution of BSA Adsorption tests were carried out at 100 rpm to avoid the influence of aggregation in BSA solutions. The adsorption quantity had an increasing trend with increasing BSA concentration, while the adsorption ratio had a descending trend; as shown in Figure 5(a), there was a plateau from 1 to 3.0 mg/mL, and then adsorption ratio decreased. When BSA on the surface of BC spheres reached the saturation point, the BC spheres were unable to adsorb continuously even as BSA concentration rises.[32] BSA-adsorbed BC spheres were eluted by 0.1 mol/L NaOH, the recovery ratio was more than 90%, indicating that BC spheres could be reused after elution (Figure 5(a)). Then regenerated BC spheres can be used repeatedly for bioseparation and immobilized reactions expanding their potential applications. According to Ashjaran et al. [30] and Hwang and Chen [26], BC membranes adsorbed approximately 1.7–1.8 mg of protein per mg of BC, while only 0.01 mg of protein adsorbed per mg of polytetrafluroethylene (ePTFE) which is a synthetic polymer as the reference material. BC exhibits great adsorption capacity for cellobiose dehydrogenase, which is a protein showing an affinity to cellulose when compared to wood pulp cellulose.[33] It is well known that the protein adsorption is significantly influenced by surface characteristics, such as hydrophilicity, topography, charge or chemistry.[34] The large surface area of the nanofibrous network of BC allows the binding of a comparatively large quantity of protein.[35] Andrade et al. [36] reported that Plasmsa proteins adhere on BC in relatively high amounts, due to the high surface area of the material and could further be modified into improvement in blood compatibility which seems to be an interesting strategy for the development of BC grafts. The effect of pH and temperature on BSA bioadsorption on BC was studied as shown in Figure 5(b). At low pH of 2.0–3.0, the adsorption capacity was low down which is due to very high positive charge at low pH values resulting into charge repulsion effect.[37] Hence, the maximum surface of protein is available for adsorption at this pH. As the pH increases from 4.0–5.0 acidic residues, the surface of the molecule become protonated and overall protein net charge decreases. If pH is near to 5.0 (pH 4.8 is Isoelectric point of BSA), charge repulsion effect faded and very high-adsorption capacity was achieved. The adsorption capacity was decreased at pH values higher than
Figure 5. (a) Adsorption and elution of BSA by BC; (b) Effect of pH and (c) temperature on bioadsorption of BSA on BC surface.
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5.0. Temperature affects the protein structure, solubility and diffusion at the interface, hence we studied the effect of temperature on BSA bioadsorption, The adsorption capacity for BSA was at 37 °C more than room temperature and further increase to high temperature resulted in decrease in adsorption capacity due to inactivation of protein [38] (Figure 5(c)). The difference in protein adsorption will be due to the change in surface properties of protein. Spherical BC may be applied in bioseparation, immobilized reaction, cell suspension culture, and as an adsorbent for sewage treatment. Application of BC for heavy metal adsorption and elution The methods for heavy metal removal from wastewater streams with heavy metal concentration adsorption gets increasing people’s attention since adsorbents have highspecific surface, strong adsorptive ability, and are suitable for treating a variety of heavy metal ions of low concentration of wastewater.[36,39,40] However, adsorption is confined because adsorbents used for this purpose are too expensive. Therefore, the developing of economic, effective and new adsorbents has become urgent demand for social and economic development. The present study investigates BC for Pb2+, Cd2+ and Ni2+ removal as representatives of heavy metals. Initially, the bioadsorption ability of BC was investigated at low concentration (10 mg/mL) of heavy metals. The bioadsorption and elution ratio was greater for Pb2+ compared with Cd2+ and Ni2+ (Figure 6). Hence to study the effect of BC at higher concentrations of Pb2+ was selected. The adsorbed Pb2+ quantity by BC spheres increased with the (increase) of Pb2+ concentration (Figure 7). Maximum adsorption capacity of Pb2+ was affected by its initial concentration. BC spheres with adsorbed Pb2+ were eluted by 0.1 M/L sodium citrate; the first elution ratio was 90% to 95%, which suggests that recovery of BC spheres is accessible (Figure 7). Thus regenerated BC sphere could be used repeatedly. In contrast to BC, wood cellulose sorbed metal ions in the range of 6.57-75.08% [41] with adsorption capacity of 61.74 mg/g for Pb2+. Various other sorbent materials like coal, activated carbon, microbial biomass, natural polysaccharide gels and functionalized synthetic polymers were used for removal of Pb2+.
Figure 6.
Comparision of adsorption and elution profile of heavy metals by BC treament.
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Figure 7.
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Adsorption and elution of pb2+ by BC.
In contrast, the adsorption capacity of BC for Pb2+ was reached to 87 mg/g, a value comparable with the values reported for other sorbents (Table 1). It is notably significant than the adsorption capacity of well-known adsorbents for Pb2+ such as activated carbon (30 mg/g),[42] Blast furnace sludge (79 mg/g) [43] and Blast furnace slag (40 mg/g).[44] According to Zahra [45], the equilibrium time of adsorption was attained after 120 min and the maximum removal percentage was achieved at an adsorbent loading weight of 1.5 gram for Pb2+. Application of BC for soil augmentation Addition of BC increased the MWHC of sandy loam soil compared with control, while porosity of soil also increased significantly (Table 2). In dry and semiarid environment, water retention capacity or moisture retention plays a key role in the growth and establishment of crops. The synthetic soil conditioners like polyacrylamide, glycols and hydrogel have major drawbacks like cost, carcinogenicity, sources of origin, etc. Natural polymers like BC may be more suitable as soil conditioners due to its non-toxic and biodegradable nature. The improvement in soil physical properties like porosity accompanied by increase in water-holding capacity correlated with microbial activity, respiration.[47] The amendment in porosity with higher water-holding capacity proves BC as natural soil conditioner. Table 1.
Adsorption capacity of BC for Pb2+ removal compared with different adsorbents.
Adsorbent for Pb2+
Adsorption capacity (mg/g)
Activated carbon Blast furnace slag Blast furnace sludge Plant cellulose BC
30 40 79 61 87
Reference [42] [44] [43] [46] Present study
Journal of Biomaterials Science, Polymer Edition Table 2.
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Effect of BC on soil properties.
MWHC (%) Porosity (%)
Control soil
Soil with BC
19.5 ± 0.1 65.4 ± 1.2
61% ± 0.23 83.1 ± 0.4
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Conclusions In summary, an effort has been made to show versatile nature of BC produced by G. hansenii NCIM 2529 in diverse green environment application which can provide a platform for its further application as green technology tool. Our preliminary data from the present study demonstrate that BC decolorised 90% of Aniline blue in 10 min at low concentrations of 100 mg/L and further it could be used for other important synthetic dyes bioremediation. Further studies are underway to see the effect of BC at varying higher concentration of Aniline blue and other types of azo dyes. It can be employed as an environmentally friendly adsorbent for the removal of heavy metals like lead, cadmium and nickel from industrial wastewater. BC sphere can be used repeatedly, expanding their potential applications in bioseparation which was proved by adsorption and elution of BSA and heavy metals. The study of adsorption isotherm of BSA and heavy metals could be the future issues to investigate. Acknowledgement The research fellowship to Bhavna Mohite from University Grants Commission, New Delhi under the Research Fellowship in Sciences for meritorious students’ scheme is greatly acknowledged.
References [1] Pansar PS, Chavan YV, Bera MB, Chand O, Kumar H. Evaluation of Acetobacter strain for the production of microbial cellulose. Asian J. Chem. 2009;10:99–102. [2] Richmond PA. Biosynthesis and biodegradation of cellulose, occurrence and functions of native cellulose. In: Haigler CH, Weimer PJ, editors. Biosynthesis and biodegradation of cellulose. New York, NY: Marcel Dekker; 1999. p. 5–23. [3] Fu Y, Viraraghavan T. Fungal decolorization of dye wastewaters: a review. Bioresource Technol. 2001;79:251–262. [4] Banat IM, Nigam P, Singh D, Marchant R. Microbial decolorization of textile-dye containing effluents – a review. Bioresource Technol. 1996;58:217–227. [5] Robinson T, Chandran B, Nigam P. Studies on the production of enzymes by white-rot fungi for the decolorization of textile dyes. Enzyme Microb. Technol. 2001;29:575–579. [6] Bailey SE, Olin TJ, Bricka RM. A review of potentially low-cost adsorbents for heavy metals. Water Res. 1999;33:2469–2479. [7] Chakravarty S, Bhattacharjee S, Gupta KK, Singh M, Chaturvedi HT, Maity S. Adsorption of zinc from aqueous solution using chemically treated newspaper pulp. Bioresource Technol. 2007;98:3136–3141. [8] Rezaee A, Derayat J, Godini H, Pourtaghi G. Adsorption of mercury from synthetic solutions by an Acetobacter xylinum biofilm. Res J. Environ. Sci. 2008;2:401–407. [9] Al-Omran AM, Mustafa MA, Shalaby AA. Intermittent evaporation from soil columns as affected by a gel farming conditioners. Sol. Sci. Soc. Am. J. 1987;51:1593–1599. [10] Cohen M. Environmental toxins and health: the health impact of pesticides. Aust. Fam. Physician. 2007;36:1002–1004.
Downloaded by [University of Tennessee, Knoxville] at 01:59 15 November 2014
2064
B.V. Mohite and S.V. Patil
[11] Svensson A, Nicklasson E, Harrah T, Panilaitis B, Kaplan DL, Brittberg M, Gatenholm P. Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials. 2005;26:419–431. [12] Klemm D, Schumann D, Udhardt U, Marsch S. Bacterial synthesized cellulose — artificial blood vessels for microsurgery. Prog. Polym. Sci. 2001;26:1561–1603. [13] Mohite B, Kamalja K, Patil S. Statistical optimization of culture conditions for enhanced bacterial cellulose production by Gluconoacetobacter hansenii NCIM 2529. Cellulose. 2012;19:1655–1666. [14] Mohite BV, Salunke BK, Patil SV. Enhanced production of bacterial cellulose by using Gluconacetobacter hansenii NCIM 2529 strain under shaking conditions. Appl. Biochem. Biotechnol. 2013;169:1497–1511. [15] Hestrin S, Schramm M. Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose. Biochem. J. 1954;58:345–350. [16] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 1976;72:248–254. [17] Zhu H, Jia S, Yang H, Jia Y, Yan L, Li J. Preparation and application of bacterial cellulose sphere: A novel biomaterial. Biotechnol. Eq. 2011;25:2233–2236. [18] Piper CS. Soil and plant analysis. New York: Interscience; 1950. [19] Mujumdar SP, Singh RA. Particle size distribution analysis. In: Mujumdar SP, Singh RA, editors. Analysis of soil physical properties. Jodhpur: Agrobios; 2000. p. 51–52. [20] Czaja W, Romanovicz D, Brown RM Jr. Structural investigations of microbial cellulose produced in stationary and agitated culture. Cellulose. 2004;11:403–411. [21] Cheng H, Wang P, Chen J, Wu WT. Cultivation of Acetobacter xylinum for bacterial cellulose production in a modified airlift reactor. Biotechnol. Appl. Biochem. 2002;35:125–132. [22] D’Souza DT, Tiwari R, Sah AK, Raghukumar C. Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes. Enzyme Microbe. Technol. 2006;38:504–511. [23] Eykholt GR, Davenport DT. Dechlorination of the chloroacetanilide pesticides alachlor and metolachlor by iron metal. Environ. Sci. Technol. 1998;32:1482–1487. [24] Vyjayanthi JP, Suresh S. Decolorization of drimarene red dye using palladized bacterial cellulose in a reactor. Water Environ. Res. 2010;82:601–609. [25] Hwang MC, Chen KM. The removal of color from effluents using polyamide-epichlorohydrin-cellulose polymer. I. Preparation and use in direct dye removal. J. Appl. Polym. Sci. 1993a;48:299–311. [26] Hwang MC, Chen KM. Removal of color from effluents using polyamideepichlorohydrincellulose polymer. II. Use in acid dye removal. J. Appl. Polym. Sci. 1993b;49:975–998. [27] Hwang MC, Chen KM. The removal of color from effluents using polyamide-epichlorohydrln-cellulose polymer. III. Use in anionic dye removal in a batch process. J. Appl. Polym. Sci. 1993c;50:735–744. [28] Rezaee A, Drayat J, Mortazavi SB. Removal of mercury from chlor-alkali industry wastewater using Acetobacter xylinum cellulose. Am. J. Environ. Sci. 2005;1:102–105. [29] Zhao YP, Wang JF, Chen FH. Combined removal of arsenic (V) and fluoride from drinking water by Fe (III)-loading ligand exchange cotton adsorbent. Chem. J. Chin. Univ. 2003;24:643–647. [30] Ashjaran A, Yazdanshenas MH, Rashidi A, Rezaee RKA. Biosorption thermodynamic and kinetic of direct dye from aqueous solutions on bacterial cellulose. Afr. J. Microbiol. Res. 2012;6:1270–1278. [31] Nada AMA, El-Wakil NA, Hassan ML. Differential adsorption of heavy metal ions by cotton stalk cation-exchangers containing multiple functional groups. J. Appl. Polym. Sci. 2006;101:4124–4132. [32] Liu MH, Deng Y, Zhan HY. Adsorption and desorption of copper (II) from solutions on a new spherical cellulose adsorbent. J. Appl. Polym. Sci. 2002;84:478–485. [33] Ougiya H, Hioki N, Watanabe K, Morinaga Y, Yoshinaga F, Samejima M. Relationship between the physical properties and surface area of cellulose derived from adsorbents of various molecular sizes. Biosci. Biotechnol. Biochem. 1880;62:1880–1884. [34] Kalme SD, Parshetti GK, Jadhav SU, Govindwar SP. Biodegradation of benzidine based dye direct blue-6 by pseudomonas desmolyticum NCIM 2112. Bioresource Technol. 2006;98:1405–1410.
Downloaded by [University of Tennessee, Knoxville] at 01:59 15 November 2014
Journal of Biomaterials Science, Polymer Edition
2065
[35] Lazaridis NK, Charalambous C. Adsorptive removal of trivalent and hexavalent chromium from binary aqueous solutions by composite alginate-goethite beads. Water Res. 2005;39:4385–4396. [36] Andrade FK, Silva JP, Carvalho M, Castanheira EMS, Soares R, Gama M. Studies on the hemocompatibility of bacterial cellulose. J. Biomed. Mater. Res. A. 2011;98A:554–566. [37] Wrzosek K, Polakovic M. Effect of pH on protein adsorption capacity of strong cation exchangers with grafted layer. J. Chromatogr. A. 2011;1218:6987–6994. [38] Cheng X, Canavan HE, Graham DJ, Castner DGB, Ratner D. Temperature dependent activity and structure of adsorbed proteins on plasma polymerized N-isopropyl acrylamide. Biointerphases. 2006;1:61–72. [39] Hoffman AS. Blood-biomaterials interactions: an overview. In: Cooper SL, Peppas NA, editors. Biomaterials: interfacial phenomena and applications. Vol. 199, Advances in Chemistry Series. Washington: ACS, 1982. p. 3–8. [40] Sagvolden G, Giaever I, Feder J. Characteristic protein adhesion forces on glass and polystyrene substrates by atomic force microscopy. Langmuir. 1998;14:5984–5987. [41] Nwosu FO, Adediran GO, Kazeem SR. Adsorption of wood metal ions of wood cellulose modified with urea and thiourea. IFE J. Sci. 2007;9:145–148. [42] Reed BE, Arunachalam SJJ. Use of granular activated carbon columns for lead removal. Environ. Eng.-ASCE. 1994;120:416–436. [43] López-Delgado A, Pérez C, López SA. Adsorption of heavy metals on blast furnace sludge. Water Res. 1998;32:989–996. [44] Srivastava SK, Gupta VK, Mohan D. Removal of lead and chromium by activated slag – a blast furnace waste. J. Environ. Eng. ASCE. 1997;123:461–468. [45] Zahra N. Lead removal from water by low Cost adsorbents: a review. Pak. J. Anal. Environ. Chem. 2012;13:01–08. [46] O’Connell DW, Birkinshaw C, O’Dwyer TF. Design of a novel cellulose-based adsorbent for use in heavy metal recovery from aqueous waste streams. In: Brebbia CA, Antunes do Carmo JS, editors. Water pollution VIII: modelling, monitoring and management. Vol. 95, WIT transactions on ecology and the environment. Southampton: WIT Press; 2006, p. 489–498. [47] Patil SV, Salunke BK, Patil CD, Salunkhe RB. Studies on amendment of different biopolymers in sandy loam and their effect on germination, seedling growth of Gossypium herbaceum L. Appl. Biochem. Biotechnol. 2011;163:780–791.
