Materials Letters 66 (2012) 216–218

Contents lists available at SciVerse ScienceDirect

Materials Letters

j ourna l homepage: www.e lsev ie r .com/ locate /mat le t

Utilization of lignocellulosic natural fiber (jute) components during a microbialpolymer production

Sougata Roy Chowdhury a, Ratan Kumar Basak a, Ramkrishna Sen b,⁎, Basudam Adhikari a

a Materials Science Centre, Indian Institute of Technology Kharagpur, WB 721302, Indiab Department of Biotechnology, Indian Institute of Technology Kharagpur, WB 721302, India

⁎ Corresponding author. Tel.: +91 3222 283752.E-mail address: rksen@yahoo.com (R. Sen).

0167-577X/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.matlet.2011.08.040

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 June 2011Accepted 10 August 2011Available online 23 August 2011

Keywords:B. megaterium RB-05JuteCompositionCellulosePolymerFiber technology

Lignocellulosic fiber (jute), a low cost natural complex carbon source, was introduced in a fermentation mediumto observe the effects of its constituents on the production of a commercially potent bacterial extracellular poly-saccharide (EPS) synthesized by Bacillus megaterium RB-05. It has been found that among all the fiber compo-nents the bacterium has utilized cellulose most for the EPS production. Maximum polymer yield of 0.297 g g−1

substrate was found after 72 h fermentation. Consumption of fiber components was typically driven by in situbacterial enzyme activity as EPS production was found significantly (pb0.05) accelerated from 36 h onwardswith considerable cellulase activity. Utilization of fiber components during different fermentation hours werecharacterized using scanning electron microscopy, FT-IR spectroscopy, X-ray diffraction, tensile property, andcontact angle measurement.

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Structural diversity and variability in functional moieties haveopened up a wide array of scopes in target oriented applications forthe novel bioactive bacterial extracellular polysaccharides (EPSs).Pseudoplastic rheology and high viscosity of these macromoleculesoffer opportunity to be used as emulsifier, stabilizer, thickener, andtexurizer [1]. Available functional consortia also develop immensepotential for these macromolecules in pharmaceutical and healthcareapplications. These polymers, usually primary or secondarymetabolitesof the microorganisms, can be found either attached to the microbialcell surface or be released as extracellular slime in cell surroundings [2].

Since jute is composed of 58–63% cellulose, 20–24% hemicellulose,12–15% lignin and about 2% pectin, water-soluble compounds, fats andwaxes [3], it is a potential carbon source formicroorganisms able to pro-duce hydrolytic enzymes such as endo-acting endoglucanase and exo-acting cellobiose. The hydrolysis products, especially xylose and glu-cose, can be used for the production of secondarymicrobial metabolitessuch as extracellular polymers [4,5]. Though lignocellulosicmaterial is akind of carbon source that is difficult to exploit during microbial fer-mentation [6] different microorganisms, preferably fungi and bacteriahave been found to utilize high molecular weight lignocellulosic bio-mass. Culture components and conditions are the most important setof parameters to achieve a high yield of polymer production during mi-crobial fermentation. So it is possible to control the polymer production

bymanipulating the culture conditions [7]. Introduction of solid surfaceas a carbon source in a culture medium may inspire such microorgan-isms to adhere and exploit the substrate components inway to produceextracellular polymers. Bacillus megaterium RB-05 [GenBank accessionno HM371417], a commercially potent EPS producing strain of sedi-ment origin [8], was used in this experiment for its ability to adhere toa solid surface. Here in this study our focus was to determine how thejute fiber constituents as a nutrient resource are affecting the polysac-charide production of RB-05.

2. Experimentals

For polymer production in presence of jute, 1 g of 30 mm long TD 5grade jute fiberswas immersed in 15–20 ml ofmineral salts solution [8]in a conical flask. To support initial growth, glucose was added to a finalconcentration of 0.1%. After autoclave for 15 min at 15 psi (121 °C), themediumwas inoculated with a 10% inoculum from an overnight grownculture of RB-05. The cultures were incubated at 33 °C for 90 h. Solidsubstrate including adhered cells and extracellular polymers were sep-arated from the medium manually, placed in 20 ml of deionized waterand vortexed vigorously for 5–10 min to remove cells and extracellularmaterials from the surface of the fibers. Fibers were removed by filtra-tion, and the filtrate, containing cells and extracellular materials, wassubjected to centrifugation at 4 °C and 12,000 g for 20 min. The liquidportion of the culture was centrifuged under the same conditions andthe supernatants from both centrifugationswere combined and filteredthrough a 0.22 μmmembrane filter. This mixture was subjected to cen-trifugation at 25,000 g for 20 min, and polysaccharides were precipitat-ed by adding four volumes of prechilled 95% ethanol. After 24 h at 4 °C,

Fig. 1. Polymer production behavior of RB-05 in jute culture and cellulase activity of thestrain affecting tensile strength of jute.⁎⁎pb0.05 considered as significant NS Not signif-icant [ANOVA Tukey test].

217S. Roy Chowdhury et al. / Materials Letters 66 (2012) 216–218

the mixture was centrifuged at 25,000 g for 20 min at 4 °C and the pel-lets were lyophilized. Carbohydrate and protein contents of the polymerwere determined according to Dubois et al. [9] and Lowry et al. [10], re-spectively. Cellulase production was estimated by the Dinitrosalicylicacid method [11].

Microbial adhesion, growth, and polymer production on the pre-conditioned (dried at 60 °C for 24 h) fiber surface were analyzedunder scanning electron microscope (VEGA TESCAN) at 10 kV in lowvacuum condition. In order to study the chemical changes in jute duringfermentation FT-IR was performed accordingly with Thermo NicoletNexus 870 Spectrophotometer in absorbancemode. Compressed pelletswere prepared by mixing 2 mg of preconditioned fiber dust with100 mg of KBr. Spectrum was recorded in the range 4000–400 cm−1

Fig. 2. (a) FT-IR spectra, (b) X-ray diffractograms, and (c) contact angle values of ju

using 32 scans. Tensile measurements (Hounsfield H10KS UTM) ofjute fibers (n=30,) were done at single fiber level applying maximumload of 100 Nwith the crosshead speed of 3 mm/min andmaintaining agage length of 25 mm. Sessile drop contact angle study of fiber surfacewasmeasured with SEO Contact Angle Meter. The samples were exam-inedwith RIGAKUX-ray diffractometer in powder formmaintaining theoperating 2θ range between 10° and 50° at a scanning speed of 2°/minusing CuKα radiation. Crystallinity indiceswere calculated using the re-lation, CrI=[(I22.5°− I18.5°)/ I22.5°×100] (in percent), where I22.5° andI18.5° were the maximum intensities at 22.5° and 18.5°.

All data were reported as the mean±SD. Statistical analyses wereperformed using ANOVA, Tukey test (LS=0.05), comparison ofmeans, considering pb0.05 as significant.

3. Results and discussion

Lignocellulosic fiber is a toughmaterial to be consumed bymicroor-ganisms as nutrient unless any enzymatic activity of the microorgan-isms hydrolyzes the β-1,4 glycosidic linkages within the cellulosemolecules [6]. In jute culture, RB-05 was unable to display such signifi-cant enzymatic activity till 36 h fermentation, thus not capable of con-suming cellulosic material of the jute. This phenomenon reflected onthe polymer yield, which was on the lower side throughout this periodand found to be 0.049±0.021 g g−1 substrate after 36 h. But from 36 honwards cellulase activity of RB-05 gradually increased (Fig. 1) thatpossibly hydrolyzes the cellulose molecules of the jute in order toavail carbon source for bacterial growth. This in other way induces thepolymer yield (0.297 g EPS g−1 substrate after 72 h), which wasfound to be paralleled to the bacterial growth (data not shown).

In early hours of fermentation with insignificant cellulase activityRB-05 was found to exploit hemicellulose and protein constituents ofjute as nutrient resource. Fig. 2a shows that peak at 1739 cm−1 forstretching of C_O bonds in carboxylic acid and ester componentsand 1240 cm−1for C\O stretching in the acetyl groups in hemicellulose

te at different time intervals (d) SEM image of control and utilized jute surface.

Table 1Microstructure analysis of jute at different time intervals.

Properties 0 h 36 h 60 h 72 h 90 h

Crystallite size (nm)⁎ 3.50±0.07 3.98±0.09 3.71±0.07 3.48±0.06 2.98±0.06FWHM of 002 (2θ)⁎ 2.31±0.09 2.03±0.11⁎⁎ 2.18±0.06NS 2.32±0.05NS 2.71±0.08⁎⁎

Peak maxima (002) (2θ)⁎ 22.51±0.07 22.37±0.04 22.27±0.08 22.35±0.07 22.40±0.07Crystalline index (%)⁎ 60.24±0.46 61.55±0.54 59.35±0.72 57.63±0.66 55.20±0.48

⁎Means±Standard deviations (n=3)⁎⁎pb0.05 considered as significant NS Not significant [ANOVA Tukey test].

218 S. Roy Chowdhury et al. / Materials Letters 66 (2012) 216–218

were either missing or present with lower relative intensity after 36 hfermentation. Relative intensity of the peak at 1240 cm−1 region wasalso attributed to C\N stretching of amides in protein, which too dimin-ishedwith increased fermentation time. Cellulose consumption of RB-05was evidenced by gradual declination of peak intensities at 865 cm−1

for β-glycosidic linkage of cellulose molecule, at 2897 cm−1 for CH3

and CH2 stretching, at 1410 cm−1 for CH3 and CH2 bending, and at1023 cm−1 for C\O\C stretching of cellulose and hemicellulose up to90 h fermentation. Fig. 2b shows the X-ray diffraction pattern of jutefiber samples after 36 h, 60 h, 72 h. and 90 h bacterial exposure with0 h negative control. The pattern of these diffractograms showed thatthe crystalline 002 peak was slightly shifted towards a lower angle(Table 1), albeit the d_002 remained statistically about 0.40 nm. After36 h bacterial exposure, CrI and crystallite size of cultured jute fiberswere found higher in comparison to that of the control sample(Table 1). This could be due to the loss of amorphousmaterials (hemicel-lulose, protein etc.) of jute. With substantial increase in cellulase activityof RB-05 from 36 h onwards, CrI and crystallite size were found in de-creasing order (36 hN60 hN72 hN90 h). Similar trend was observed incase of analyzing full width half maxima (FWHM) (Table 1). After initialsharpening, 002 peak gradually broadened up with time and a signifi-cant broadening (pb0.05) was observed after 90 h. Usually defects incrystal lattice, compositional inhomogeneity, and presence of fine parti-cles in between grains are responsible for broadening of any peak. In thiscase, hydrolysis of β-1,4 glycosidic linkages of cellulosemay have causedbroadening of peak and decreased the degree of crystallinity [12].

Cellulose, along with the binding matter lignin, imparts mechanicalstrength to any lignocellulosic fiber. After 36 h bacterial exposure ten-sile strength of the fibers were found almost unchanged as comparedto the initial that indicates uptake of the non-cellulosic materialsother than lignin did not affect much the fiber strength (pN0.05).

Fig. 3. Adherence, growth, and polymer production of the bacterium on jute surface.

Considerable decrease (pb0.05) in the uniaxial tensile strength from36 h onwards describes the exploitation of cellulose by the bacterium(Fig. 1). Almost around 70% of initial tensile strength of the fiber waslost at the end of 90 h fermentation.

The average contact angle value of the control jute sample (53±5°)was found less than that of the fermented jute of 36 h (71±4°). Thisagain signifies the initial bacterial consumption of water sensitivenon-cellulosic materials. But this trend reversed with time as averagecontact angle values were found around 55±5° and 47±6° after 60 hand 90 h fermentation respectively (Fig. 2c). Enzymatic break down ofcellulose to smaller water soluble saccharides probably made the fibersurface more hydrophilic.

Microbial adhesion was found bit patchy as was evidenced by inhib-ited bacterial growth and low polymer production up to 36 h (Fig. 1). Astime progressed, RB-05 formed a filmy structure over the fiber surface(Fig. 3). Cells were viewed associated with some slime-like material,probably the crude polymer produced by RB-05. As far as substratewas concerned, it became rougher (Fig. 2d)with increasing fermentationtime due to exploitation of cellular components by RB-05 for its growth.

4. Conclusion

Enzymatic decomposition of cellulose molecules among all thecomponents of jute fiber has proved to be the most influential for mi-crobial growth and polymer production. Initially up to 36 h the poly-mer production was found to be low (0.049±0.021 g g−1 substrate)and nutrient uptake was primarily limited to the non-cellulosic mate-rials in absence of considerable bacterial cellulase activity. Polymeryield rate accelerated parallel to the bacterial cellulase activity andwas found significant (pb0.05) at the end of 72 h fermentation onlyafter consuming bulk of the decomposed cellulose materials of jute.

References

[1] Kodali VP, Das S, Sen RK. An exopolysaccharide from a probiotic: biosynthesis dy-namics, composition and emulsifying activity. Food Res Int 2009;42:695–9.

[2] Knoshaug EP, Ahlgrent JA, Trempy JE. Growth associated exopolysaccharide ex-pression in Lactococcuc lactis ssp. cremoris Ropy352. J Dairy Sci 2000;83:633–40.

[3] Pan NC, Day A, Mahalanabis KK. Chemical composition of jute and its estimation.Man-Made Tex India 1999;9:467–73.

[4] Sedlak M, Ho NW. Characterization of the effectiveness of hexose transporters fortransporting xylose during glucose and xylose co-fermentation by a recombinantSaccharomyces yeast. Yeast 2004;21:671–84.

[5] Hahn-Hägerdal B, Karhumaa K, Larsson CU, Gorwa-Grauslund M, Görgens J, vanZyl WH. Role of cultivation media in the development of yeast strains for largescale industrial use. Microb Cell Fact 2005;4:31.

[6] Chen H, Xu XQ, Zhu Y. Optimization of hydroxyl radical scavenging activity of exo-polysaccharides from Inonotus obliquus in submerged fermentation using re-sponse surface methodology. J Microbiol Biotechnol 2010;20:835–43.

[7] Cerning J. Production of exopolysaccharides by lactic acid bacteria and dairy pro-pionibacteria. Lait 1995;75:463–72.

[8] Roy Chowdhury S, Basak RK, Sen R, Adhikari B. Characterization and emulsifyingproperty of a carbohydrate polymer produced by Bacillus pumilus UW-02 isolatedfrom waste water irrigated agricultural soil. Int J Biol Macromol 2011;48:705–12.

[9] Dubois M, Gilles KA, Hamilton J, Rebers PA, Smith F. Colorimetric method for de-termination of sugar and relative substances. Anal Chem 1956;28:350–66.

[10] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with theFolin phnol reagent. J Biol Chem 1951;193:265–75.

[11] Miller G. Use of dinitrosalicylic reagent for the determination of reducing sugar.Anal Chem 1959;31:426–8.

[12] Sinha E, Rout SK, Barhai PK. Study of the structural and thermal properties of plas-ma treated jute fibre. Appl Phys A 2008;92:283–90.