- Email: [email protected]
Distillery effluent as a potential medium for bacterial cellulose production: A biopolymer of great commercial importance
Accepted Manuscript Short Communication Distillery effluent as a potential medium for bacterial cellulose production: a biopolymer of great commercial importance Firdaus Jahan, Vinod Kumar, R.K.Saxena PII: DOI: Reference:
S0960-8524(17)31659-0 http://dx.doi.org/10.1016/j.biortech.2017.09.094 BITE 18922
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 July 2017 12 September 2017 15 September 2017
Please cite this article as: Jahan, F., Kumar, V., R.K.Saxena, Distillery effluent as a potential medium for bacterial cellulose production: a biopolymer of great commercial importance, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.09.094
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Distillery effluent as a potential medium for bacterial cellulose production: a biopolymer of great commercial importance Firdaus Jahana,c* , Vinod Kumara,b* and R.K.Saxenaa *
equal contribution, aDepartment of Microbiology, University of Delhi South Campus, New Delhi 110021, India
b
Center of Innovative and Applied Bioprocessing (CIAB), Sector-81 (Knowledge City), PO Manauli, S.A.S.
Nagar, Mohali-140306, Punjab, India c
Food Safety and Standards Authority of India (FSSAI), FDA Bhawan, New Delhi 110002, India
Corresponding author: Prof. R.K.Saxenaa a
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India
Email: [email protected]
1
Abstract In the present study, an attempt was made to utilize the distillery effluent for the production of bacterial cellulose by a novel bacterial species, Gluconacetobacter oboediens. Maximum bacterial cellulose production of 0.85 g/100 ml was achieved in crude distillery effluent. The production was successfully scaled up to 1.0 L size producing 8.1 g of bacterial cellulose. Morphological, structural and thermal characterization of purified bacterial cellulose by SEM, FT-IR and TGA analysis showed that it is pure cellulose having good properties. Henceforth, the present study proved a concept that distillery effluent could be utilized for the production of bacterial cellulose, a biopolymer of immense importance, which in turn may be used for producing different value added products. Keywords: Distillery effluent, Gluconacetobacter oboediens, Bacterial cellulose Introduction The management of wastewater is one of the most important environmental problems faced all over the world. The effluent generated from distilleries is one of the most hazardous water pollutants (Nagaraj and Kumar, 2008). Distilleries generate (i) spentwash from distillation column; (ii) spent lees from analyser column and (iii) wastewaters like fermenter washings, fermenter cooling, floor washings, spillage and cooling. Among these, spentwash is of the major environmental concern owing to its quantity and quality. It is one of the most complex, toxic and strongest organic industrial effluents, having extremely high COD and BOD levels (Chauhan and Dikshit, 2012). About 10-15 liters of spent wash is generated for every litre of alcohol produced and is characterized by high percentage of dissolved organic (52,000–58,000 mg/L BOD; 92,600-1,00,000 mg/L COD) and inorganic matter (1,660–4,200 mg/L form of nitrogen, 225– 3,038 mg/L phosphorus, and 9,600–17,475 mg/L potassium etc.), dark brown colour (2,38,000– 2,52,000 Pt-Co units), high temperature (70-100oC) and low pH (4-4.5) (Chauhan and Dikshit, 2
2012). Nowadays, discharge of the distillery effluent is the most important problems faced by distilleries. Although there has been a good amount of progress and improvement in the status, yet it is evident that distilleries are lagging far behind in achieving the zero discharge norms (AIDA, 2008). Treatment of these effluents is a very tedious and costly process making it impractical. In the present investigation, an attempt was made to utilize distillery effluent for the production of bacterial cellulose (a value added product) by a novel bacterial species, Gluconacetobacter oboediens. Bacterial cellulose is a biopolymer of tremendous industrial importance owing to its numerous unique properties (Torres et al., 2012; Cherian et al., 2013, Yim et al., 2015; Lin et al., 2016). To a great extent, this study may prove beneficial in reducing the pollution caused by the release of distillery effluent which is one of the most important problems faced by ethanol producing industries. 2. Materials and Methods 2.1
Materials Distillery effluent was collected from All India Distillers’ Association (AIDA), New
Delhi, India. Analysis of the effluent showed presence of approximately 4.0 % carbon and 0.15 % nitrogen source and a pH of 4.75. It also contained a number of metal ions at high concentrations and some other suspended solids. Microcrystalline cellulose used for comparison studies was purchased from Sigma-Aldrich Chemical Co., USA. All the other chemicals and reagents used in the present study were of analytical grade. 2.2
Microorganism The bacterial species used in the present study was previously isolated from mixed fruit
residue (Saxena and Jahan, 2014). The isolate was identified as Gluconacetobacter oboediens on 3
the basis of 16 S rDNA analysis (The Centre for Genomic Applications, New Delhi, India). The neighbour joining tree of this bacterium is presented in Fig. 1. This bacterium was deposited to Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India under the Budapest treaty with MTCC number 5610. The organism was maintained on Hestrin-Schramm (HS) medium plates (Schramm and Hestrin, 1954) at 4 ºC and subcultured at regular intervals of 2 weeks. 2.3.
Inoculum preparation The inoculum was prepared in 50 ml of HS medium by growing the bacterium at
30 ºC at 200 rpm in a temperature controlled incubator shaker (New Brunswick Shaker, USA) for 72 h. Under shake culture, this bacterium formed flocs/pellets having bacterial cells embedded in the cellulose matrix. Here, 6.0 % v/v of this inoculum containing 1.5 x108 cfu/ml (approx.) was used for bacterial cellulose production. 2.4.
Production of bacterial cellulose in distillery effluent medium Different sets of production medium were prepared for cellulose production using
distillery effluent. In the first set, the crude distillery effluent (pH 4.75) was used as such without any modification. In the second set, pH of the distillery effluent was adjusted to 4.3 (optimized pH for Gluconacetobacter oboediens) with 1.0 N HCl. In the third set, sucrose (as carbon source) and corn steep liquor (as nitrogen source) were added at 2.0 % and 1.0 %, respectively without pH adjustment. In the fourth set, sucrose and corn steep liquor were added in the similar way as in the third set and pH was adjusted to 4.3. Cellulose production was carried out in 100 ml of each of these four sets of the production medium (sterilized at 10 psi for 20 mins) in 500 ml conical flasks at 30 ºC under static culture conditions for 8 days. All the experiments were carried out in triplicates and the results are presented as mean of the replicates. 4
2.5
Scale up of bacterial cellulose production The cellulose production was scaled upto 1.0 L of the distillery effluent in the medium
composition wherein maximum bacterial cellulose yield (g/g) was observed. For this, different volumes i.e. 250, 500 and 1000 ml of the medium (effluent) sterilized at 10 psi for 20 min was poured aseptically in sterilized trays of size 18 x 14 x 5 cm3, 28 x 23 x 5 cm3 and 33.5 x 28 x 4.5 cm3, respectively. These trays were inoculated with 6.0% v/v of the inoculum and incubated at 30 ºC for 8 days under static culture conditions for cellulose production. 2.6
Purification and quantification of bacterial cellulose The bacterial cellulose produced was purified by using the method as described in our
earlier report (Jahan et al., 2012) with some modifications. Before processing, the cellulose mat was squeezed so as to remove maximum amount of the entrapped medium components and bacterial cells. At the final steps, bleaching was carried out with 1.0 % calcium hypochlorite to remove any leftover colour. Thereafter, the purified bacterial cellulose was dried at room temperature on a rough porous slab and weighed. The amount of bacterial cellulose produced was estimated as dry weight in g/L. 2.7
Electron microscopy The morphology of bacterial cellulose was studied by Scanning Electron Microscopy
(SEM) and Transmission Electron Microscopy (TEM) using method as described by Castro et al (2013), with minor modifications. For SEM, thin layers of bacterial cellulose samples were coated with gold/palladium using an ion sputter coater. The coated samples were observed at 2000x magnification with LEO- 435 VP (U.K.) scanning electron microscope operating at 20 kV at the Scanning electron Microscope Central Facility, Department of Textile Technology, IIT, New Delhi, India.
5
Bacterial cellulose samples were homogenized, diluted in distilled water and sonicated to obtain a good dispersion. Drops of the suspensions were deposited onto glow-discharged, carbon coated TEM grids and negatively stained with 2 wt% uranyl acetate. Samples were observed at 5000x by JEOL 2100 F Transmission electron microscope operating at 200 kV at the Advance Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India. 2.8
Fourier transform infrared (FT-IR) spectroscopy The structure of bacterial cellulose and microcrystalline plant cellulose with respect to the
position of different bond stretches in their chemical structure was studied by Fourier TransformInfrared Spectrophotometer (Perkin Elmer 16 PC spectrophotometer, Perkin Elmer, Boston, USA) at University Science Instrumentation Centre, Delhi University, Delhi, India. For this, method used by Castro et al (2013) was employed with some modifications. Powdered samples of bacterial cellulose were prepared in KBr at a proportion of 1:100 (m/m) and analyzed over a range of 500 – 4000 cm-1 using 8 scans with resolution equal to 4 cm-1. 2.9
Thermogravimetric analysis (TGA) The thermal degradation behavior of bacterial cellulose and microcrystalline plant
cellulose (standard) was examined by Thermogravimetric analysis (TGA) (Perkin Elmer, STA6000) carried out at Department of Textile Technology, IIT Delhi, New Delhi, India. Method reported by Surma-Slusarska et al (2008) was used with certain modifications. To achieve this, 20 mg of cellulose samples were subjected to thermal treatment in the temperature range of 50 - 600 °C under a N2 atmosphere, using a heating rate and flow rate of 5 °C/min and 70 mL/min, respectively. 3.
Results and Discussion
3.1
Bacterial cellulose production in distillery effluent
6
Results of bacterial cellulose production presented in Table 1 showed that in the crude distillery effluent (without any modification), 0.85 g/ 100 ml (i.e. 8.5 g/L) of bacterial cellulose was produced. Further, in the second set of production media where the pH was adjusted to 4.3, there was no cellulose production. In the third set, where sucrose (2.0 %) and CSL (1.0 %) were added and pH was not adjusted (pH 4.75), 1.08 g/ 100 ml (10.8 g/L) of bacterial cellulose was produced. In the last set wherein sucrose and CSL were added and pH was also adjusted, no cellulose production was observed. It is clearly evident from these observations that bacterial cellulose was produced only in those compositions of distillery effluent wherein the pH was not adjusted. However, when the pH of the effluent was adjusted using 1.0 N HCl, no bacterial growth and cellulose production was observed. This might be due to the formation of some inhibitors which resulted in the inhibition of the growth of the bacterium and cellulose production. It was also observed that when carbon and nitrogen sources were supplemented in the distillery effluent there was a slight increase in bacterial cellulose production (1.08 g/100 ml). This shows that the bacterium can efficiently utilize the additional carbon and nitrogen sources supplemented in the effluent for cellulose production. However, it can be inferred from these results that it is better to utilize crude distillery effluent for bacterial cellulose production as it will be much more economical involving zero raw material cost. Also, crude effluent resulted in better cellulose yield (0.21 gcellulose/gcarbon) as compared to that achieved after supplementation of carbon source (0.18 gcellulose/gcarbon). Most of the studies of bacterial cellulose production have been carried out using conventional carbon sources (Pourramezan et al., 2009; Mohite et al., 2013). Also, there are few studies on the production of bacterial cellulose utilizing agro-industrial wastes (Uraki et al., 2002; Hong et al., 2012 and Cavka et al., 2013). However, to the best of our knowledge, there are only one or two reports on bacterial cellulose production using distillery effluent. In a study 7
by Wu and Liu (2013), 6.26 g/L of BC was produced in 7 days by Gluconacetobacter xylinus, which is lesser as compared to the present study. The present work is the first successful study wherein distillery effluent has efficiently been used for bacterial cellulose production by a novel bacterial species, Gluconacetobacter oboediens. 3.2
Scale up of bacterial cellulose production Having observed better cellulose yield in crude distillery effluent, scale up of cellulose
production in 250, 500 and 1000 ml of the effluent was attempted in trays of different dimensions. Results presented in Table 2 showed that cellulose production was successfully scaled upto 1.0 L of the effluent producing 8.11 g of bacterial cellulose. Further, the cellulose production in 1.0 L of the effluent medium was attempted in 10 trays to validate the bacterial cellulose yield. The total wet weight of the cellulose mats produced from 10 L of the crude distillery effluent was 4720 g which after processing yielded 80 g of dry bacterial cellulose. This shows that on an average 8.0 g of bacterial cellulose was produced per liter of the effluent, which is almost similar to bacterial cellulose production in 1.0 L effluent. 3.3
Morphological analysis by Scanning and Transmission Electron Microscopy Scanning electron microscopy revealed that bacterial cellulose produced is composed of a
dense network of interwoven ultrafine fibrils, which is similar to the observations of Klemm et al (2001), Krystynowicz et al (2002) and Castro et al (2013). TEM results revealed that the width of bacterial cellulose fibrils is approx. 41.9 – 68.1 nm which is in agreement with the findings of Castro et al (2013). 3.4
Structural analysis by FT-IR spectroscopy Results obtained by FT-IR spectroscopy showed characteristic peaks which represent the
bond stretches present in the sugar ring. The distinguish peaks of 3350 cm-1 indicates O-H stretching, 2960 cm-1 indicates C-H stretching, 1650 cm-1 indicates C-O-C stretching and 1058 8
cm-1 indicates C-O stretching. Peaks at similar positions were obtained in the FT-IR spectra of the plant cellulose thus, confirming the chemical structure of this bacterial cellulose. Similar FTIR spectra for bacterial cellulose were reported by Surma-Ślusarska et al (2008), Castro et al (2012), Goh et al (2012) and Halib et al (2012). 3.5
Thermal stability by thermo-gravimetric analysis The thermo gravimetric analysis (TGA) of bacterial cellulose presented in Fig. 2A
revealed that it was almost stable upto 250 °C and beyond this temperature it started decomposing. In the temperature range from 260 – 600 °C, about 56% of bacterial cellulose was decomposed and upto 600 °C, about 31 % of bacterial cellulose was left undecomposed. These results are in agreement with the findings of Surma-Slusarska et al (2008) and Cherian et al (2013) who reported that it degrades at higher temperatures (300 ºC). However, the TGA curve of plant cellulose (Fig. 2B) showed that there was more than 70 % decomposition in the temperature range between 50 - 500 °C and upto 600 °C temperature only 18 % of plant cellulose was left undecomposed. These results clearly show that bacterial cellulose is more thermostable than plant cellulose. Similar to our results, Halib et al (2012) reported that bacterial cellulose is more thermostable than plant cellulose. 4.0
Conclusions Distillery effluent was successfully utilized for the production of bacterial cellulose
resulting in 8.11 g/L of cellulose. Results also demonstrated the scalability of the production process. This approach will benefit various distilleries where the effluent is released in large amounts which they can utilize for the production of this commercially important bacterial cellulose. In addition, the effluent left over after cellulose production may be less toxic to be discharged. Though, this is a preliminary study but it proved a concept and opened directions to
9
utilize distillery effluent for producing value added products like bacterial cellulose at zero raw material cost. Supplementary Material Composition of distillery effluent supplemented as Table S1. Electron micrographs are provided as Fig S1. FTIR spectra of bacterial and plant cellulose are supplemented as Fig. S2. Acknowledgements Authors acknowledge the financial support from Council of Scientific and Industrial Research (CSIR), India. Firdaus Jahan acknowledges with thanks the receipt of CSIR–SRF to carry out this work. Vinod Kumar thanks Ministry of New and Renewable Energy (MNRE), India for providing fellowship. Scale up studies have been carried out with the support of Technology Based Incubator, UDSC, New Delhi and is sincerely acknowledged. References 1. AIDA
(2008).
Ethanol
opportunities
and
challenges.
URL
http://aidaindia.org/its08/topics_covered.html, visited on 24th March 2008. 2. Castro, C., Zuluaga, R., Alvareza, C., Putaux, JL., Caro, G., Rojas, O.J., Mondragon, I., Ganan, P., 2013. Bacterial cellulose produced by a new acid-resistant strain of Gluconacetobacter genus. Carbohydr. Poly. 89, 1033-1037. 3. Cavka, A., Guo, X., Tang, S.J., Winestrand, S., Jonsson, L.J., Hong, F., 2013. Production of bacterial cellulose and enzyme from waste fibre sludge. Biotechnol. Biofuels. 6, 25. 4. Chauhan, M.S., Dikshit, A.K., 2012. Indian Distillery Industry: Problems and Prospects of Decolourisation of Spentwash. 2012 International Conference on Future Environment and Energy, IPCBEE Vol.28, IACSIT Press, Singapore.
10
5. Cherian, B.M., Leão, A.L., de Souza, S.F., de Olyveira, G.M, Costa, L.M.M., Brandão, C.V.S., Narine, S.S., 2013. Advances in Natural Polymers, Advanced Structured Materials. Thomas et al. (eds.), 18, Springer-Verlag Berlin Heidelberg. 6. Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A., Bhat, R., 2012. Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (Kombucha). II. Int. Food Res. J. 19, 153–158. 7. Halib, N., Amin, M.C.I.M., Ishak, A., 2012. Physicochemical Properties and Characterization of Nata de Coco from Local Food Industries as a Source of Cellulose. Sains Malaysiana. 41, 205–211. 8. Hong, F., Guo, X., Zhang, S., Han, S., Yang, G., Jönsson, L.J., 2012. Bacterial cellulose production from cotton-based waste textiles: enzymatic saccharification enhanced by ionic liquid pretreatment. Bioresour. Technol. 104, 503–508. 9. Jahan, F., Kumar, V., Rawat, G., Saxena, R.K., 2012. Production of microbial cellulose by a bacterium isolated from fruit. Appl. Biochem. Biotechnol. 167, 1157- 1171. 10. Klemm, D., Shumann, D., Udhardt, U., Marsch, S., 2001. Bacterial synthesized cellulose – Artificial blood vessels for microsurgery. Prog. Polym. Sci. 26, 1561–1603. 11. Krystynowicz, A., Czaja, W., Wiktorowska-Jezierska, A., Gonçalves-Mioekiewicz, M., Turkiewicz, M., Bielecki, S., 2002. Factors affecting the yield and properties of bacterial cellulose. J. Ind. Microbiol. Biotechnol. 29, 189–195. 12. Mohite, B.V., Salunke, B.K., Patil, S.V., 2013. Enhanced Production of Bacterial Cellulose by using Gluconacetobacter hansenii NCIM 2529 Strain under shaking conditions. Appl. Biochem. Biotechnol. 169, 1497-1511. 13. Nagaraj, M., Kumar, A., 2008. Distillery wastewater treatment and disposal. Indian Institute of Technology, Roorkee. 11
14. Pourramezan, G., Roayaei, A., Qezelbash, Q., 2009. Optimization of culture conditions for bacterial cellulose production by Acetobacter sp. 4B-2. Biotechnol. 8, 150–154. 15. Lin, S.P., Liu, C.T., Hsu, K.D., Hung, Y.T., Shih, T.Y., Chen, K.C., 2016. Production of bacterial cellulose with various additives in a PCS rotating disk bioreactor and its material property analysis. Cellulose. 23, 367-377. 16. Saxena, R.K., Jahan, F., 2014. Novel isolated bacterial strain of Gluconacetobacter oboediens and an optimized economic process for microbial cellulose production Therefrom. US2014/0080184 A1. 17. Schramm, M., Hestrin, S., 1954. Factors affecting production of cellulose at the air/liquid interface of a culture of Acetobacter xylinum. J. Gen. Microbiol. 11, 123-129. 18. Surma-ślusarska, B., Presler, S., Danielewicz, D., 2008. Characteristics of bacterial cellulose obtained from Acetobacter xylinum culture for application in papermaking. Fibres and Textiles in Eastern Europe. 16, 108–111. 19. Torres, F., Commeaux, S., Troncoso, O., 2012. Biocompatibility of Bacterial Cellulose Based Biomaterials. J. Func. Biomat. 3, 864–878. 20. Uraki, Y., Morito, M., Kishimoto, T., Sano, Y., 2002. Bacterial cellulose production using monosaccharides derived from hemicellulose in water-soluble fraction of water liquor from atmosphere acetic acid pulping. Holzforschung. 56, 341–347. 21. Wu, J.M., Liu, R.H., 2013. Cost-effective production of bacterial cellulose in static cultures using distillery wastewater. J. Biosci. Bioeng. 115, 284-290. 22. Yim, S.M., Song, J.E., Kim, H.R., 2015. Production and characterization of bacterial cellulose fabrics by nitrogen sources of tea and carbon sources of sugar. Process Biochem. 117, 518–23.
12
23.
24.
13
25.
14
Highlights: Production of bacterial cellulose at zero raw material cost utilizing distillery effluent.
15
Table 1: Amount of cellulose produced in different medium compositions prepared from distillery effluent
Effluent medium
Bacterial cellulose (g/100 ml)
Crude effluent (without any modification, pH 4.75) Crude effluent (pH adjusted to 4.3) Effluent + sucrose (2.0 %) + corn steep liquor (1.0 %) (pH not adjusted) Effluent + sucrose (2.0 %) + corn steep liquor (1.0 %) (pH adjusted to 4.3)
0.85
Bacterial cellulose* (g/L) 8.5
-
-
1.08
-
* calculated yield
16
10.8
-
Table 2: Scale up of cellulose production upto 1.0 L of crude distillery effluent in different tray sizes Crude effluent (ml)
Tray size (cm3)
Bacterial cellulose (g)
250
18 x14 x 5
2.31
500
28 x 23 x 5
4.25
1000
33.5 x 28 x 4.5
8.11
17
26.
18
S0960-8524(17)31659-0 http://dx.doi.org/10.1016/j.biortech.2017.09.094 BITE 18922
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 July 2017 12 September 2017 15 September 2017
Please cite this article as: Jahan, F., Kumar, V., R.K.Saxena, Distillery effluent as a potential medium for bacterial cellulose production: a biopolymer of great commercial importance, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.09.094
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Distillery effluent as a potential medium for bacterial cellulose production: a biopolymer of great commercial importance Firdaus Jahana,c* , Vinod Kumara,b* and R.K.Saxenaa *
equal contribution, aDepartment of Microbiology, University of Delhi South Campus, New Delhi 110021, India
b
Center of Innovative and Applied Bioprocessing (CIAB), Sector-81 (Knowledge City), PO Manauli, S.A.S.
Nagar, Mohali-140306, Punjab, India c
Food Safety and Standards Authority of India (FSSAI), FDA Bhawan, New Delhi 110002, India
Corresponding author: Prof. R.K.Saxenaa a
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India
Email: [email protected]
1
Abstract In the present study, an attempt was made to utilize the distillery effluent for the production of bacterial cellulose by a novel bacterial species, Gluconacetobacter oboediens. Maximum bacterial cellulose production of 0.85 g/100 ml was achieved in crude distillery effluent. The production was successfully scaled up to 1.0 L size producing 8.1 g of bacterial cellulose. Morphological, structural and thermal characterization of purified bacterial cellulose by SEM, FT-IR and TGA analysis showed that it is pure cellulose having good properties. Henceforth, the present study proved a concept that distillery effluent could be utilized for the production of bacterial cellulose, a biopolymer of immense importance, which in turn may be used for producing different value added products. Keywords: Distillery effluent, Gluconacetobacter oboediens, Bacterial cellulose Introduction The management of wastewater is one of the most important environmental problems faced all over the world. The effluent generated from distilleries is one of the most hazardous water pollutants (Nagaraj and Kumar, 2008). Distilleries generate (i) spentwash from distillation column; (ii) spent lees from analyser column and (iii) wastewaters like fermenter washings, fermenter cooling, floor washings, spillage and cooling. Among these, spentwash is of the major environmental concern owing to its quantity and quality. It is one of the most complex, toxic and strongest organic industrial effluents, having extremely high COD and BOD levels (Chauhan and Dikshit, 2012). About 10-15 liters of spent wash is generated for every litre of alcohol produced and is characterized by high percentage of dissolved organic (52,000–58,000 mg/L BOD; 92,600-1,00,000 mg/L COD) and inorganic matter (1,660–4,200 mg/L form of nitrogen, 225– 3,038 mg/L phosphorus, and 9,600–17,475 mg/L potassium etc.), dark brown colour (2,38,000– 2,52,000 Pt-Co units), high temperature (70-100oC) and low pH (4-4.5) (Chauhan and Dikshit, 2
2012). Nowadays, discharge of the distillery effluent is the most important problems faced by distilleries. Although there has been a good amount of progress and improvement in the status, yet it is evident that distilleries are lagging far behind in achieving the zero discharge norms (AIDA, 2008). Treatment of these effluents is a very tedious and costly process making it impractical. In the present investigation, an attempt was made to utilize distillery effluent for the production of bacterial cellulose (a value added product) by a novel bacterial species, Gluconacetobacter oboediens. Bacterial cellulose is a biopolymer of tremendous industrial importance owing to its numerous unique properties (Torres et al., 2012; Cherian et al., 2013, Yim et al., 2015; Lin et al., 2016). To a great extent, this study may prove beneficial in reducing the pollution caused by the release of distillery effluent which is one of the most important problems faced by ethanol producing industries. 2. Materials and Methods 2.1
Materials Distillery effluent was collected from All India Distillers’ Association (AIDA), New
Delhi, India. Analysis of the effluent showed presence of approximately 4.0 % carbon and 0.15 % nitrogen source and a pH of 4.75. It also contained a number of metal ions at high concentrations and some other suspended solids. Microcrystalline cellulose used for comparison studies was purchased from Sigma-Aldrich Chemical Co., USA. All the other chemicals and reagents used in the present study were of analytical grade. 2.2
Microorganism The bacterial species used in the present study was previously isolated from mixed fruit
residue (Saxena and Jahan, 2014). The isolate was identified as Gluconacetobacter oboediens on 3
the basis of 16 S rDNA analysis (The Centre for Genomic Applications, New Delhi, India). The neighbour joining tree of this bacterium is presented in Fig. 1. This bacterium was deposited to Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India under the Budapest treaty with MTCC number 5610. The organism was maintained on Hestrin-Schramm (HS) medium plates (Schramm and Hestrin, 1954) at 4 ºC and subcultured at regular intervals of 2 weeks. 2.3.
Inoculum preparation The inoculum was prepared in 50 ml of HS medium by growing the bacterium at
30 ºC at 200 rpm in a temperature controlled incubator shaker (New Brunswick Shaker, USA) for 72 h. Under shake culture, this bacterium formed flocs/pellets having bacterial cells embedded in the cellulose matrix. Here, 6.0 % v/v of this inoculum containing 1.5 x108 cfu/ml (approx.) was used for bacterial cellulose production. 2.4.
Production of bacterial cellulose in distillery effluent medium Different sets of production medium were prepared for cellulose production using
distillery effluent. In the first set, the crude distillery effluent (pH 4.75) was used as such without any modification. In the second set, pH of the distillery effluent was adjusted to 4.3 (optimized pH for Gluconacetobacter oboediens) with 1.0 N HCl. In the third set, sucrose (as carbon source) and corn steep liquor (as nitrogen source) were added at 2.0 % and 1.0 %, respectively without pH adjustment. In the fourth set, sucrose and corn steep liquor were added in the similar way as in the third set and pH was adjusted to 4.3. Cellulose production was carried out in 100 ml of each of these four sets of the production medium (sterilized at 10 psi for 20 mins) in 500 ml conical flasks at 30 ºC under static culture conditions for 8 days. All the experiments were carried out in triplicates and the results are presented as mean of the replicates. 4
2.5
Scale up of bacterial cellulose production The cellulose production was scaled upto 1.0 L of the distillery effluent in the medium
composition wherein maximum bacterial cellulose yield (g/g) was observed. For this, different volumes i.e. 250, 500 and 1000 ml of the medium (effluent) sterilized at 10 psi for 20 min was poured aseptically in sterilized trays of size 18 x 14 x 5 cm3, 28 x 23 x 5 cm3 and 33.5 x 28 x 4.5 cm3, respectively. These trays were inoculated with 6.0% v/v of the inoculum and incubated at 30 ºC for 8 days under static culture conditions for cellulose production. 2.6
Purification and quantification of bacterial cellulose The bacterial cellulose produced was purified by using the method as described in our
earlier report (Jahan et al., 2012) with some modifications. Before processing, the cellulose mat was squeezed so as to remove maximum amount of the entrapped medium components and bacterial cells. At the final steps, bleaching was carried out with 1.0 % calcium hypochlorite to remove any leftover colour. Thereafter, the purified bacterial cellulose was dried at room temperature on a rough porous slab and weighed. The amount of bacterial cellulose produced was estimated as dry weight in g/L. 2.7
Electron microscopy The morphology of bacterial cellulose was studied by Scanning Electron Microscopy
(SEM) and Transmission Electron Microscopy (TEM) using method as described by Castro et al (2013), with minor modifications. For SEM, thin layers of bacterial cellulose samples were coated with gold/palladium using an ion sputter coater. The coated samples were observed at 2000x magnification with LEO- 435 VP (U.K.) scanning electron microscope operating at 20 kV at the Scanning electron Microscope Central Facility, Department of Textile Technology, IIT, New Delhi, India.
5
Bacterial cellulose samples were homogenized, diluted in distilled water and sonicated to obtain a good dispersion. Drops of the suspensions were deposited onto glow-discharged, carbon coated TEM grids and negatively stained with 2 wt% uranyl acetate. Samples were observed at 5000x by JEOL 2100 F Transmission electron microscope operating at 200 kV at the Advance Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India. 2.8
Fourier transform infrared (FT-IR) spectroscopy The structure of bacterial cellulose and microcrystalline plant cellulose with respect to the
position of different bond stretches in their chemical structure was studied by Fourier TransformInfrared Spectrophotometer (Perkin Elmer 16 PC spectrophotometer, Perkin Elmer, Boston, USA) at University Science Instrumentation Centre, Delhi University, Delhi, India. For this, method used by Castro et al (2013) was employed with some modifications. Powdered samples of bacterial cellulose were prepared in KBr at a proportion of 1:100 (m/m) and analyzed over a range of 500 – 4000 cm-1 using 8 scans with resolution equal to 4 cm-1. 2.9
Thermogravimetric analysis (TGA) The thermal degradation behavior of bacterial cellulose and microcrystalline plant
cellulose (standard) was examined by Thermogravimetric analysis (TGA) (Perkin Elmer, STA6000) carried out at Department of Textile Technology, IIT Delhi, New Delhi, India. Method reported by Surma-Slusarska et al (2008) was used with certain modifications. To achieve this, 20 mg of cellulose samples were subjected to thermal treatment in the temperature range of 50 - 600 °C under a N2 atmosphere, using a heating rate and flow rate of 5 °C/min and 70 mL/min, respectively. 3.
Results and Discussion
3.1
Bacterial cellulose production in distillery effluent
6
Results of bacterial cellulose production presented in Table 1 showed that in the crude distillery effluent (without any modification), 0.85 g/ 100 ml (i.e. 8.5 g/L) of bacterial cellulose was produced. Further, in the second set of production media where the pH was adjusted to 4.3, there was no cellulose production. In the third set, where sucrose (2.0 %) and CSL (1.0 %) were added and pH was not adjusted (pH 4.75), 1.08 g/ 100 ml (10.8 g/L) of bacterial cellulose was produced. In the last set wherein sucrose and CSL were added and pH was also adjusted, no cellulose production was observed. It is clearly evident from these observations that bacterial cellulose was produced only in those compositions of distillery effluent wherein the pH was not adjusted. However, when the pH of the effluent was adjusted using 1.0 N HCl, no bacterial growth and cellulose production was observed. This might be due to the formation of some inhibitors which resulted in the inhibition of the growth of the bacterium and cellulose production. It was also observed that when carbon and nitrogen sources were supplemented in the distillery effluent there was a slight increase in bacterial cellulose production (1.08 g/100 ml). This shows that the bacterium can efficiently utilize the additional carbon and nitrogen sources supplemented in the effluent for cellulose production. However, it can be inferred from these results that it is better to utilize crude distillery effluent for bacterial cellulose production as it will be much more economical involving zero raw material cost. Also, crude effluent resulted in better cellulose yield (0.21 gcellulose/gcarbon) as compared to that achieved after supplementation of carbon source (0.18 gcellulose/gcarbon). Most of the studies of bacterial cellulose production have been carried out using conventional carbon sources (Pourramezan et al., 2009; Mohite et al., 2013). Also, there are few studies on the production of bacterial cellulose utilizing agro-industrial wastes (Uraki et al., 2002; Hong et al., 2012 and Cavka et al., 2013). However, to the best of our knowledge, there are only one or two reports on bacterial cellulose production using distillery effluent. In a study 7
by Wu and Liu (2013), 6.26 g/L of BC was produced in 7 days by Gluconacetobacter xylinus, which is lesser as compared to the present study. The present work is the first successful study wherein distillery effluent has efficiently been used for bacterial cellulose production by a novel bacterial species, Gluconacetobacter oboediens. 3.2
Scale up of bacterial cellulose production Having observed better cellulose yield in crude distillery effluent, scale up of cellulose
production in 250, 500 and 1000 ml of the effluent was attempted in trays of different dimensions. Results presented in Table 2 showed that cellulose production was successfully scaled upto 1.0 L of the effluent producing 8.11 g of bacterial cellulose. Further, the cellulose production in 1.0 L of the effluent medium was attempted in 10 trays to validate the bacterial cellulose yield. The total wet weight of the cellulose mats produced from 10 L of the crude distillery effluent was 4720 g which after processing yielded 80 g of dry bacterial cellulose. This shows that on an average 8.0 g of bacterial cellulose was produced per liter of the effluent, which is almost similar to bacterial cellulose production in 1.0 L effluent. 3.3
Morphological analysis by Scanning and Transmission Electron Microscopy Scanning electron microscopy revealed that bacterial cellulose produced is composed of a
dense network of interwoven ultrafine fibrils, which is similar to the observations of Klemm et al (2001), Krystynowicz et al (2002) and Castro et al (2013). TEM results revealed that the width of bacterial cellulose fibrils is approx. 41.9 – 68.1 nm which is in agreement with the findings of Castro et al (2013). 3.4
Structural analysis by FT-IR spectroscopy Results obtained by FT-IR spectroscopy showed characteristic peaks which represent the
bond stretches present in the sugar ring. The distinguish peaks of 3350 cm-1 indicates O-H stretching, 2960 cm-1 indicates C-H stretching, 1650 cm-1 indicates C-O-C stretching and 1058 8
cm-1 indicates C-O stretching. Peaks at similar positions were obtained in the FT-IR spectra of the plant cellulose thus, confirming the chemical structure of this bacterial cellulose. Similar FTIR spectra for bacterial cellulose were reported by Surma-Ślusarska et al (2008), Castro et al (2012), Goh et al (2012) and Halib et al (2012). 3.5
Thermal stability by thermo-gravimetric analysis The thermo gravimetric analysis (TGA) of bacterial cellulose presented in Fig. 2A
revealed that it was almost stable upto 250 °C and beyond this temperature it started decomposing. In the temperature range from 260 – 600 °C, about 56% of bacterial cellulose was decomposed and upto 600 °C, about 31 % of bacterial cellulose was left undecomposed. These results are in agreement with the findings of Surma-Slusarska et al (2008) and Cherian et al (2013) who reported that it degrades at higher temperatures (300 ºC). However, the TGA curve of plant cellulose (Fig. 2B) showed that there was more than 70 % decomposition in the temperature range between 50 - 500 °C and upto 600 °C temperature only 18 % of plant cellulose was left undecomposed. These results clearly show that bacterial cellulose is more thermostable than plant cellulose. Similar to our results, Halib et al (2012) reported that bacterial cellulose is more thermostable than plant cellulose. 4.0
Conclusions Distillery effluent was successfully utilized for the production of bacterial cellulose
resulting in 8.11 g/L of cellulose. Results also demonstrated the scalability of the production process. This approach will benefit various distilleries where the effluent is released in large amounts which they can utilize for the production of this commercially important bacterial cellulose. In addition, the effluent left over after cellulose production may be less toxic to be discharged. Though, this is a preliminary study but it proved a concept and opened directions to
9
utilize distillery effluent for producing value added products like bacterial cellulose at zero raw material cost. Supplementary Material Composition of distillery effluent supplemented as Table S1. Electron micrographs are provided as Fig S1. FTIR spectra of bacterial and plant cellulose are supplemented as Fig. S2. Acknowledgements Authors acknowledge the financial support from Council of Scientific and Industrial Research (CSIR), India. Firdaus Jahan acknowledges with thanks the receipt of CSIR–SRF to carry out this work. Vinod Kumar thanks Ministry of New and Renewable Energy (MNRE), India for providing fellowship. Scale up studies have been carried out with the support of Technology Based Incubator, UDSC, New Delhi and is sincerely acknowledged. References 1. AIDA
(2008).
Ethanol
opportunities
and
challenges.
URL
http://aidaindia.org/its08/topics_covered.html, visited on 24th March 2008. 2. Castro, C., Zuluaga, R., Alvareza, C., Putaux, JL., Caro, G., Rojas, O.J., Mondragon, I., Ganan, P., 2013. Bacterial cellulose produced by a new acid-resistant strain of Gluconacetobacter genus. Carbohydr. Poly. 89, 1033-1037. 3. Cavka, A., Guo, X., Tang, S.J., Winestrand, S., Jonsson, L.J., Hong, F., 2013. Production of bacterial cellulose and enzyme from waste fibre sludge. Biotechnol. Biofuels. 6, 25. 4. Chauhan, M.S., Dikshit, A.K., 2012. Indian Distillery Industry: Problems and Prospects of Decolourisation of Spentwash. 2012 International Conference on Future Environment and Energy, IPCBEE Vol.28, IACSIT Press, Singapore.
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5. Cherian, B.M., Leão, A.L., de Souza, S.F., de Olyveira, G.M, Costa, L.M.M., Brandão, C.V.S., Narine, S.S., 2013. Advances in Natural Polymers, Advanced Structured Materials. Thomas et al. (eds.), 18, Springer-Verlag Berlin Heidelberg. 6. Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A., Bhat, R., 2012. Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (Kombucha). II. Int. Food Res. J. 19, 153–158. 7. Halib, N., Amin, M.C.I.M., Ishak, A., 2012. Physicochemical Properties and Characterization of Nata de Coco from Local Food Industries as a Source of Cellulose. Sains Malaysiana. 41, 205–211. 8. Hong, F., Guo, X., Zhang, S., Han, S., Yang, G., Jönsson, L.J., 2012. Bacterial cellulose production from cotton-based waste textiles: enzymatic saccharification enhanced by ionic liquid pretreatment. Bioresour. Technol. 104, 503–508. 9. Jahan, F., Kumar, V., Rawat, G., Saxena, R.K., 2012. Production of microbial cellulose by a bacterium isolated from fruit. Appl. Biochem. Biotechnol. 167, 1157- 1171. 10. Klemm, D., Shumann, D., Udhardt, U., Marsch, S., 2001. Bacterial synthesized cellulose – Artificial blood vessels for microsurgery. Prog. Polym. Sci. 26, 1561–1603. 11. Krystynowicz, A., Czaja, W., Wiktorowska-Jezierska, A., Gonçalves-Mioekiewicz, M., Turkiewicz, M., Bielecki, S., 2002. Factors affecting the yield and properties of bacterial cellulose. J. Ind. Microbiol. Biotechnol. 29, 189–195. 12. Mohite, B.V., Salunke, B.K., Patil, S.V., 2013. Enhanced Production of Bacterial Cellulose by using Gluconacetobacter hansenii NCIM 2529 Strain under shaking conditions. Appl. Biochem. Biotechnol. 169, 1497-1511. 13. Nagaraj, M., Kumar, A., 2008. Distillery wastewater treatment and disposal. Indian Institute of Technology, Roorkee. 11
14. Pourramezan, G., Roayaei, A., Qezelbash, Q., 2009. Optimization of culture conditions for bacterial cellulose production by Acetobacter sp. 4B-2. Biotechnol. 8, 150–154. 15. Lin, S.P., Liu, C.T., Hsu, K.D., Hung, Y.T., Shih, T.Y., Chen, K.C., 2016. Production of bacterial cellulose with various additives in a PCS rotating disk bioreactor and its material property analysis. Cellulose. 23, 367-377. 16. Saxena, R.K., Jahan, F., 2014. Novel isolated bacterial strain of Gluconacetobacter oboediens and an optimized economic process for microbial cellulose production Therefrom. US2014/0080184 A1. 17. Schramm, M., Hestrin, S., 1954. Factors affecting production of cellulose at the air/liquid interface of a culture of Acetobacter xylinum. J. Gen. Microbiol. 11, 123-129. 18. Surma-ślusarska, B., Presler, S., Danielewicz, D., 2008. Characteristics of bacterial cellulose obtained from Acetobacter xylinum culture for application in papermaking. Fibres and Textiles in Eastern Europe. 16, 108–111. 19. Torres, F., Commeaux, S., Troncoso, O., 2012. Biocompatibility of Bacterial Cellulose Based Biomaterials. J. Func. Biomat. 3, 864–878. 20. Uraki, Y., Morito, M., Kishimoto, T., Sano, Y., 2002. Bacterial cellulose production using monosaccharides derived from hemicellulose in water-soluble fraction of water liquor from atmosphere acetic acid pulping. Holzforschung. 56, 341–347. 21. Wu, J.M., Liu, R.H., 2013. Cost-effective production of bacterial cellulose in static cultures using distillery wastewater. J. Biosci. Bioeng. 115, 284-290. 22. Yim, S.M., Song, J.E., Kim, H.R., 2015. Production and characterization of bacterial cellulose fabrics by nitrogen sources of tea and carbon sources of sugar. Process Biochem. 117, 518–23.
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Highlights: Production of bacterial cellulose at zero raw material cost utilizing distillery effluent.
15
Table 1: Amount of cellulose produced in different medium compositions prepared from distillery effluent
Effluent medium
Bacterial cellulose (g/100 ml)
Crude effluent (without any modification, pH 4.75) Crude effluent (pH adjusted to 4.3) Effluent + sucrose (2.0 %) + corn steep liquor (1.0 %) (pH not adjusted) Effluent + sucrose (2.0 %) + corn steep liquor (1.0 %) (pH adjusted to 4.3)
0.85
Bacterial cellulose* (g/L) 8.5
-
-
1.08
-
* calculated yield
16
10.8
-
Table 2: Scale up of cellulose production upto 1.0 L of crude distillery effluent in different tray sizes Crude effluent (ml)
Tray size (cm3)
Bacterial cellulose (g)
250
18 x14 x 5
2.31
500
28 x 23 x 5
4.25
1000
33.5 x 28 x 4.5
8.11
17
26.
18