Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus

Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus

Bioresource Technology 98 (2007) 2010–2017 Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptom...

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Bioresource Technology 98 (2007) 2010–2017

Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus Parag S. Saudagar, Rekha S. Singhal

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Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Received 6 May 2006; received in revised form 4 August 2006; accepted 4 August 2006 Available online 2 October 2006

Abstract The present work reports the nutritional requirements and environmental conditions for submerged culture of Streptomyces clavuligerus for clavulanic acid production using orthogonal matrix method (Taguchi L16 design) and also fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. Clavulanic acid production was increased by 18% with the span of feeding glycerol and reached a maximum at 1.30 mg/ml with 120 h glycerol feeding as compared to 1.10 mg/ml in the control. The production also increased with the span of feeding amino acids and reached a maximum of 1.31 and 1.86 mg/ml with feeding arginine and threonine, respectively in 120 h. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls (1.10 and 1.70 mg/ml, respectively).  2006 Elsevier Ltd. All rights reserved. Keywords: Clavulanic acid; Fed-batch fermentation; Streptomyces clavuligerus; Threonine; Arginine

1. Introduction Specific nutritional requirements of microorganisms used in industrial fermentation processes are as complex and varied as the microorganisms in question. Besides the microbial type, the species and strains are very specific as to their requirements for biosynthesis and growth from their environment in a variety of ways. Many investigators have attempted to optimize submerged cultures for antibiotic production from different fungi such as erythromycin from Saccharospora erythraea (McDermott et al., 1993; Bhattactacharjee et al., 2002), cephamycin from Streptomyces clavuligerus (Park et al., 1994), and clavulanic acid from S. clavuligerus (Ives and Bushell, 1997). The productivity of microbial metabolites is closely related to the fermentation process used. Medium optimization by one-factor-at-time method involves changing one variable (nutrients, pH, tempera-

*

Corresponding author. Tel.: +91 22 24145616; fax: +91 22 24145614. E-mail address: [email protected] (R.S. Singhal).

0960-8524/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.08.003

ture, etc.) while fixing the others at a certain arbitrary levels (Xu et al., 2003; Survase et al., 2006). Since most industrial experiments usually involve a significant number of factors, a full factorial design results in a large number of experiments. To reduce the number of experiments to a practical level, only a small set from all the possibilities is selected. Taguchi design involves a special set of general design guidelines for factorial experiments that cover many applications (Xu et al., 2003). Fed-batch fermentation is an approach aimed at efficiently carrying out fermentation for production of biomolecules. Industrial fermentation of most amino acids is accomplished with this method. Fed-batch culture has been used extensively to increase the productivity of microbial processes such as production of jenseniin G (Ekinci and Barefoot, 2006), ergosterol (Shang et al., 2006), poly-bhydroxybutyrate (Patnaik, 2006), and chlorophyll (Rangel-Yagui et al., 2004). Ives and Bushell (1997) and Teodoro et al. (2006) applied this approach to clavulanic acid production in S. clavuligerus. Glycerol is one of the best carbon sources for clavulanic acid fermentation (Baggaley et al., 1997; Ives and Bushell,

P.S. Saudagar, R.S. Singhal / Bioresource Technology 98 (2007) 2010–2017

1997; Elson and Oliver, 1978). In the absence of glycerol, S. clavuligerus does not produce clavulanic acid, but produces cephamycin C, another metabolite of the same organism. Elson and Oliver (1978) used labeled 13C precursor and clearly demonstrated that glycerol fed in the fermentation medium was incorporated into the b-lactam ring of clavulanic acid. Townsend and Ho (1985) suggested L-glycerate to act as an intermediate between glycerol and clavulanic acid. Chen et al. (2002) reported that glycerol at 10–20 g/l increased clavulanic production by S. clavuligerus in shake flask cultures. The biosynthesis of clavulanic acid was prolonged with feeding glycerol and the production increased to 0.27 mg/ml as compared to 0.115 mg/ml without feeding. In fermenter batch culture, degradation of clavulanic acid began after 72 h. With glycerol feeding in fed-batch culture, clavulanic acid production was not only increased to about 0.280 mg/ml, but also remained stable up to 130 h. In fed-batch culture, glycerol feeding rather than ornithine feeding has been demonstrated to be the rate limiting for the clavulanic acid synthesis (Chen et al., 2003). The biosynthesis of clavulanic has been well described (Townsend and Ho, 1985; Valentine et al., 1993 and Stirling and Elson, 1979). It is well documented that pyruvate acts as C3 precursor, whereas arginine acts as the C5 precursor for clavulanic acid production (Ives and Bushell, 1997). Radiolabeled feeding experiments have indicated arginine and ornithine to be efficiently incorporated into clavulanic acid structure (Townsend and Ho, 1985; Romero et al., 1986). Chen et al. (2003) reported no enhancement of clavulanic acid biosynthesis from the arginine feeding, and hence, it was not regarded as a rate-limiting substrate. Romero et al. (1986) reported ornithine to strongly inhibit cephamycin biosynthesis. The objective of the present study was to study the nutritional requirements and environmental conditions for submerged culture of S. clavuligerus for clavulanic acid production using orthogonal matrix method and further

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study fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. 2. Methods 2.1. Media components All the media chemicals were purchased from Hi-Media, Mumbai. HPLC solvents were purchased from SD Fine Chemicals Ltd., Mumbai. 2.2. Microbial culture and maintenance Strain of S. clavuligerus MTCC 1142 was procured from MTCC, Chandigarh, India and was maintained on a defined medium containing (%) 0.4 yeast extract, 1 malt extract, 0.4 glucose and 2 agar with a pH adjusted to 7.2 ± 0.2. The slants grown at 25 C for 4 days were used for inoculation into a seed culture medium (2% glycerol, 1% bacteriological peptone, and 1% malt extract with pH adjusted to 7.0 ± 0.2). For the preliminary studies, 2% of seed culture grown for 48 h in an incubator shaker at 25 C and 200 rpm was used for inoculation into the production medium. 2.3. Fermentation The medium designed by Gouveia et al. (1999) was modified and used in the present study. It contained (g/l) 15 glycerol, 20 sucrose, 22.4 proline, 16.8 glutamic acid, 0.4 calcium chloride, 0.1 ferric chloride, 2 potassium dihydrogen phosphate, 5 sodium chloride, 0.1 manganese chloride, 0.05 zinc chloride and 1 magnesium sulphate with a pH adjusted to 7.0 ± 0.2. L16 orthogonal array (Table 1) was used to optimize the concentrations of the media components. The design for the L16-orthogonal array was

Table 1 L16 orthogonal array for clavulanic acid production by Streptomyces clavuligerus using MINITAB 13.3 S. no.

A

B

C

D

E

A

B

C

D

E

CA, mg/ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

1 2 3 4 2 1 4 3 3 4 1 2 4 3 2 1

1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3

1 2 3 4 4 3 2 1 2 1 4 3 3 4 1 2

0.375 0.375 0.375 0.375 0.750 0.750 0.750 0.750 1.500 1.500 1.500 1.500 2.250 2.250 2.250 2.250

0.500 1.000 2.000 3.000 0.500 1.000 2.000 3.000 0.500 1.000 2.000 3.000 0.500 1.000 2.000 3.000

0.560 1.120 2.240 3.360 1.120 0.560 3.360 2.240 2.240 3.360 0.560 1.120 3.360 2.240 1.120 0.560

0.420 0.840 1.680 2.520 1.680 2.520 0.420 0.840 2.520 1.680 0.840 0.420 0.840 0.420 2.520 1.680

0.05 0.1 0.2 0.3 0.3 0.2 0.1 0.05 0.1 0.05 0.3 0.2 0.2 0.3 0.05 0.1

0.114 0.373 0.317 0.354 0.328 0.530 0.306 0.290 0.328 0.269 0.274 0.218 0.218 0.226 0.463 0.475

Where, A is glycerol, B is sucrose, C is proline, D is glutamic acid and E is K2HPO4.

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developed and analyzed using ‘‘MINITAB 13.30’’ software. All experiments were performed in at least triplicates. 2.4. Effect of process parameters on clavulanic acid production Effect of pH on clavulanic acid production was studied by varying the initial pH of the fermentation medium optimized using L16 orthogonal array in the pH range of 5.0– 8.5. The pH of the fermentation broth was found to change with time of fermentation and was maintained at the desired level by addition of 0.1 N NaOH/0.1 N HCl after every 6 h of fermentation. In order to study the effect of phosphate concentration, K2HPO4 in the fermentation medium was varied in the concentration range of 10– 200 mM. The effect of variation of anaplerotic flux on the C3 (pyruvate) and C5 (arginine) precursor on clavulanic acid production by S. clavuligerus was determined by supplementing the production media with different amino acids (viz. L-arginine, L-ornithine, L-proline, L-valine, L-leucine, L-tryptophan, L-cystiene, L-glutamine and L-threonine), pyruvic acid and a-ketoglutarate at 1–100 mM. 2.5. Fed batch fermentation The effect of glycerol concentration on clavulanic acid production was studied by supplementing the fermentation medium with glycerol in the concentration range of 5–25 g/ l. Similarly arginine and threonine were supplemented in the fermentation medium at an initial concentration of 5–100 mM. To monitor the effect of feeding, experiments were started after 60 h of fermentation. Two milliliter of feed solutions (15 g/l glycerol/0.1 M arginine/1 M threonine) were added every 12 h. Samples were withdrawn after every 12 h and analyzed for clavulanic acid production, glycerol utilization and growth of S. clavuligerus.

Two milliliter of 0.1% rhamnose was then added to each tube in order to remove excess of periodate ions. After mixing, 4 ml of Nash reagent (20 mM ammonium acetate buffer and 20 mg acetyl acetone) was added to the mixture. The yellow colored product formed after keeping the reaction mixture on water bath at 53 C has a strong absorbance at 412 nm. The linear calibration curve of glycerol was obtained in the range of 0–25 lg. 2.8. Quantification of phosphate in the broth To 1 ml of suitably diluted fermentation broth added 1 ml of 6 N H2SO4, 2 ml of distilled water, 1 ml of 2.5% ammonium molybdate and 1 ml of 10% ascorbic acid. The reaction mixture was then incubated at 37 C for 2 h. The blue colored complex thus formed was quantified by measuring the absorbance at 820 nm. The linear calibration curve of phosphate was obtained in the range of 0–25 lg (Chen et al., 1956). 2.9. Growth curve of S. clavuligerus Fermentation broth (1 ml) was withdrawn after every 24 h, centrifuged at 8000g for 20 min at 4 C and the cell pellet so obtained was then washed twice with distilled water and dried to a constant weight at 80 C (DCW). The values for l were calculated using the formula lt ¼ lnðX =X 0 Þ where X – biomass concentration at time t of the logarithmic or exponential growth (mg/ml); X0 – biomass initial concentration at time t = t0; l – specific growth rate at the logarithmic or exponential growth (h1); t – time that corresponds to biomass concentration X (h). 3. Results and discussion

2.6. Quantification of clavulanic acid in the broth

3.1. Evaluation of nutritional effects of media components

Clavulanic acid in the fermentation broth was estimated by HPLC using the procedure as described by Foulstone and Reading (1982). Waters Spherisorb 5l ODS2 column (4.6 mm · 250 mm) was used. The mobile phase was methanol/(50 mM) KH2PO4 (40:60) with a pH value adjusted to pH 3.08 by adding H3PO4. The flow rate was maintained at 0.8 ml/min. UV detection was at 312 nm. Augmentin (intravenous injection containing 100 mg of clavulanic acid and 500 mg of amoxicillin) was used as the standard.

The concentrations for the media components were selected based on the basal media composition that was optimized using one factor-at-a-time (Saudagar and Singhal, unpublished work). Table 2 represents the response table for means (larger is better) and for signal to noise ratio obtained with L16 orthogonal array. The last two rows in the tables document the delta values and ranks for the system. A higher delta value indicates greater effect of that component. ‘Rank’ orders the factors from the greatest effect (based on the delta values) to the least effect on the response characteristic. The order in which the individual components selected in the present study affected the fermentation were glutamic acid > sucrose > K2HPO4 > glycerol > proline suggesting that glutamic acid had a major effect, and proline had least effect on clavulanic acid production by S. clavuligerus. Figs. 1 and 2 represent the main effect plots for the system. A main effect is present when different levels of a factor affect the characteristic differently.

2.7. Quantification of glycerol in the broth Glycerol in the fermentation broth was estimated by a colorimetric procedure as described by Lynch and Yang (2004). The method is based on the acidic periodate oxidation of alditols to produce formaldehyde. To 1 ml of the suitably diluted fermentation broth added 1 ml of 0.015 M sodium metaperiodate in 0.12 M HCl in test tubes.

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2013

Table 2 Response table for means and S/N ratio Level

A

B

C

D

E

Mean

S/N ratio

Mean

S/N ratio

Mean

S/N ratio

Mean

S/N ratio

Mean

S/N ratio

1 2 3 4 Delta

0.289 0.364 0.272 0.346 0.092

11.606 9.058 11.391 9.825 2.548

0.247 0.349 0.340 0.334 0.102

12.864 9.600 9.550 9.867 3.31

0.348 0.345 0.290 0.287 0.061

10.529 9.542 10.832 10.985 1.443

0.216 0.289 0.347 0.419 0.203

13.824 10.948 9.383 7.726 6.10

0.284 0.371 0.321 0.295 0.087

11.930 8.750 10.490 10.720 1.180

Rank

3

2

5

1

4

Fig. 1. Main effect plot for mean.

Fig. 2. Main effect plot for S/N ratio.

MINITAB creates the main effects plot by plotting the characteristic average for each factor level. These averages were the same as those displayed in the response Table 2. In the present study, for each of the 5 variables at 5 levels, one level increased the mean compared to the other level. This difference was a main effect i.e. glycerol, sucrose, proline and K2HPO4 at level 2 and glutamic acid at level 4. These levels also represented the optimal concentrations of the individual components in the medium. Response tables can also be used to predict the optimal levels of each component used in the study. To obtain the optimized levels or composition of each factor, the analysis based on statistical calculations is shown in Table 2. Table 3 documents the final medium for clavulanic acid pro- duction by S. clavuligerus. To confirm these results,

ex- periments were carried out using these nutrient concentrations and it was observed that the mean value obtained was 0.640 mg/ml as compared to 0.580 mg/ml predicted using Minitab for the same composition. The final optimized medium produced 0.640 mg/ml clavulanic acids at the end of 96 h as compared to 0.200 mg/ml before optimization. This implied that the selected conditions were the most suitable in practice. 3.2. Effect of phosphate repression on clavulanic acid production Phosphate in concentrations ranging from 0.3 to 300 mM generally supported extensive cell growth, but a concentration of 10 mM and above suppressed the

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Table 3 Final optimized medium for production of clavulanic acid by Streptomyces clavuligerus

1.8

S. no.

Components

Quantity, g/l

1.6

1 2 3 4 5 6 7 8 9 10 11

Glycerol Sucrose L-Proline L-glutamic acid CaCl2 FeCl3 Æ 6H2O K2HPO4 NaCl MnCl2 Æ 4H2O ZnCl2 MgSO4 Æ 7H2O

15.00 20.00 22.40 16.60 0.40 0.10 2.00 5.00 0.10 0.05 0.50

Clavulanic acid, mg/ml

1.4 1.2 1 0.8 0.6 0.4 0.2

gi ni ne Lpr ol i Lor ne ni th in e Lly sin e Lle L- ucin gl ut am Lin e th re o Lni n try pt e op ha Ln cy ste in e Lva lin e

0

Table 4 Effect of phosphate repression on growth of Steptomyces clavuligerus and clavulanic acid production K2HPO4, mM

Clavulanic acid, mg/ml

DCW, mg/ml

1 10 100 200

0.734 ± 0.035 0.878 ± 0.046 0.779 ± 0.040 0.524 ± 0.062

20.0 ± 2 24.0 ± 2.3 21.5 ± 1.8 20.6 ± 1.1

biosynthesis of many antibiotics (Martin, 1976). Aharonowitz and Demain (1978) reported that clavulanic acid production was reduced by 80% when the phosphate concentration in the fermentation medium was more than 100 mM whereas cephamycin production was reduced by 50% when the phosphate concentration in the fermentation medium was 50 mM. Romero et al. (1984) have shown the same inhibition of production of clavulanic acid by S. clavuligerus. Table 4 documents the effect of phosphate concentration on growth of S. clavuligerus and clavulanic acid production. Evidently a critical phosphate concentration was necessary for clavulanic acid production. Increasing the phosphate concentration from 100 to 200 mM resulted in a decrease in clavulanic acid production from 0.779 to 0.526 mg/ml at the end of 120 h, although no major change in the growth of S. clavuligerus at higher phosphate concentrations was observed. On the contrary, decreasing the phosphate concentration to 10 mM resulted in an increase in clavulanic acid production to 0.878 mg/ml at the end of 96 h. A decrease in yield was observed at 120 h due to de- gradation of the product. The above results were in accordance to those reported by Aharonowitz and Demain (1978), Romero et al. (1984), Lubbe et al. (1985), and Lebrihi et al. (1987). 3.3. Effect of amino acids on production of clavulanic acid Fig. 3 illustrates the effect of supplementation of varying concentrations (1–100 mM) of different amino acids on clavulanic acid production by S. clavuligerus MTCC 1142. It was observed that all the amino acids except

L-

ar

MINITAB predicted yield = 0.580 mg/ml; experimental yield = 0.640 mg/ml.

2

1mM

10 mM

100 mM

Fig. 3. Effect of amino acids on clavulanic acid production.

L-valine

increased the yield of clavulanic acid at optimal concentrations. At higher concentrations, a decrease in antibiotic production was observed, thus explaining the concentration dependent stimulation of clavulanic acid. Effects of amino acids such as arginine, ornithine, proline have been well documented by several authors (Jenson and Paradkar, 1999; Chen et al., 2003; Romero et al., 1986). However this is the first report suggesting stimulatory effect of threonine on clavulanic acid production. In the present study L-arginine (10 mM), L-proline (100 mM), L-threonine (100 mM), L-glutamine (10 mM) and pyruvic acid (100 mM) had a greater stimulatory effect on clavulanic acid production as compared to L-ornithine (100 mM), a-ketoglutarate (100 mM), L-isoleucine (100 mM) and L-valine (100 mM). The possible reasons for the observed results can be explained with respect to the biosynthetic pathway for clavulanic acid production. It is well documented that pyruvate acts as C3 precursor, whereas arginine acts as the C5 precursor for clavulanic acid production (Ives and Bushell, 1997). Radiolabeled feeding experiments have indicated arginine and ornithine to be efficiently incorporated into clavulanic acid structure (Townsend and Ho, 1985; Romero et al., 1986). Addition of L-proline, L-arginine, L-glutamine, and L-threonine decreased the anaplerotic flux on pyruvate (C3 precursor) for the synthesis of these amino acids, thus diverting the pathway to clavulanic acid production. L-Threonine and L-arginine were found to be the most efficient precursor for clavulanic acid production and increased the yield of clavulanic acid from 0.500 lg/ml to 1.700 mg/ml and 1.100 mg/ml (Fig. 3) suggesting maximum flux to flow through synthesis of L-threonine and L-arginine from pyruvate during the normal metabolism in S. clavuligerus. Supplementing the medium with L-valine decreased the yield of

3.4. Effect of varying initial concentration of glycerol, arginine and threonine on clavulanic acid production in shake-flask culture

16

1.4

14

1.2

12 Glycerol, mg/m

clavulanic acid, which may be attributed to a simultaneous increase in the yield of cephamycin (another b-lactam produced by S. clavuligerus) as L-valine is reported to be the precursor for cephamycin production (Shu-Jen and Basch, 1999).

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1

10 0.8 8 0.6 6 0.4

4

0.2

2

Effect of varying initial concentrations of glycerol, arginine and threonine on clavulanic acid production was studied. It was observed that maximum yields were obtained with 15 mg/l of glycerol (1.100 mg/ml), 10 mM arginine (1.100 mg/ml) and 100 mM threonine (1.700 mg/ml), respectively. Further increasing the concentration of glycerol resulted in inhibition of clavulanic acid synthesis. Decreasing the concentration glycerol below 10 mg/ml showed a decrease in clavulanic acid production. Kirk et al. (2000) studied the effect of growth limiting substrates on clavulanic acid production and associated metabolic fluxes in S. clavuligerus. They concluded that glycerol limitation restricts the capability of S. clavuligerus for anaplerotic metabolism on minimizing the potential for extensive tricarboxilic acid cycle derived from biosynthesis and thus restricting the biosynthesis of clavulanic acid. Chen et al. (2002) reported that glycerol at 10–15 mg/ml increased clavulanic production by S. clavuligerus in shake flask cultures, while Romero et al. (1984) reported glycerol above 15 mg/ml to inhibit clavulanic acid biosynthesis. The results observed in the present study are in accordance to these reports. Clavulanic acid concentration reached a maximum of 1.100 mg/ml when 10 mM of arginine was used, beyond which a decrease in clavulanic acid production was observed. On the other hand, supplementing arginine with threonine in production medium showed an remarkable increase in the clavulanic acid from 1.100 mg/ml to 1.700 mg/ml at 100 mM. This suggested the addition of L-threonine to decrease the anaplerotic flux on pyruvate (C3 precursor) as compared to arginine for the synthesis of these amino acids (i.e. threonine and arginine) diverting the pathway to clavulanic acid production. Once the initial limiting concentrations of glycerol, arginine and threonine were known, further studies were undertaken to study the effect of intermittent feeding of these constituents on clavulanic acid production. 3.5. Effect of intermittent feeding of glycerol, arginine and threonine on clavulanic acid production The effect of feeding glycerol on clavulanic acid production by S. clavuligerus in shake flask culture was studied (Fig. 4). In the control culture with no feeding strategy used, glycerol was consumed almost completely after 84 h and clavulanic acid production reached a maximum of 1.100 mg/ml. In the feeding cultures, glycerol feeding was

Clavulanic acid, mg/ml

P.S. Saudagar, R.S. Singhal / Bioresource Technology 98 (2007) 2010–2017

0 0

20

40

60

80

100

120

0 140

Time, h Glycerol (120 h)

Glycerol (96 h)

Glycerol (72 h)

Glycerol (60 h)

Control Glycerol

CA production (120 h)

CA production (96 h)

CA production (72 h)

Control CA production (60 h)

Fig. 4. Effect of feeding glycerol on clavulanic acid production by Streptomyces clavuligerus MTCC 1142. [Glycerol [120 h, 72 h & 60 h] signifies the batch in which glycerol was fed up to 120 h, 72 h & 60 h, respectively. Similarly CA [120 h, 72 h & 60 h] signify the results for clavulanic acid production in the batches with feeding glycerol up to 120 h, 72 h & 60 h, respectively].

performed from 60 h of cultivation when the cells start to enter the stationary phase. Two milliliter of glycerol solution (100 g/l) were added every 12 h. The glycerol concentration was selected to avoid catabolite repressive effect of glycerol. The feeding was stopped at 60 h, 72 h, 96 h and 120 h, respectively. Clavulanic acid production was found to increase with the span of feeding glycerol and was maximum (1.300 mg/ml) in the flask with 120 h glycerol feeding. Glycerol feeding also increased the biomass as compared to the control. There was an 18% increase in overall clavulanic acid production with glycerol feeding as compared to the control. The results observed for clavulanic acid production with glycerol feeding (1.300 mg/ml) are much higher than 0.220 mg/ml (220 lg/ml) as reported by Chen et al. (2003) and 0.380 mg/ml as reported by Teodoro et al. (2006) using fed-batch culture technique. The possible reason for the observed results can be explained with respect to the increased utilization of glycerol with feeding strategies. The rate of clavulanic acid production increased with the increase in the glycerol feeding time and was found to be maximum (0.032 mg/ml h) at 120 h of glycerol feeding as compared to 0.021 mg/ml h observed in control (Table 4). Also it can be seen that value of lmax increased with glycerol feeding and was found to be maximum (0.066 h1) as compared to 0.047 h1 observed in control. The above results also indicate that part of glycerol is being used for both growth of the organism as well as for clavulanic acid production. The effect of feeding arginine and threonine on clavulanic acid production in shake flask culture was also investigated (Figs. 5 and 6). In the control culture without feeding arginine and threonine, clavulanic acid production reached a maximum of 1.100 mg/ml and 1.700 mg/ml,

0.4

4

0

24

48

72 Time, h

96

120

0 144

Control Glycerol, mg/ml

Glycerol, mg/ml

Control DCW, mg/ml

DCW, mg/ml

Control CAproduction, mg/ml

CA Production, mg/ml

Kp (CA, mg/ml h)

0

Arginine feeding

0.2

2

2

14

1.8

Threonine feeding

16

1.6 1.4

10

1.2

8

1

6

0.8 0.6

4

Arginine feeding

12

Clavulanic acid mg/ml

Glycerol, mg/ml DCW, mg/ml

Fig. 5. Effect of feeding arginine at 120 h of fermentation on clavulanic acid production by Streptomyces clavuligerus MTCC 1142.

0.4

2

0.2

0

0.029 0.031 0.036 0.039 0.044 0.0210 0.028 0.031 0.033 0.035

0.6

6

0.0210 0.025 0.026 0.037 0.031

0.8

8

0.052 0.052 0.053 0.054 0.055

1

10

0.050 0.052 0.057 0.057 0.058

1.2

12

Glyecrol feeding

1.4

14

Clavulanic acid mg/ml

16

Threonine feeding

P.S. Saudagar, R.S. Singhal / Bioresource Technology 98 (2007) 2010–2017

Glycerol, mg/m DCW, mg/ml

2016

0 0

20

40

60

80

100

120

140

0.047 0.058 0.060 0.064 0.066 1.700 ± 0.026 1.715 ± 0.034 1.730 ± 0.058 1.782 ± 0.049 1.863 ± 0.071 CA: Clavulanic acid. a Mean ± SD of three determinations.

1.110 ± 0.019 1.159 ± 0.012 1.200 ± 0.037 1.268 ± 0.049 1310 ± 0.060 1.100 ± 0.011 1.180 ± 0.038 1.200 ± 0.039 1.260 ± 0.026 1.300 ± 0.049 Control (0) 60 72 96 120

Glycerol feeding Threonine feeding

respectively. In the feeding cultures, 2 ml of amino acid solution (arginine 0.1 M/threonine 1.0 M) was added every 12 h after 60 h. The feeding was stopped at 60 h, 72 h, 96 h and 120 h, respectively. Clavulanic acid production was found to increase with the span of feeding amino acids and reached a maximum of 1.310 mg/ml and 1.863 mg/ml with feeding arginine and threonine, respectively, with 120 h feeding. No remarkable change was observed in glycerol consumption and the growth of S. clavuligerus with amino acid feeding thus indicating a direct stimulatory effect of these amino acids on clavulanic acid production. Arginine and threonine did not contribute much to the growth of S. clavuligerus, as seen from the lmax values of 0.057 h1 and 0.054 h1 as compared to respective values of 0.050 h1 and 0.052 h1 in control. However the rate of clavulanic acid production showed a remarkable increase from 0.021 mg/ml h to 0.035 mg/ml h with arginine feeding and from 0.0295 mg/ml h to 0.0438 mg/ml h with threonine feeding (Table 5). The results also reveal that the amino acids could be utilized continuously by S. clavuligerus for the biosynthesis of

Arginine feeding

Fig. 6. Effect of feeding threonine at 120 h of fermentation on clavulanic acid production by Streptomyces clavuligerus MTCC 1142.

Glycerol feeding

CA Production, mg/ml

lmax, h1

Control CA production

Clavulanic acid productiona, mg/ml

Control DCW, mg/ml

DCW, mg/ml

Time of feeding (h)

Glycerol, mg/ml

Table 5 Comparison of clavulanic acid production utilizing different feeding strategies

Time, h Control Glycerol, mg/ml

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clavulanic acid, and that the feeding strategies can enhance clavulanic acid production. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls. This increase is significant from the point of view of process economics, since clavulanic acid is a high-valued pharmaceutical. Besides, the yield of clavulanic acid reported in this work is the highest so far using the wild type strain of S. clavuligerus. The exact mechanism for stimulatory action of threonine on clavulanic acid production is unknown and further work needs to be initiated. Threonine does appear to be an important link in the biosynthesis of clavulanic acid in S. clavuligerus. 4. Conclusion Using one factor at-a-time method and the orthogonal matrix method, it was possible to determine optimal operating conditions to obtain maximum production of clavulanic acid by S. clavuligerus. Phosphate repression had a positive effect on clavulanic acid. An appropriate feeding strategy for enhanced production of clavulanic acid by S. clavuligerus was developed. Supplementing threonine to the production medium resulted in a remarkable increase in clavulanic acid production as in comparison with other amino acids. References Aharonowitz, Y., Demain, A.L., 1978. Carbon catabolite repression of cephalosporin production in Streptomyces clavuligerus. Antimicrob. Agents Chemother. 14, 159–164. Baggaley, K.H., Brown, A.G., Schofield, C.J., 1997. Chemistry and biosynthesis of clavulanic acid and other clavams. Nat. Prod. Rep. 14 (4), 309–333. Bhattactacharjee, S., Ananta, K., Mandal, S.K., 2002. Alkaline phosphatase and erythromycin production by Saccharospora erythraea. Indian J. Microbiol. 42, 67–72. Chen, P.S., Toribara, T.Y., Warner, H., 1956. Microdetermination of phosphorous. Anal. Chem. 28 (11), 1756–1758. Chen, K.C., Lin, Y.H., Tsai, C.M., Hsieh, C.H., Houng, J.Y., 2002. Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding. Biotech. Lett. 24, 455–458. Chen, K.C., Lin, Y.H., Wu, J.Y., Hwang, S.C.J., 2003. Enhancement of clavulanic acid production in Streptomyces clavuligerus with ornithine feeding. Enz. Micro. Tech. 32, 152–156. Ekinci, F.Y., Barefoot, S.F., 2006. Fed-batch enhancement of jenseniin G, a bacteriocin produced by Propionibacterium thoenii (jensenii) P126. Food Microbiol. 23 (4), 325–330. Elson, S.W., Oliver, R.S., 1978. Studies on the biosynthesis of clavulanic acid I. Incorporation of 13C-labeled precursors. J. Antibiot. 31, 586–592. Foulstone, M., Reading, C., 1982. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids by high performance liquid chromatography. Antimicrob. Agents Chemother. 22, 753–762. Gouveia, E.R., Baptista-Neto, A., Azevedo, A.G., Badino Jr., A.C., Hokka, C.O., 1999. Improvement of clavulanic acid production by Streptomyces clavuligerus in medium containing soybean derivatives. World. J. Microbiol. Biotechnol. 15, 623–627.

2017

Ives, P.R., Bushell, M.E., 1997. Manipulation of physiology of clavulanic acid production in Streptomyces clavuligerus. Microbiology 143, 3573– 3579. Jenson, S.E., Paradkar, A.S., 1999. Biosynthesis and Molecular genetics of Clavulanic acid. Antonie van Leeuwenhoek. 75, 125–133. Kirk, S., Avignone-Rossa, C.A., Bushell, M.E., 2000. Growth limiting substrates affected antibiotic production and associated metabolic fluxes in S. clavuligerus. Biotech. Lett. 22, 1803–1809. Lebrihi, A., Gemain, P., Lefebvre, G., 1987. Phosphate repression of cephamycin and clavulanic acid production by Streptomycesclavuligerus. Appl. Microbiol. Biotechnol. 26, 130–135. Lubbe, C., Wolfe, S., Demain, A.L., 1985. Repression and Inhibition of cephalosporin synthetases in Streptomyces clavuligerus by inorganic phosphate. Arch. Microbiol. 140, 317–320. Lynch, H.C., Yang, Y., 2004. Degradation products of clavulanic acid promote clavulanic acid production in cultures of Streptomyces clavuligerus. Enz. Microb. Tech. 34, 48–54. Martin, J.F., 1976. Phosphate regulation of gene expression of candicidin biosynthesis. In: Schlessinger, D. (Ed.), Microbiology. American Society for Microbiology, Washington, DC, pp. 548–552. McDermott, J.F., Lethbridge, G., Bushell, M.E., 1993. Estimation of kinetics constants and elucidation of trends in growth and erythromycin production in batch and continuous cultures of Saccharospora erythraea using curve-fitting technique. Enz. Microb. Tech. 15, 657– 663. Park, S., Momose, I., Tsunoda, K., Okabe, M., 1994. Enhancement of cephamycin C production using soybean oil as a sole carbon source. Appl. Microbiol. Biotechnol. 40, 773–779. Patnaik, P., 2006. Dispersion optimization to enhance PHB production in fed-batch cultures of Ralstonia eutropha. Biores. Technol. 97 (16), 1994–2001. Rangel-Yagui, C.O., Danesi, E.D., Carvalho, J.C., Sato, S., 2004. Chlorophyll production from Spirulina platensis: cultivation with urea addition by fed-batch process. Biores. Technol. 92 (2), 133–141. Romero, J., Liras, P., Martin, J.F., 1984. Dissociation of cephamycin C and clavulanic acid production in Streptomyces clavuligerus. Appl. Microbiol. Biotechnol. 20, 318–325. Romero, J., Liras, P., Martin, J.F., 1986. Utilization of ornithine and arginine as specific precursors of clavulanic acid. Appl. Environ. Microb. 52, 892–897. Shang, F., Wen, S., Wang, X., Tan, T., 2006. Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae. J. Biotech. 122 (3), 285–292. Shu-Jen, D., Basch, J., 1999. Cephalosporin. In: Encyclopedia of Bioprocess Technology. A Wiley-Interscience Publication, Vol. 1. John Wiley & Sons, Inc., USA, pp. 560–569. Stirling, I., Elson, S.W., 1979. Studies on the biosynthesis of clavulanic acid. II. Chemical degradation of 13C-labeled clavulanic acid. J. Antibiot. 32, 1125–1129. Survase, S.S., Saudagar, P.S., Singhal, R.S., 2006. Production of scleroglucan from Sclerotium rolfsii MTCC 2156. Biores. Technol. 97 (8), 989–993. Teodoro, J.C., Neto, A.B., Cruz-Herna´ndez, I.L., Hokka, C.O., Badino, A.C., 2006. Influence of feeding conditions on clavulanic acid production in fed-batch cultivation with medium containing glycerol. Appl. Microbiol. Biotechnol. 72 (3), 450–455. Townsend, C.A., Ho, M.F., 1985. Biosynthesis of clavulanic acid: origin of C5 unit. J. Am. Chem. Soc. 107, 1065–1066. Valentine, B.P., Bailey, C.R., Doherty, A., Morris, J., Elson, S.W., Bagley, K.H., Nicholson, N.H., 1993. Evidence that arginine is later metabolic intermediate than ornithine in the biosynthesis of clavulanic acid by Streptomyces clavuligerus. J. Chem. Soc. Chem. Comm., 1210– 1211. Xu, C.P., Kim, S.W., Hwang, H.J., Choi, J.W., Yun, J.W., 2003. Optimization of submerged culture conditions for mycelial growth and exobiopolymer production by Paecilomyces tenuipes C240. Proc. Biochem. 38, 1025–1030.