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Enhanced production of scleroglucan by Sclerotium rolfsii MTCC 2156 by use of metabolic precursors
Bioresource Technology 98 (2007) 410–415
Enhanced production of scleroglucan by Sclerotium rolfsii MTCC 2156 by use of metabolic precursors Shrikant A. Survase, Parag S. Saudagar, Rekha S. Singhal
*
Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Received 17 October 2005; received in revised form 5 December 2005; accepted 7 December 2005 Available online 27 June 2006
Abstract The aim of this work was to study the effect of addition of different amino acids and sugar nucleotides as metabolic precursors on the production of scleroglucan. A maximum yield of 20.00 g/l and 22.32 g/l was obtained with optimized media supplemented with L-lysine (1.1 mM) and uridine mono-phosphate (UMP), respectively as compared to 16.52 g/l scleroglucan achieved with the control in the absence of metabolic precursors. 2006 Elsevier Ltd. All rights reserved. Keywords: Scleroglucan; Sclerotium rolfsii; Precursors; Sugar nucleotides; Amino acids; Molecular weight
1. Introduction Scleroglucan, a non-ionic, water-soluble homopolysaccharide, is produced by fermentation from filamentous fungi Sclerotium rolfsii ATCC 201126. It consists of a linear chain of b-D-(1-3)-glucopyranosyl groups and b-D-(1-6)-glucopyranosyl groups (Farina et al., 1998). It has remarkable rheological properties and stability over a wide range of pH, salinities, and temperature (Wang and McNeil, 1996). This polysaccharide has potential applications in the food and pharmaceutical industry (Halleck, 1967; McNeil and Harvey, 1993). It also finds applications in chemically enhanced oil recovery and in cosmetics. The development of proper fermentation media is a requirement to obtain high yields of desired product at set specification (in terms of purity, solubility, gel strength, etc.). These goals can be achieved by optimizing media composition, fermentation conditions and fermentor design, as well by developing superior strains by mutation (Margaritis and Pace, 1985). * Corresponding author. Tel.: +91 022 24145616; fax: +91 022 24145614. E-mail addresses: [email protected], [email protected] (R.S. Singhal).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.12.013
There is very little information available on the biosynthesis of scleroglucan by Sclerotium glucanicum and Sclerotium rolfsii, but generally this should resemble the biosynthetic steps encountered in the production of other glucans (Rodgers, 1973). First glucose is transferred into the cells via a hexokinase, and is then phosphorylated by the action of phosphoglucomutase (PGM) and phosphoglucoisomerase (PGI). A pyrophosphorylase (UGP) catalyzes the formation of uridine diphosphate glucose (UDP-Glucose) which reacts with lipid carrier and begins to polymerize. Addition of precursor molecules is of considerable importance in the polysaccharide synthesis in terms of metabolic driving force. For the synthesis of most of the polysaccharides, sugar nucleotides serve as the glycosyl donors. In case of polysaccharides, higher intracellular levels of nucleotide phosphate sugars under nitrogen-limitation reportedly enhance metabolite flux for exopolysaccharide synthesis. Higher intracellular levels of UMP under nitrogen-limited conditions enhance metabolite flux of curdlan synthesis in Agrobacterium species (Kim et al., 1999). Gellan precursors were detected by enzyme assays, and they were found to be nucleotide phosphate sugars (Giavasis et al., 2000).
S.A. Survase et al. / Bioresource Technology 98 (2007) 410–415
411
phate 0.13, citric acid 0.07, potassium chloride 0.05 and ferrous sulphate 0.005, pH 4.5 ± 0.2 (Survase et al., 2006).
Nampoothiri et al. (2003) have used amino acids as nitrogen source or as stimulator for improving biopolymer yields such as for gellan gum, although there is inadequate information available on the use of amino acids as precursor for scleroglucan production. L-Threonine was used in the optimized medium, although it did not show an increased yield (Farina et al., 1998). To the best of our knowledge, there are no reports documenting the effect of sugar nucleotides on the production of scleroglucan, whereas information on use of amino acids as metabolic precursors for scleroglucan production is scant. The effect of addition of various nucleotides and amino acids, alone and in combination on scleroglucan and biomass production was undertaken. The time of addition and concentration of precursors and the effect of precursors on the molecular weight of scleroglucan were also evaluated.
Scleroglucan was isolated from the broth by the procedure described by Farina et al. (2001). Fermented broths were neutralized with NaOH or HCl as required, diluted 3–4-fold with distilled water, heated at 80 C for 30 min, homogenized and then centrifuged (10,000g, 30 min). The pellet so obtained was washed with distilled water and dried at 105 C. The supernatant was used for estimation of scleroglucan production. Two volumes of 96% (v/v) ethanol was added to precipitate the scleroglucan from clear supernatant. The mixture was allowed to stand for 8 h at 4 C for complete precipitation. Scleroglucan was recovered by filtration under vacuum and dried at 105 C.
2. Methods
2.5. Growth profile for Sclerotium rolfsii MTCC 2156
2.1. Chemicals
The biomass was isolated by the procedure as described above, dried at 105 C and reported as dry cell weight (DCW). The precipitated scleroglucan was recovered and measured after drying at 105 C.
Medium components such as sucrose, magnesium sulphate, ferrous sulphate, ammonium sulphate, yeast extract, L-valine, L-tryptophan, L-proline, L-threonine, L-glutamic acid, L-lysine, L-arginine and L-aspartic acid were purchased from Hi-Media Limited, Mumbai, India. Dipotassium hydrogen phosphate, sodium nitrate, guanosine-5 0 -monophosphate (GMP), uridine-5 0 -diphosphate (UDP), uridine-5 0 -monophosphate (UMP), adenosine-5 0 diphosphate (ADP), cytidine-5 0 -monophosphate (CMP), nicotinamide adenine dinucleotide (NADH) and adenosine-5 0 -triphosphate (ATP) were purchased from S.D. Fine Chemicals Limited, Mumbai. 2.2. Maintenance of culture and seed culture preparation A strain of Sclerotium rolfsii MTCC 2156 was used in this study. The culture was grown on potato dextrose agar at 28 C for five days. A 3 ml cell suspension prepared from plates was used to inoculate 50 ml of sterile seed culture medium in 250 ml conical flasks, which was incubated at 28 C, 180 rpm for two days on rotary shaker. 2.3. Fermentative production of scleroglucan Scleroglucan production was carried out in two stages. In the first stage, cells were grown in the seed culture; and in the second, seed culture was inoculated into the fermentation medium for scleroglucan production. The seed culture was homogenized and 5 ml of the same was used to inoculate into 50 ml of the fermentation medium and the flasks were incubated every 24 h at room temperature for 84 h on a rotary shaker at 180 rpm. The medium for scleroglucan production by S. rolfsii MTCC 2156 contained (%) sucrose 8, sodium nitrate 0.3, yeast extract 0.1, magnesium sulphate 0.025, di-potassium hydrogen phos-
2.4. Isolation of scleroglucan from the fermentation broth
2.6. Effect of nucleotide phosphate sugars on scleroglucan production To study the effect of addition of different precursors on scleroglucan and biomass production, GMP, UDPG, UMP, ADP, CMP, NADH and ATP were added individually in media at concentrations of 0.1, 0.5 and 1.0 mM. 2.6.1. Effect of varying concentrations of UDPG and UMP concentration Effect of varying concentration of nucleotide phosphate sugars on scleroglucan production was studied by supplementing the production medium with 0.4 mM, 0.5 mM, 0.6 mM of UDPG and UMP. The effect of addition of these nucleotides at various stages of fermentation viz. at 0 h, 48 h and 60 h and their effect on scleroglucan and biomass production were studied. 2.7. Effect of addition of amino acids on scleroglucan and biomass production To study the effect of addition of amino acids on scleroglucan production, L-valine, L-tryptophan, L-proline, L- threonine, L-glutamic acid, L-lysine, L-arginine and Laspartic acid were added individually at 1 mM and 10 mM in the optimized media. 2.7.1. Effect of L-lysine concentration Different concentrations of L-lysine (0.5, 0.8, 1.1 and 1.4 mM) were added to the optimized medium and its effect on scleroglucan production and biomass production was studied.
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2.8. Effect of UMP and L-lysine addition in combination on scleroglucan and biomass production The effect of L-lysine and UMP in combination on scleroglucan production and biomass yield was studied by supplementing the fermentation medium with L-lysine (1.1 mM) and UMP (0.4 mM) after 48 h of fermentation. 2.9. Effect of UMP and L-lysine addition on molecular weight of scleroglucan
used for the estimation of residual sucrose. A suitably diluted 0.1 ml aliquot of each sample was analyzed by phenol sulphuric acid method 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 tubes were mixed by shaking after 10 min, and cooled at 25 C for 30 min. The extinction was read at 490 nm. The standard curve was plotted using glucose in the concentration range of 10– 100 lg/ml (Dubois et al., 1956). 3. Results and discussion
Molecular weight of the scleroglucan sample isolated after 72 h of fermentation was determined by using intrinsic viscosity data and Mark-Houwink equation (Moresi et al., 2001). The effect of addition of UMP and L-lysine on molecular weight after 72 h fermentation was studied. The molecular weight was calculated using Mark-Houwink equation given as follows ½g ¼ KsM aw where, [g] is intrinsic viscosity; Mw is average molecular weight and Ks is the Mark-Houwink relation constant traditionally deduced from measurements of Mw and ‘a’ the Mark-Houwink exponent, relates the power law dependence of molecular weight of the intrinsic viscosity. For scleroglucan, Ks is 3.93 · 104 and a = 1.1 were used (Noik and Lecourtier, 1993; Kulicke et al., 1997). Intrinsic viscosity [g] was calculated as intercept of the plot of concentration of scleroglucan vs. specific viscosity Fig. 6. Specific viscosity (gsp) was calculated as gsp ¼
ðgsolution gsolvent Þ gsolvent
2.10. Analytical determinations 2.10.1. Estimation of biomass Fermented broths (10 ml) were neutralized with NaOH or HCl as required, diluted 3–4-fold with distilled water, heated at 80 C for 30 min, homogenized and then centrifuged (10,000g, 30 min). The pellet so obtained was washed with distilled water and dried at 105 C. The supernatant was used for estimation of scleroglucan production.
UDP and UMP are reported to be precursors for synthesis of various microbial polysaccharides. There is no such report with respect to scleroglucan production. Hence the effect of various nucleotides on scleroglucan production was studied. Fig. 1 documents the effect of different precursors on scleroglucan production. UMP and UDPG at 0.5 mM increased the yield from 16.5 g/l to a maximum of 20.36 g/l and 20.78 g/l, respectively. Other precursors did not increase the yields significantly, although they did increase the biomass production suggesting their probable use as nitrogen source for growth. UDPG and UMP at different concentrations were added at various stages of fermentation. The results are shown in Table 1. UDPG (0.5 mM) and UMP (0.4 mM) added after 48 h of fermentation gave maximum scleroglucan yield of 21.12 g/l and 22.08 g/l, respectively. UMP (0.4 mM) added after 48 h increased scleroglucan production significantly. Fig. 2 documents the batch study for scleroglucan production after addition of UMP. Phillips and Lawford (1983) showed UMP to be converted to UDP, which then acts as a precursor for polysaccharide biosynthesis. Monomer (glucose) is activated via attachment of a nucleotide diphosphate (UDP). This sugar nucleotide (UDP glucose) is a potential precursor of either scleroglucan, or the other closely related cell wall glucans. This may provide the mechanistic basis for the observed results of increased yield of scleroglucan.
45
25
40 35 30 15
25 20
10
15
DCW (g/l)
2.10.2. Estimation of scleroglucan production Two volumes of 96% (v/v) ethanol were added to precipitate the scleroglucan from clear supernatant. The mixture was allowed to stand for 8 h at 4 C for complete precipitation. Scleroglucan was recovered by filtration under vacuum and dried at 105 C.
Scleroglucan (g/l)
20
10
5
5
2.10.3. Sugar utilization during fermentation by S. rolfsii MTCC 2156 For this, 1 ml of broth was taken after every 12 h during the course of 72 h fermentation, centrifuged at 10,000g for 15 min at 4 C. After removing the scleroglucan by ethanol precipitation from the cell-free broth, the supernatant was
0
0 UDPG
UMP
ADP
0.1mM
0.5mM
1.0mM
GMP
CMP
0.1mM DCW
NADH 0.5mM DCW
ATP 1.0mM DCW
Fig. 1. Effect of nucleotide precursors on scleroglucan and biomass production by Sclerotium rolfsii MTCC 2156.
S.A. Survase et al. / Bioresource Technology 98 (2007) 410–415
413
Table 1 Effect of UMP and UDPG concentration and time of addition on the scleroglucan and biomass by Sclerotium rolfsii MTCC 2156 Time of addition (h)
Concentration (mM)
Scleroglucan yielda (g/l)
DCWa (g/l)
UMP
UDPG
UMP
UDPG
UMP
UDPG
0
0.33 0.40 0.55 0.66
– 0.4 0.5 0.6
17.09 ± 0.58 21.50 ± 0.24 19.59 ± 0.64 19.29 ± 0.54
– 18.37 ± 0.26 20.48 ± 0.15 20.80 ± 0.51
30.01 ± 1.39 29.80 ± 2.65 28.21 ± 2.47 28.12 ± 2.84
– 37.25 ± 1.23 38.20 ± 1.57 37.66 ± 0.87
48
0.33 0.40 0.55 0.66
– 0.4 0.5 0.6
19.78 ± 0.66 22.08 ± 0.98 21.77 ± 0.87 21.02 ± 0.22
– 17.70 ± 0.45 21.12 ± 0.32 20.54 ± 0.30
35.83 ± 1.20 31.50 ± 1.65 25.61 ± 2.98 25.31 ± 1.52
– 32.25 ± 0.68 31.43 ± 0.97 30.14 ± 1.48
60
0.33 0.40 0.55 0.66
– 0.4 0.5 0.6
18.36 ± 0.47 19.62 ± 0.57 17.84 ± 0.62 17.21 ± 0.42
– 16.33 ± 0.42 21.07 ± 0.24 19.67 ± 0.46
33.00 ± 0.95 31.73 ± 0.87 27.72 ± 1.87 26.42 ± 2.69
– 25.65 ± 1.65 27.05 ± 2.10 26.78 ± 0.56
Results are mean ± SD of three determinations.
90
35
80 70
30
60
25
50 20
40
15
30
10
20
5
10
0
0 0
12
24
36 48 Time (h)
DCW (g/l) pH (-)
60
72
40
Scleroglucan (g/l); DCW (g/l)
40
Residual sucrose (g/l)
Scleroglucan (g/l); DCW (g/l); pH (-)
a
35 30 25 20 15 10
0
84
0.5
Scleroglucan (g/l)
30
15 10
5
5 0 ha n LLy si Lne As pa rti c ac id LPr ol in e LAr gi ni ne
id
op
ic m ta
LG lu 1mM
LTr yp t
ac
in Va l L-
on
in
e
e
0
10mM
1mM DCW
10mM DCW
Fig. 3. Effect of amino acids on scleroglucan production and biomass by Sclerotium rolfsii MTCC 2156.
40
80
35
70
30
60
25
50
20
40
15
30
10
20
5
10
0
Residual sucrose (g/l)
20 10
DCW (g/l)
25
15
Scleroglucan (g/l); DCW (g/l); pH (-)
20
re
DCW (g/l)
Fig. 4. Effect of concentration of L-lysine on the scleroglucan production and biomass by Sclerotium rolfsii MTCC 2156.
35
Scleroglucan (g/l)
1.4
40
25
Th
0.8 1.1 L-Lysine concentration (mM)
Scleroglucan (g/l) Residual sucrose (g/l)
Fig. 2. Production profile of scleroglucan on the optimized media containing UMP (0.4 mM) added after 48 h of fermentation.
L-
5
0 0
12
24
36
48
60
72
84
Time (h) DCW (g/l) pH (-)
Scleroglucan (g/l) Residual sucrose (g/l)
Fig. 5. Production profile for scleroglucan with optimized media supplemented with L-lysine (1.1 mM).
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Table 2 Effect of metabolic precursors on the carbon balancea for batch cultivation of scleroglucan by S. rolfsii MTCC 2156 Parameters
Values at 72 h of fermentation (g/l) Pure carbon (g/l) Carbon portion (%)
Sucrose consumption
Scleroglucan
CO2 + oxalic acid
DCW
A
B
C
A
B
C
A
B
C
A
B
C
59.0 24.7 100
59.0 24.7 100
48.0 20.5 100
19.3 8.4 33.8
21.8 9.5 39.4
16.5 7.0 34.0
31.0 12.3 49.6
30.0 11.9 49.1
26.0 10.3 50.4
– – 16.4
12.6
15.5
A: With L-lysine addition. B: With UMP addition. C: Without adding any precursors (Survase et al., 2006). a Results based on carbon in scleroglucan, sucrose and biomass as 44%, 42%, and 39.7%.
40
y
Opti
35
= 170.64x + 13.548
y
UMP
Specific viscosity (-)
Fig. 3 documents the effect of addition of different amino acids at different concentrations. Only L-lysine was promising, addition of which gave scleroglucan production of 19.88 g/l and biomass of 32.15 g/l at 1 mM concentration. The concentration of L-lysine was further optimized to 1.1 mM, which yielded 20.02 g/l scleroglucan and 32.80 g/l biomass (Fig. 4). It is known that sucrose is catabolized through two pathways. Different amino acids are biosynthesized from end products or intermediates of glycolysis pathway and citric acid cycle. Glucose-6-phosphate, the intermediate of glycolysis pathway gets converted to glucose-1-phosphate and then to UDP glucose. Addition of amino acids in fermentation media may have increased the carbon flux towards scleroglucan synthesis. There were earlier reports (Farina et al., 1998) where L-threonine was used in the production media for scleroglucan production with no increase in the yields. Nampoothiri et al. (2003) reported increase in yield for gellan gum with addition of amino acids. Fig. 5 documents the results for production profile for scleroglucan with optimized media supplemented with L-lysine. The addition of UMP and L-lysine in combination did not increase the yield further. The combination of the two gave the maximum yield of 21.20 g/l of scleroglucan. The carbon balance after addition of L-lysine and UMP separately was compared with that of the system without any added precursors. The results are shown in Table 2. It can be seen that incorporation of metabolic precursors enhance the utilization of sucrose to scleroglucan. The conversion of sucrose to carbon dioxide and oxalic acid is in the range of 12.6–16.4%, which is lower than 25% reported by Schilling et al. (2000). This could be due to differences in the culture strains, and also to the fact that Schilling et al. (2000) used glucose as the substrate. In this study, sucrose rather than glucose was used as the carbon source. The molecular weight of the polymer obtained after 72 h of fermentation was determined using intrinsic viscosity data by using Mark-Houwink equation. The intrinsic viscosity was extrapolated from the plot between concentration of polymer and specific viscosity. The Y intercept is the intrinsic viscosity (Fig. 6). The intrinsic viscosity for the scleroglucan samples isolated from media optimized by orthogonal array was 13.54 g/dl, for sample obtained
R2 = 0.99 = 166.28x + 14.209
30
R2 = 0.99 y Lys = 166.88x + 14.314
25
R2 = 0.99
20 15 10 5 0 0
0.025
Optimized Media
0.05 Concentration (%) Media with UMP
0.075
0.1
Media with L-lysine
Fig. 6. Effect of concentration on specific viscosity, of scleroglucan sample from optimized media, media with UMP and media with L-lysine.
from media with UMP was 14.20 g/dl, and with L-lysine was 14.31 g/dl. The intrinsic viscosities were found to be within the range of 0.4–120 g/dl, which is in accordance to the results reported by Norisuye et al. (1980) but the values are much lower than 21.53 g/dl reported by Grassi et al. (1996), and 95.6 g/dl reported by Farina et al. (2001). Thus, the average molecular weight of scleroglucan with optimized medium, with optimized medium containing UMP, and with optimized medium containing L-lysine were 8.76 · 105, 9.15 · 105, 9.22 · 105 Da, respectively. 4. Conclusion Sugar nucleotides such as UMP, UDPG and amino acid such as L-lysine could serve as the metabolic precursors for the scleroglucan production. Addition of precursors improved the yield, but not the molecular weight of scleroglucan significantly. References Dubois, M., Gilles, K.A., Hamilton, J.K., Robers, P.A., Smith, F., 1956. Colorimetric methods for determination of sugars and related substances. Anal. Chem. 28, 350–356. Farina, J.I., Sineriz, F., Molina, O.E., Peratti, N.I., 1998. High scleroglucan production by Sclerotium rolfsii: influence of media composition. Biotech. Letts. 20 (9), 825–831.
S.A. Survase et al. / Bioresource Technology 98 (2007) 410–415 Farina, J.I., Sineriz, F., Molina, O.E., Perotti, N.I., 2001. Isolation and physicochemical characterization of soluble scleroglucan from Sclerotium rolfsii, Rheological properties, molecular weight and conformational characteristics. Carbohydr. Polym. 44, 41–50. Giavasis, I., Harvey, L.M., McNeil, B., 2000. Gellan Gum. Crit. Rev. Biotechnol. 20, 177–211. Grassi, M., Lapasin, R., Pricl, S., 1996. A study of rheological behavior of scleroglucan weak gels. Carbohydr. Polym. 29, 169–181. Halleck, F.E., 1967. Polysaccharides and production thereof, US patent 3,301,848. Kim, M.K., Lee, I.Y., Ko, J.H., Rhee, Y.H., Park, Y.H., 1999. Higher intracellular levels of uridine monophosphate under nitrogen-limited conditions enhance the metabolic flux of curdlan synthesis in Agrobacterium species. Biotech. Bioeng. 62, 317–323. Kulicke, W.M., Lettau, A.I., Thielking, H., 1997. Correlation between immunological activity, molecular mass, and molecular structure of different b-D-glucans. Carbohydr. Res. 297, 135–143. Margaritis, A., Pace, G.W., 1985. Microbial Polysaccharides. In: MooYoung, M., Robinson, C.W. (Eds.), Advances in Biotechnology, vol. 2, pp. 1005–1044. McNeil, B., Harvey, L.M., 1993. Viscous fermentation products. Crit. Rev. Biotechnol. 13 (4), 275–304. Moresi, M., Presti, S.L., Mancini, M., 2001. Rheology of scleroglucan dispersions. J. Food Eng. 50, 235–245.
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Nampoothiri, K.M., Singhania, R.R., Sabarinath, C., Pandey, A., 2003. Fermentative production of gellan using Sphingomonas paucimobilis. Process Biochem. 38, 1513–1519. Noik, C., Lecourtier, J., 1993. Studies on scleroglucan conformation by rheological measurements versus temperature up to 150 C. Polymer. 34, 150–157. Norisuye, T., Yanaki, T., Fugita, H., 1980. A triple helix of Schizophyllum commune polysaccharides in aqueous solutions. J. Polymer Science: Polymer Physics Edition. 18, 547–558. Phillips, K.R., Lawford, H.G., 1983. Curdlan: Its properties and production in batch and continuous fermentations. In: Bushell, D.E. (Ed.), Progress Industrial Microbiology. Elsevier Scientific Publishing Co., Amsterdam, pp. 201–229. Rodgers, N.E., 1973. Scleroglucan. In: Industrial Gums, second ed. Academic Press, New York, pp. 499–511. Schilling, B.M., Henning, A., Rau, U., 2000. Repression of oxalic acid biosynthesis in the unsterile scleroglucan production process with Sclerotium rolfsii ATCC 15205. Biopro. Eng. 22, 51–55. Survase, S.A., Saudagar, P.S., Singhal, R.S., 2006. Production of scleroglucan from Sclerotium rolfsii MTCC 2156. Biores. Tech. 97 (8), 989–993. Wang, Y., McNeil, B., 1996. Scleroglucan. Crit. Rev. Biotechnol. 16 (3), 185–215.
Enhanced production of scleroglucan by Sclerotium rolfsii MTCC 2156 by use of metabolic precursors Shrikant A. Survase, Parag S. Saudagar, Rekha S. Singhal
*
Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Received 17 October 2005; received in revised form 5 December 2005; accepted 7 December 2005 Available online 27 June 2006
Abstract The aim of this work was to study the effect of addition of different amino acids and sugar nucleotides as metabolic precursors on the production of scleroglucan. A maximum yield of 20.00 g/l and 22.32 g/l was obtained with optimized media supplemented with L-lysine (1.1 mM) and uridine mono-phosphate (UMP), respectively as compared to 16.52 g/l scleroglucan achieved with the control in the absence of metabolic precursors. 2006 Elsevier Ltd. All rights reserved. Keywords: Scleroglucan; Sclerotium rolfsii; Precursors; Sugar nucleotides; Amino acids; Molecular weight
1. Introduction Scleroglucan, a non-ionic, water-soluble homopolysaccharide, is produced by fermentation from filamentous fungi Sclerotium rolfsii ATCC 201126. It consists of a linear chain of b-D-(1-3)-glucopyranosyl groups and b-D-(1-6)-glucopyranosyl groups (Farina et al., 1998). It has remarkable rheological properties and stability over a wide range of pH, salinities, and temperature (Wang and McNeil, 1996). This polysaccharide has potential applications in the food and pharmaceutical industry (Halleck, 1967; McNeil and Harvey, 1993). It also finds applications in chemically enhanced oil recovery and in cosmetics. The development of proper fermentation media is a requirement to obtain high yields of desired product at set specification (in terms of purity, solubility, gel strength, etc.). These goals can be achieved by optimizing media composition, fermentation conditions and fermentor design, as well by developing superior strains by mutation (Margaritis and Pace, 1985). * Corresponding author. Tel.: +91 022 24145616; fax: +91 022 24145614. E-mail addresses: [email protected], [email protected] (R.S. Singhal).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.12.013
There is very little information available on the biosynthesis of scleroglucan by Sclerotium glucanicum and Sclerotium rolfsii, but generally this should resemble the biosynthetic steps encountered in the production of other glucans (Rodgers, 1973). First glucose is transferred into the cells via a hexokinase, and is then phosphorylated by the action of phosphoglucomutase (PGM) and phosphoglucoisomerase (PGI). A pyrophosphorylase (UGP) catalyzes the formation of uridine diphosphate glucose (UDP-Glucose) which reacts with lipid carrier and begins to polymerize. Addition of precursor molecules is of considerable importance in the polysaccharide synthesis in terms of metabolic driving force. For the synthesis of most of the polysaccharides, sugar nucleotides serve as the glycosyl donors. In case of polysaccharides, higher intracellular levels of nucleotide phosphate sugars under nitrogen-limitation reportedly enhance metabolite flux for exopolysaccharide synthesis. Higher intracellular levels of UMP under nitrogen-limited conditions enhance metabolite flux of curdlan synthesis in Agrobacterium species (Kim et al., 1999). Gellan precursors were detected by enzyme assays, and they were found to be nucleotide phosphate sugars (Giavasis et al., 2000).
S.A. Survase et al. / Bioresource Technology 98 (2007) 410–415
411
phate 0.13, citric acid 0.07, potassium chloride 0.05 and ferrous sulphate 0.005, pH 4.5 ± 0.2 (Survase et al., 2006).
Nampoothiri et al. (2003) have used amino acids as nitrogen source or as stimulator for improving biopolymer yields such as for gellan gum, although there is inadequate information available on the use of amino acids as precursor for scleroglucan production. L-Threonine was used in the optimized medium, although it did not show an increased yield (Farina et al., 1998). To the best of our knowledge, there are no reports documenting the effect of sugar nucleotides on the production of scleroglucan, whereas information on use of amino acids as metabolic precursors for scleroglucan production is scant. The effect of addition of various nucleotides and amino acids, alone and in combination on scleroglucan and biomass production was undertaken. The time of addition and concentration of precursors and the effect of precursors on the molecular weight of scleroglucan were also evaluated.
Scleroglucan was isolated from the broth by the procedure described by Farina et al. (2001). Fermented broths were neutralized with NaOH or HCl as required, diluted 3–4-fold with distilled water, heated at 80 C for 30 min, homogenized and then centrifuged (10,000g, 30 min). The pellet so obtained was washed with distilled water and dried at 105 C. The supernatant was used for estimation of scleroglucan production. Two volumes of 96% (v/v) ethanol was added to precipitate the scleroglucan from clear supernatant. The mixture was allowed to stand for 8 h at 4 C for complete precipitation. Scleroglucan was recovered by filtration under vacuum and dried at 105 C.
2. Methods
2.5. Growth profile for Sclerotium rolfsii MTCC 2156
2.1. Chemicals
The biomass was isolated by the procedure as described above, dried at 105 C and reported as dry cell weight (DCW). The precipitated scleroglucan was recovered and measured after drying at 105 C.
Medium components such as sucrose, magnesium sulphate, ferrous sulphate, ammonium sulphate, yeast extract, L-valine, L-tryptophan, L-proline, L-threonine, L-glutamic acid, L-lysine, L-arginine and L-aspartic acid were purchased from Hi-Media Limited, Mumbai, India. Dipotassium hydrogen phosphate, sodium nitrate, guanosine-5 0 -monophosphate (GMP), uridine-5 0 -diphosphate (UDP), uridine-5 0 -monophosphate (UMP), adenosine-5 0 diphosphate (ADP), cytidine-5 0 -monophosphate (CMP), nicotinamide adenine dinucleotide (NADH) and adenosine-5 0 -triphosphate (ATP) were purchased from S.D. Fine Chemicals Limited, Mumbai. 2.2. Maintenance of culture and seed culture preparation A strain of Sclerotium rolfsii MTCC 2156 was used in this study. The culture was grown on potato dextrose agar at 28 C for five days. A 3 ml cell suspension prepared from plates was used to inoculate 50 ml of sterile seed culture medium in 250 ml conical flasks, which was incubated at 28 C, 180 rpm for two days on rotary shaker. 2.3. Fermentative production of scleroglucan Scleroglucan production was carried out in two stages. In the first stage, cells were grown in the seed culture; and in the second, seed culture was inoculated into the fermentation medium for scleroglucan production. The seed culture was homogenized and 5 ml of the same was used to inoculate into 50 ml of the fermentation medium and the flasks were incubated every 24 h at room temperature for 84 h on a rotary shaker at 180 rpm. The medium for scleroglucan production by S. rolfsii MTCC 2156 contained (%) sucrose 8, sodium nitrate 0.3, yeast extract 0.1, magnesium sulphate 0.025, di-potassium hydrogen phos-
2.4. Isolation of scleroglucan from the fermentation broth
2.6. Effect of nucleotide phosphate sugars on scleroglucan production To study the effect of addition of different precursors on scleroglucan and biomass production, GMP, UDPG, UMP, ADP, CMP, NADH and ATP were added individually in media at concentrations of 0.1, 0.5 and 1.0 mM. 2.6.1. Effect of varying concentrations of UDPG and UMP concentration Effect of varying concentration of nucleotide phosphate sugars on scleroglucan production was studied by supplementing the production medium with 0.4 mM, 0.5 mM, 0.6 mM of UDPG and UMP. The effect of addition of these nucleotides at various stages of fermentation viz. at 0 h, 48 h and 60 h and their effect on scleroglucan and biomass production were studied. 2.7. Effect of addition of amino acids on scleroglucan and biomass production To study the effect of addition of amino acids on scleroglucan production, L-valine, L-tryptophan, L-proline, L- threonine, L-glutamic acid, L-lysine, L-arginine and Laspartic acid were added individually at 1 mM and 10 mM in the optimized media. 2.7.1. Effect of L-lysine concentration Different concentrations of L-lysine (0.5, 0.8, 1.1 and 1.4 mM) were added to the optimized medium and its effect on scleroglucan production and biomass production was studied.
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2.8. Effect of UMP and L-lysine addition in combination on scleroglucan and biomass production The effect of L-lysine and UMP in combination on scleroglucan production and biomass yield was studied by supplementing the fermentation medium with L-lysine (1.1 mM) and UMP (0.4 mM) after 48 h of fermentation. 2.9. Effect of UMP and L-lysine addition on molecular weight of scleroglucan
used for the estimation of residual sucrose. A suitably diluted 0.1 ml aliquot of each sample was analyzed by phenol sulphuric acid method 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 tubes were mixed by shaking after 10 min, and cooled at 25 C for 30 min. The extinction was read at 490 nm. The standard curve was plotted using glucose in the concentration range of 10– 100 lg/ml (Dubois et al., 1956). 3. Results and discussion
Molecular weight of the scleroglucan sample isolated after 72 h of fermentation was determined by using intrinsic viscosity data and Mark-Houwink equation (Moresi et al., 2001). The effect of addition of UMP and L-lysine on molecular weight after 72 h fermentation was studied. The molecular weight was calculated using Mark-Houwink equation given as follows ½g ¼ KsM aw where, [g] is intrinsic viscosity; Mw is average molecular weight and Ks is the Mark-Houwink relation constant traditionally deduced from measurements of Mw and ‘a’ the Mark-Houwink exponent, relates the power law dependence of molecular weight of the intrinsic viscosity. For scleroglucan, Ks is 3.93 · 104 and a = 1.1 were used (Noik and Lecourtier, 1993; Kulicke et al., 1997). Intrinsic viscosity [g] was calculated as intercept of the plot of concentration of scleroglucan vs. specific viscosity Fig. 6. Specific viscosity (gsp) was calculated as gsp ¼
ðgsolution gsolvent Þ gsolvent
2.10. Analytical determinations 2.10.1. Estimation of biomass Fermented broths (10 ml) were neutralized with NaOH or HCl as required, diluted 3–4-fold with distilled water, heated at 80 C for 30 min, homogenized and then centrifuged (10,000g, 30 min). The pellet so obtained was washed with distilled water and dried at 105 C. The supernatant was used for estimation of scleroglucan production.
UDP and UMP are reported to be precursors for synthesis of various microbial polysaccharides. There is no such report with respect to scleroglucan production. Hence the effect of various nucleotides on scleroglucan production was studied. Fig. 1 documents the effect of different precursors on scleroglucan production. UMP and UDPG at 0.5 mM increased the yield from 16.5 g/l to a maximum of 20.36 g/l and 20.78 g/l, respectively. Other precursors did not increase the yields significantly, although they did increase the biomass production suggesting their probable use as nitrogen source for growth. UDPG and UMP at different concentrations were added at various stages of fermentation. The results are shown in Table 1. UDPG (0.5 mM) and UMP (0.4 mM) added after 48 h of fermentation gave maximum scleroglucan yield of 21.12 g/l and 22.08 g/l, respectively. UMP (0.4 mM) added after 48 h increased scleroglucan production significantly. Fig. 2 documents the batch study for scleroglucan production after addition of UMP. Phillips and Lawford (1983) showed UMP to be converted to UDP, which then acts as a precursor for polysaccharide biosynthesis. Monomer (glucose) is activated via attachment of a nucleotide diphosphate (UDP). This sugar nucleotide (UDP glucose) is a potential precursor of either scleroglucan, or the other closely related cell wall glucans. This may provide the mechanistic basis for the observed results of increased yield of scleroglucan.
45
25
40 35 30 15
25 20
10
15
DCW (g/l)
2.10.2. Estimation of scleroglucan production Two volumes of 96% (v/v) ethanol were added to precipitate the scleroglucan from clear supernatant. The mixture was allowed to stand for 8 h at 4 C for complete precipitation. Scleroglucan was recovered by filtration under vacuum and dried at 105 C.
Scleroglucan (g/l)
20
10
5
5
2.10.3. Sugar utilization during fermentation by S. rolfsii MTCC 2156 For this, 1 ml of broth was taken after every 12 h during the course of 72 h fermentation, centrifuged at 10,000g for 15 min at 4 C. After removing the scleroglucan by ethanol precipitation from the cell-free broth, the supernatant was
0
0 UDPG
UMP
ADP
0.1mM
0.5mM
1.0mM
GMP
CMP
0.1mM DCW
NADH 0.5mM DCW
ATP 1.0mM DCW
Fig. 1. Effect of nucleotide precursors on scleroglucan and biomass production by Sclerotium rolfsii MTCC 2156.
S.A. Survase et al. / Bioresource Technology 98 (2007) 410–415
413
Table 1 Effect of UMP and UDPG concentration and time of addition on the scleroglucan and biomass by Sclerotium rolfsii MTCC 2156 Time of addition (h)
Concentration (mM)
Scleroglucan yielda (g/l)
DCWa (g/l)
UMP
UDPG
UMP
UDPG
UMP
UDPG
0
0.33 0.40 0.55 0.66
– 0.4 0.5 0.6
17.09 ± 0.58 21.50 ± 0.24 19.59 ± 0.64 19.29 ± 0.54
– 18.37 ± 0.26 20.48 ± 0.15 20.80 ± 0.51
30.01 ± 1.39 29.80 ± 2.65 28.21 ± 2.47 28.12 ± 2.84
– 37.25 ± 1.23 38.20 ± 1.57 37.66 ± 0.87
48
0.33 0.40 0.55 0.66
– 0.4 0.5 0.6
19.78 ± 0.66 22.08 ± 0.98 21.77 ± 0.87 21.02 ± 0.22
– 17.70 ± 0.45 21.12 ± 0.32 20.54 ± 0.30
35.83 ± 1.20 31.50 ± 1.65 25.61 ± 2.98 25.31 ± 1.52
– 32.25 ± 0.68 31.43 ± 0.97 30.14 ± 1.48
60
0.33 0.40 0.55 0.66
– 0.4 0.5 0.6
18.36 ± 0.47 19.62 ± 0.57 17.84 ± 0.62 17.21 ± 0.42
– 16.33 ± 0.42 21.07 ± 0.24 19.67 ± 0.46
33.00 ± 0.95 31.73 ± 0.87 27.72 ± 1.87 26.42 ± 2.69
– 25.65 ± 1.65 27.05 ± 2.10 26.78 ± 0.56
Results are mean ± SD of three determinations.
90
35
80 70
30
60
25
50 20
40
15
30
10
20
5
10
0
0 0
12
24
36 48 Time (h)
DCW (g/l) pH (-)
60
72
40
Scleroglucan (g/l); DCW (g/l)
40
Residual sucrose (g/l)
Scleroglucan (g/l); DCW (g/l); pH (-)
a
35 30 25 20 15 10
0
84
0.5
Scleroglucan (g/l)
30
15 10
5
5 0 ha n LLy si Lne As pa rti c ac id LPr ol in e LAr gi ni ne
id
op
ic m ta
LG lu 1mM
LTr yp t
ac
in Va l L-
on
in
e
e
0
10mM
1mM DCW
10mM DCW
Fig. 3. Effect of amino acids on scleroglucan production and biomass by Sclerotium rolfsii MTCC 2156.
40
80
35
70
30
60
25
50
20
40
15
30
10
20
5
10
0
Residual sucrose (g/l)
20 10
DCW (g/l)
25
15
Scleroglucan (g/l); DCW (g/l); pH (-)
20
re
DCW (g/l)
Fig. 4. Effect of concentration of L-lysine on the scleroglucan production and biomass by Sclerotium rolfsii MTCC 2156.
35
Scleroglucan (g/l)
1.4
40
25
Th
0.8 1.1 L-Lysine concentration (mM)
Scleroglucan (g/l) Residual sucrose (g/l)
Fig. 2. Production profile of scleroglucan on the optimized media containing UMP (0.4 mM) added after 48 h of fermentation.
L-
5
0 0
12
24
36
48
60
72
84
Time (h) DCW (g/l) pH (-)
Scleroglucan (g/l) Residual sucrose (g/l)
Fig. 5. Production profile for scleroglucan with optimized media supplemented with L-lysine (1.1 mM).
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Table 2 Effect of metabolic precursors on the carbon balancea for batch cultivation of scleroglucan by S. rolfsii MTCC 2156 Parameters
Values at 72 h of fermentation (g/l) Pure carbon (g/l) Carbon portion (%)
Sucrose consumption
Scleroglucan
CO2 + oxalic acid
DCW
A
B
C
A
B
C
A
B
C
A
B
C
59.0 24.7 100
59.0 24.7 100
48.0 20.5 100
19.3 8.4 33.8
21.8 9.5 39.4
16.5 7.0 34.0
31.0 12.3 49.6
30.0 11.9 49.1
26.0 10.3 50.4
– – 16.4
12.6
15.5
A: With L-lysine addition. B: With UMP addition. C: Without adding any precursors (Survase et al., 2006). a Results based on carbon in scleroglucan, sucrose and biomass as 44%, 42%, and 39.7%.
40
y
Opti
35
= 170.64x + 13.548
y
UMP
Specific viscosity (-)
Fig. 3 documents the effect of addition of different amino acids at different concentrations. Only L-lysine was promising, addition of which gave scleroglucan production of 19.88 g/l and biomass of 32.15 g/l at 1 mM concentration. The concentration of L-lysine was further optimized to 1.1 mM, which yielded 20.02 g/l scleroglucan and 32.80 g/l biomass (Fig. 4). It is known that sucrose is catabolized through two pathways. Different amino acids are biosynthesized from end products or intermediates of glycolysis pathway and citric acid cycle. Glucose-6-phosphate, the intermediate of glycolysis pathway gets converted to glucose-1-phosphate and then to UDP glucose. Addition of amino acids in fermentation media may have increased the carbon flux towards scleroglucan synthesis. There were earlier reports (Farina et al., 1998) where L-threonine was used in the production media for scleroglucan production with no increase in the yields. Nampoothiri et al. (2003) reported increase in yield for gellan gum with addition of amino acids. Fig. 5 documents the results for production profile for scleroglucan with optimized media supplemented with L-lysine. The addition of UMP and L-lysine in combination did not increase the yield further. The combination of the two gave the maximum yield of 21.20 g/l of scleroglucan. The carbon balance after addition of L-lysine and UMP separately was compared with that of the system without any added precursors. The results are shown in Table 2. It can be seen that incorporation of metabolic precursors enhance the utilization of sucrose to scleroglucan. The conversion of sucrose to carbon dioxide and oxalic acid is in the range of 12.6–16.4%, which is lower than 25% reported by Schilling et al. (2000). This could be due to differences in the culture strains, and also to the fact that Schilling et al. (2000) used glucose as the substrate. In this study, sucrose rather than glucose was used as the carbon source. The molecular weight of the polymer obtained after 72 h of fermentation was determined using intrinsic viscosity data by using Mark-Houwink equation. The intrinsic viscosity was extrapolated from the plot between concentration of polymer and specific viscosity. The Y intercept is the intrinsic viscosity (Fig. 6). The intrinsic viscosity for the scleroglucan samples isolated from media optimized by orthogonal array was 13.54 g/dl, for sample obtained
R2 = 0.99 = 166.28x + 14.209
30
R2 = 0.99 y Lys = 166.88x + 14.314
25
R2 = 0.99
20 15 10 5 0 0
0.025
Optimized Media
0.05 Concentration (%) Media with UMP
0.075
0.1
Media with L-lysine
Fig. 6. Effect of concentration on specific viscosity, of scleroglucan sample from optimized media, media with UMP and media with L-lysine.
from media with UMP was 14.20 g/dl, and with L-lysine was 14.31 g/dl. The intrinsic viscosities were found to be within the range of 0.4–120 g/dl, which is in accordance to the results reported by Norisuye et al. (1980) but the values are much lower than 21.53 g/dl reported by Grassi et al. (1996), and 95.6 g/dl reported by Farina et al. (2001). Thus, the average molecular weight of scleroglucan with optimized medium, with optimized medium containing UMP, and with optimized medium containing L-lysine were 8.76 · 105, 9.15 · 105, 9.22 · 105 Da, respectively. 4. Conclusion Sugar nucleotides such as UMP, UDPG and amino acid such as L-lysine could serve as the metabolic precursors for the scleroglucan production. Addition of precursors improved the yield, but not the molecular weight of scleroglucan significantly. References Dubois, M., Gilles, K.A., Hamilton, J.K., Robers, P.A., Smith, F., 1956. Colorimetric methods for determination of sugars and related substances. Anal. Chem. 28, 350–356. Farina, J.I., Sineriz, F., Molina, O.E., Peratti, N.I., 1998. High scleroglucan production by Sclerotium rolfsii: influence of media composition. Biotech. Letts. 20 (9), 825–831.
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