Food Sci. Biotechnol. 21(4): 1045-1053 (2012) DOI 10.1007/s10068-012-0136-8
RESEARCH ARTICLE
Microbial Levan from Pseudomonas fluorescens: Characterization and Medium Optimization for Enhanced Production Nagnath R. Jathore, Mahesh V. Bule, Ashwini V. Tilay, and Uday S. Annapure
Received: 11 January 2012 / Revised: 5 March 2012 / Accepted: 17 March 2012 / Published Online: 31 August 2012 © KoSFoST and Springer 2012
Abstract Levan, a polyfructan which consists of Dfructofuranosyl residues linked predominantly by β-(2,6) linkage as a core chain with some β-(2,1) branch chains have potential applications in the pharmaceutical, food, and cosmetic industries. The present work reports on characterization of levan produced using Pseudomonas fluorescens by Fourier transform infrared spectroscopy (FTIR) and NMR and static fermentation condition optimization using one factor-at-a-time followed by statistical designs. The characterization of levan by FTIR revealed that structure of levan to be homologous to the standard levan sample. 13C and 1H NMR studies further successfully confirmed the levan structure. The optimized medium composition was observed to be (in g/L) sucrose 60; ammonium chloride 1.5; sodium nitrate 2.0, and casein peptone 15.0. The yield of levan was increased significantly from 5.27 to 15.42 g/L when the fermentation was carried out using optimal medium. Keywords: levan, characterization, static culture, optimization, Pseudomonas fluorescens
Introduction Levan is a neutral homopolysaccharide composed of Dfructofuranosyl residues joined by β-(2,6) as a main chain Nagnath R. Jathore, Mahesh V. Bule, Ashvini V. Tilay, Uday S. Annapure () Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India Tel: +91-022-3361-2507, Fax: +91-022-3361-1020 E-mail:
[email protected] Mahesh V. Bule Department of Biological System Engineering, PO BOX 646120, LJ Smith Hall, Washington State University, Pullman, WA-99164-6120, USA
with some β-(2,1) branching points. Levan can be produced either from plants or microorganisms. Plant levan also called as phelins, have much lower molecular weight than bacterial levan (1). Microbial levan has several common features in soluble form, including an exceptionally low intrinsic viscosity for a polymer of high molecular weight and extreme concentration dependence of viscosity at the intermediate zone though they have highly branched molecular structure (2). These properties made levan suitable for different technical applications. Potential applications of levan cover the area of food, pharmaceutical, and cosmetics production (3-6). Besides, levan can also be used as an encapsulating agent, thickener, surface-finishing agent, emulsifier as well as a carrier for colors and flavors in the food industry (7). Earlier microbial production of levan was performed using different organisms like Zymomonas, Streptococcus, Xanthomonas, Bacillus, and Pseudomonas (7-12). Pseudomonas fluorescens is an obligate aerobe and Gramnegative bacterium that can use oxygen as well as nitrate as a hydrogen acceptor. This nitrate-reducing strain can produce levan in the total absence of air (10). Since levan possesses many properties favorable and beneficial for human health, an attempt was made to synthesize this polymer employing the strain of P. fluorescens NCIM 2059. Production of microbial levan is generally carried out with sucrose-based substrates. Keith et al. (13) reported that levan was obtained from disaccharides containing only fructose. Küçükaşik et al. (14) and de Oliveira et al. (11) reported molasses is suitable source for production of levan. Borsari et al. (15) used sugar cane juice and sucrose as carbon sources including for levan production using Zymomonas mobilis culture. Ribbons et al. (16) observed Z. mobilis to produce levan from sucrose but not from glucose or fructose alone. The energy required for the
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polymer synthesis is taken from hydrolysis of disaccharide glycosidic bond (17). The polymer that has been prepared from disaccharides not containing fructose was most likely not levan, but other acetone-precipitating microbial polysaccharides. Hence, sucrose is considered as sole carbon source of levan. The objective of the present study was to characterize polysaccharide (levan) produced by P. fluorescens NCIM 2059 and to investigate the nutritional and environmental requirements for levan production. The levan produced was purified using GPC and characterized by FTIR, 13C and 1 H NMR. The medium optimization was performed using one-factor-at-a-time (except carbon source) methodology followed by statistical designs (Plackett-Burman and RSM).
Materials and Methods Media components Glucose, beef extract, bacteriologicalpeptone (bacto-peptone), casein peptone, corn steep liquor, agar, ammonium chloride (NH4Cl), sodium nitrate (NaNO3), potassium nitrate (KNO3), potassium hydroxide (KOH), potassium di-hydrogen phosphate (KH2PO4), di-potassium hydrogen phosphate (K2HPO4), magnesium sulphate (MgSO4), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), ammonium sulphate ([(NH4)2SO4]) and ammonium dihydrogen phosphate ([(NH4)2PO4]), were procured from Hi-media Laboratories Pvt., Ltd. (Mumbai, India). Sucrose, NaCl, yeast extract, phenol, HCl, H2SO4, and ethanol were procured from s. d. Fine Chemicals Pvt., Ltd. (Mumbai, India). The standard Zymomonas mobilis derived levan used in this study was procured from Sigma-Aldrich (Mumbai, India). Microorganism and its maintenance The microbial strain Pseudomonas fluorescens NCIM 2059 was procured from National Centre for Industrial Microorganism, NCL (Pune, India). The bacterial strain was maintained on slant containing sterile defined medium (%, w/v) beef extract 1.0, bacto-peptone 1.0, NaCl 0.5, and agar 2.0 in distilled water. Sterilization of maintenance medium was carried out at 121oC in autoclave for 20 min. The strain was subcultured and grown at 28±2oC for 48 h. Sub culturing was done after every 2 weeks. The fully-grown culture was maintained at 4oC. The medium reported by Muro et al. (18) with slight modification was used for the production of polysaccharide. The medium composition used in this study was (g/L) sucrose 40.0, bacto-peptone 10.0, (NH4)2SO4 1.0, KH2PO4 1.0, and MgSO4·7H2O 1.0 in distilled water. The medium was sterilized for 15 min at 121oC.
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Inoculum preparation and fermentative production The inoculum of P. fluorescens NCIM 2059 was prepared by harvesting 2 of loopful organisms and mixing it in to 30 mL of sterile production medium taken in 100-mL capacity Erlenmeyer flask. The inoculated medium was kept for 24 h at 28±2oC under static condition. Fermentative production was carried out in 250-mL Erlenmeyer flasks with working volume of 50 mL. The production medium was inoculated with 3%(v/v) of 24 h old P. fluorescens NCIM 2059 seed culture. The inoculated flasks were kept under static condition at 28±2oC. For aerobic culture all conditions were similar except flaks were incubated in orbital shaking incubator (Remi Laboratory Instruments, Mumbai, India) at 28±2oC with 180 rpm speed. Characterization of polysaccharide produced FTIR spectrometric analysis: Levan obtained from P. fluorescens culture with above mentioned medium and was purified by GPC and was monitored using TLC (data not shown). The fractions containing levan was pulled together and freeze-dried to use for further analysis. FTIR analysis was conducted for the determination of the functional group deposition of the isolated levan (purified and unpurified) polymer originated from P. fluorescens and was compared with purified Z. mobilis standard levan sample. FTIR spectra in transmittance mode were obtained on an FTIR spectrophotometer (Shimadzu, Kyoto, Japan). Sixty-four scans were taken for each sample from 4,000 to 600/cm at a resolution of 4/cm. 1 H and 13C NMR spectrums: Thirty mg of the both purified and standard levan was dissolved in D2O (0.5 mL). The 1H NMR and 13C NMR spectra were recorded with 5-mm tubes at room temperature using NMR spectrophotometer (JNM-ECS300; Jeol, Tokyo, Japan) operating at 300 MHz. Optimization of fermentation media using one-factorat-a-time method In order to compare levan production profile of P. fluorescens culture under agitating and static culture condition, the agitated culture was incubated by shaking with 180 rpm at 28 ± 2oC and static culture was supplemented with 0.3% NaNO3 in basic medium. The production profile was monitored by analyzing dry biomass and levan production after every 12 h. The static culture condition was further investigated by supplementing NaNO3 in the range of 1-5 g/L. The influence of organic nitrogen source was studied by replacing bacteriological peptone of basal medium with yeast extract, beef extract, proteose peptone, casein peptone, and corn steep liquor at 10 g/L concentration. The effect of different inorganic nitrogen sources on levan production was studied by replacing ammonium sulphate of basal medium with KNO3,
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Characterization and Production of Microbial Levan
NH4Cl, NaNO3, and NH4NO3 at 1 g/L concentration. Effect of pH on levan production was studied by changing the initial pH of the fermentation medium in the range of 5-8. The pH at which there was maximum production of levan was continued for further determinations. Screening of most significant fermentation parameters using Plackett-Burman design The Plackett-Burman experimental design (19) is a valuable tool for the rapid evaluation of the effects of various medium components. Since this design is a preliminary optimization technique, which tests only 2 levels of each medium component, it cannot provide the optimal quantity of each component required in the medium. This technique was previously used significantly for optimization of CoQ10 production (20). Screening of most significant fermentation parameters affecting levan production was studied by Plackett-Burman design. A total of 7 (n) variables including 6 nutritional (sucrose, casein peptone, NH4Cl, KH2PO4, MgSO4, and NaNO3) and 1 dummy or unassigned variable were studied in 8 (n+1) experiments. Each variable was represented at 2 levels, high and low, denoted by (+) and (-) signs, respectively. The difference between the 2 values was taken large enough to ensure that the peak area for highest levan production is included. The number of positive and negative signs per experiment or trial are (n+1)/2 and (n− 1)/2, respectively, with each column having equal number of positive and negative signs. The effect of each variable or factor is the difference between the average of the measurements made at the high level of that factor and the average of the measurements made at the low level of that factor, which was determined by the following Eq. 1: 2(ΣPi+ – ΣPi-) EXi = -------------------------------N
(1)
where, E(Xi) is the concentration effect of tested variable, and Pi+ and Pi− represent levan production from the trials where the variables (Xi) being measured and were added to the production medium at high and low concentration, respectively, and N is the number of experiments carried out. In addition to the variables of real interest, the PlackettBurman design considers insignificant dummy variables. The dummy variables, which are not assigned any values, introduce some redundancy required by the statistical procedure. Incorporation of the dummy variables into an experiment allows an estimation of the variance (experimental error) of an effect. Concentration optimization of selected medium components by central composite design (CCD) To find out the optimum concentration of the variables
(sucrose, NH4Cl, NaNO3, and casein peptone) identified as most significant factors for levan production by PlackettBurman design were taken for CCD. Each variable in the design was studied at 5 different levels, 5 replicates at the centre point, with a total number of 21 experiments. The optimal values of the independent variables that gave theoretical maximum response in Eq. 2 were obtained by maximizing the equation within a definite boundary condition. Levan production was taken as response (Y) and a multiple regression analysis of the data was carried out for obtaining an empirical model that relates the response measured to the independent variables. The relationship of the independent variables and the response was calculated by the second order polynomial equation (Eq. 2). k
k
2
k
Y = β0 + Σi = 1βiXi + Σi = 1βiiXi + Σi = 1Σj = i + 1βijXi
(2)
where, Y is the predicted response; β0 a constant; βi the linear coefficient; βii the squared coefficient; and βij the cross-product coefficient, k is number of factors and Xi is independent variable. The second order polynomial coefficients and the response surface plots were obtained using Design Expert Version 6.0.10 trial version (State Ease, Minneapolis, MN, USA), and the model was validated for the process conditions used in this study. Experimental validation of medium optimum conditions obtained with CCD. Levan production profile, total dry biomass, total reducing sugar, and sugar utilization pattern was studied with optimized media. Analytical determinations Determination of dry biomass: The fermentation broth (50 mL) was centrifuged at 9,100×g for 30 min. The cell pellets were dried in hot air oven at 80oC for 5-6 h. Then the dry weight was calculated gravimetrically (21). Isolation and determination of levan: The supernatant obtained after broth centrifugation was boiled for 5 min to deactivate the extracellular enzymes. pH was adjusted to 10 with 0.1 M KOH and levan was precipitated by 3 volumes (of supernatant) of 75% cold ethanol. One mL of 1% CaCl2 solution was added to 10 mL of supernatant to support the precipitation. Precipitate was centrifuged at 1,400×g for 15 min and washed with 1.5 volumes of 75% ethanol. The precipitate was resuspended and hydrolyzed in 0.1 M HCl for 1 h at 100oC. After making the proper dilutions of hydrolyzed solution the content of levan was analyzed as D-fructose by the dinitrosalicylic acid method (21,22). The obtained quantity of D-fructose was divided by the factor 1.11 to calculate the amount of levan (23). All the experiments were performed in triplicates and the difference in the reading was less than 3%. Determination of reducing sugar concentration: The major reducing sugar in broth is glucose, but small amount
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of fructose is also present. The proper dilution of broth was made and concentration of reducing sugar was calculated using standard curve of glucose. The reducing sugar estimation was performed as follows: 1 mL of 3,5dinitrosalicylic acid (DNSA) was added to each sample and blank prepared with 1 mL water. The tubes were kept on the boiling water bath for 10 min after that reaction mixture was cooled at room temperature for 30 min. Ten mL of distilled water was added in each tube and extinction was read at 540 nm (24). Determination of sugar utilization by P. fluorescens NCIM 2059: In order to estimate sugar concentration, 1 mL of broth was withdrawn after every 12 h. The broth was centrifuged at 9,100×g for 15 min at 4oC. After suitable dilution sample was analyzed by phenol-sulphuric acid method. It was performed as follows, to 0.1 mL of diluted sample, 1 mL of 5%(w/v) phenol solution, and 5 mL of 95% sulphuric acid were added. The reaction mixture was incubated for 10 min and the tubes were mixed properly. This mixture was cooled at 25oC for 30 min and absorbance was measured at 490 nm. The standard curve was plotted using glucose in the concentration range of 10-100 mg/mL (25).
Results and Discussion Characterization of polysaccharide produced by P. fluorescens culture The polysaccharide was produced
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using P. fluorescens culture aerobically by incubating for 6 days. The obtained polysaccharide (unpurified and purified) and standard levan obtained from Sigma-Aldrich was characterized by FTIR. The FTIR spectra (Fig. 1) of levan was referenced according to Barone and Medynets (26) showed: characteristic broad stretching peak of O-H stretching around 3,319.26/cm, weak C-H vibration were observed at around 2,935.48/cm, carbonyl C=O stretching noted at 1,722.31/cm, H-O-H scissors at 1,645.17/cm are of residual water, C-H bending was observed at 1,423.37/ cm, C-H stretch was observed at 1,265.22/cm, C-OH bend at 1,035.70/cm. The absorptions at 1,035.70 and 1,122.48 /cm indicated a pyranose form of sugars (27). The wavelength region 950-1,200/cm is reported to be fingerprint of molecule because it allows the identification of major chemical groups in polysaccharide (28). It is also reported that this region is dominated by ring vibrations overlapped with stretching vibrations of (C-OH) side groups and the (C-O-C) glycosidic band vibration. Further confirmation of levan was performed using 13C NMR and 1H analysis. The 13C NMR spectrums of the purified and standard levan are shown in Fig. 2 shows 6 main resonances for carbon signals at (in ppm) C-1: 60.435, C-2: 104.696, C-3: 76.770, C-4: 75.783, C-5: 80.880, C-6: 63.978 (for P. fluorescens levan) and C-1: 60.767, C-2: 104.641, C-3: 77.683, C-4: 75.754, C-5: 80.783, C-6: 63.957 (for Z. mobilis levan) these assignment were performed according to Han and Clarke (2). The C2 resonance (NP. fluorescens =104.696, NStandard =104.641)
Fig. 1. FTIR spectrum of unpurified and purified levan obtained from P. fluorescens and standard levan.
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Characterization and Production of Microbial Levan
Fig. 2. 13C NMR spectrum of P. fluorescens purified levan and standard levan (x marked peaks were unwanted).
Fig. 3. 1H NMR spectrum of P. fluorescens purified levan and standard levan.
indicates the presence of β-fructofuranose. The comparison of purified P. fluorescens levan with standard levan spectra and with the published 13C chemical shifts of levan produced by Bacillus polymyxa indicated that the P. fluorescens levan had a [→6)-β-D-Fruf-(2→]n structure, a levan. 1H NMR (Fig. 3) further confirmed levan structure showing identical pattern peaks of various resonances (29). After confirmation of levan structure the P. fluorescens culture was subjected for optimization.
Optimization by one-factor-at-a-time The levan synthesizing strain identified as P. fluorescens is known to utilize oxygen and/or nitrate as hydrogen acceptor. Hence, the production profile of levan under agitating and sodium nitrate added static culture condition was studied. The results of this study are shown in Fig. 4. It was observed that the levan production was slightly higher in static culture condition (6.72 g/L after 6 days) than agitating culture condition (5.08 g/L after 6 days). After 6 days of
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incubation no significant change was observed in accumulation of levan. In both culture conditions dry cell weight was increased exponentially up to 3 days of fermentation, thereafter it was increased slowly. After 6 days of fermentation, the dry cell weight was 4.12 and 4.25 g/L with agitating and static fermentation, respectively. Total sugar concentration was decreased up to 13.34±0.47 and 12.12±0.31 g/L with agitating and static culture conditions, respectively after 10 days of incubation. Contrary to total sugar concentration trend, reducing sugar concentration was increased with fermentation time. The studies performed using 1 to 5 g/L NaNO3 revealed 3 g/L to support maximum production (data not shown). Out of different organic nitrogen sources utilized in this study, casein peptone was observed to support the highest levan (7.81 g/L) as well as biomass (4.51 g/L) production (supplementary Fig. 2). These results are in agreement with study performed by Han and Clark (2), where they observed addition of 0.2% peptone in cane juice improved yield of levan from 0.65 to 1.95%. The effect of different inorganic nitrogen sources showed NH4Cl to support maximum (8.63 g/L) levan as compared to other nitrogen sources (data not shown). Contrary to our results, Muro et al. (18) observed (NH4)2SO4 to support levan synthesis during Z. mobilis fermentation. Medium adjusted with different initial pH showed pH of 6.0 to support maximum production (8.91 g/L) of levan as well as biomass (4.60 g/ L) (data not shown). Raymond (30) and Han and Clarke (2) reported maximum levan production between 5.0 to 6.0
pH. This could be due to enzyme levansucrase having optimum activity in pH range of 5.0 to 6.0 which is necessary for levan synthesis. Screening of significant medium components After the outcome of one-factor-at-a-time method different medium components were subjected to determine order of significance by Plackett-Burman design. The importance of optimizing medium components during obtaining maximum production of levan is highlighted in Table 1. Out of 6 different medium components, which were likely to play important role in improving levan production, 4 components (sucrose, NH4Cl, NaNO3, and casein peptone) significantly affected levan production. Further, the dummy variable included in the design did not exhibit any impact on levan production. The values for the effects of sucrose, NH4Cl, casein peptone, and NaNO3 were 4.91, 0.90, 0.54, and 0.32, respectively are shown in Table 1. The mean square (variance of effect) showed higher value for sucrose (48.23). The F-test value for sucrose (0.750), NH4Cl (0.025), casein peptone (0.009), and NaNO3 (0.0003) implied them to be the most significant terms. It was also found that sucrose, NH4Cl, casein peptone, and NaNO3 showed large effects on the production of levan hence selected for response surface optimization. KH2PO4 and MgSO4 showed a very low effect as that of dummy variable hence not considered for further study.
Table 1. Plackett-Burman design and statistical calculations for 7 variables Sucrose (g/L)
Casein peptone (g/L)
NH4Cl (g/L)
KH2PO4 (g/L)
MgSO4 (g/L)
NaNO3 (g/L)
Dummy
Levan (g/L)
(+) 60 (-) 20 (-) 20 (+) 60 (-) 20 (+) 60 (+) 60 (-) 20
(+) 15 (+) 15 (-) 5 (-) 5 (+) 15 (-) 5 (+) 15 (-) 5
(+) 1.5 (+) 1.5 (+) 1.5 (-) 0.5 (-) 0.5 (+) 1.5 (-) 0.5 (-) 0.5
(-) 0.5 (+) 1.5 (+) 1.5 (+) 1.5 (-) 0.5 (-) 0.5 (+) 1.5 (-) 0.5
(+) 1.5 (-) 0.5 (+) 1.5 (+) 1.5 (+) 1.5 (-) 0.5 (-) 0.5 (-) 0.5
(-) 1.0 (+) 5.0 (-) 1.0 (+) 5.0 (+) 5.0 (+) 5.0 (-) 1.0 (-) 1.0
(-) (-) (+) (-) (+) (+) (+) (-)
12.02±0.261) 6.97±0.14 5.91±0.07 10.65±0.050 6.13±0.16 11.2±0.23 10.26±0.080 5.48±0.11
Statistical calculations for levan yield ΣH 44.13 35.38 ΣL 24.49 33.24 Difference 19.64 2.14 Effect 4.91 0.54 Diff. square 385.82 4.59 Mean square 48.23 0.57 F-test 0.7499 0.0089
36.10 32.51 3.59 0.90 12.88 1.61 0.0250
33.79 34.82 -1.03 -0.26 1.06 0.13 0.0021
34.70 33.91 0.79 0.20 0.62 0.08 0.0012
34.95 33.67 1.28 0.32 1.64 0.21 0.0032
33.50 35.12 -1.62 -0.40 2.62 0.33 0.0051
Run no. Design 1 2 3 4 5 6 7 8
1)
Values are mean±SD of 3 individual determinations.
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Characterization and Production of Microbial Levan Table 2. Matrix of independent variables in actual form with their corresponding response Run
A: Sucrose (g/L)
B: NH4Cl (g/L)
C: NaNO3 (g/L)
D: Casein peptone ( g/L)
Levan (g/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
40.00 6.36 73.64 60.00 40.00 40.00 60.00 40.00 20.00 40.00 40.00 40.00 40.00 60.00 20.00 20.00 40.00 40.00 20.00 60.00 40.00
1.00 1.00 1.00 0.50 1.00 1.00 1.50 1.00 0.50 1.84 1.00 0.16 1.00 0.50 0.50 1.50 1.00 1.00 1.50 1.50 1.00
1.32 3.00 3.00 4.00 3.00 3.00 4.00 3.00 4.00 3.00 3.00 3.00 3.00 2.00 2.00 4.00 3.00 3.00 2.00 2.00 4.68
10.00 10.00 10.00 15.00 18.41 10.00 5.00 10.00 5.00 10.00 10.00 10.00 10.00 15.00 5.00 15.00 1.59 10.00 15.00 5.00 10.00
10.28±0.241) 3.68±0.11 16.80±0.690 13.91±0.840 9.98±0.71 8.82±0.16 10.84±0.530 8.27±0.25 5.85±0.13 9.08±0.38 8.98±0.15 6.62±0.24 8.34±0.51 12.05±0.320 5.28±0.73 5.01±0.08 11.49±0.130 8.52±0.92 9.37±0.68 13.25±0.190 6.49±0.35
1)
Values are mean±SD of 3individual determinations.
Table 3. Analysis of variance for the experimental results of the central composite design (quadratic model) Factor1)
Coefficient Estimate
Sum of Squares
Standard Error
DF
F-value
Prob>F p-value
Model A B C D A2 B2 C2 D2 AB AC AD BC BD CD
14.89 3.90 0.73 -0.78 -0.45 0.55 -0.30 -0.11 0.73 -1.09 0.41 0.56 -1.15 0.84 -0.080
203.48 86.15 3.02 8.36 1.15 4.50 1.30 0.17 7.87 3.93 1.32 1.03 10.53 2.32 0.051
0.62 0.21 0.21 0.14 0.21 0.13 0.13 0.13 0.13 0.28 0.18 0.28 0.18 0.28 0.18
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1
56.25 333.39 11.70 32.42 4.43 17.42 5.04 0.65 30.45 15.19 5.12 3.97 40.77 8.96 0.20
F’ for the term casein peptone is 0.0799 which is
insignificant term. The effect of cultivation time on levan production with optimized medium (all components in g/L, sucrose 60; NH4Cl 1.5; NaNO3 2.0; and casein peptone 15.0) shown in Fig. 5. The maximum yield (15.42 g/L) was found after 6 days. In broth increased trend of reducing sugar concentration was observed up to 84 h and thereafter it was decreased. This might be due to the hydrolysis reaction catalyzed by levansucrase resulted in formation glucose and fructose (1) in initial stage of fermentation. Overall, the polysaccharide produced by P. fluorescens culture was confirmed to be levan with help of FTIR and NMR studies. Static culture of P. fluorescens in presence of NaNO3 synthesizes significant amount of levan. Utilization of statistical design had considerably increased production of
Characterization and Production of Microbial Levan
levan and significantly reduced the number of experiments required as compared to classical approach. Acknowledgments We are grateful to the Department of Biotechnology, Ministry of Science and Technology, India, for providing financial assistance during the course of this research.
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