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Biochemical Engineering Journal
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egular article
roduction of clavulanic acid by Streptomyces clavuligerus in batch culturesithout and with glycerol pulses under different temperature conditions
ecília L.L. Costa, Alberto C. Badino ∗
epartment of Chemical Engineering, Federal University of São Carlos, Cx. Postal 676, CEP 13565-905, São Carlos, SP, Brazil
r t i c l e i n f o
rticle history:eceived 7 March 2012eceived in revised form 12 July 2012ccepted 12 August 2012vailable online 19 August 2012
eywords:lavulanic acid
a b s t r a c t
Clavulanic acid (CA) is a potent beta-lactamase inhibitor produced by Streptomyces clavuligerus. Likeother beta-lactam compounds, CA is chemically unstable at high temperatures. The decomposition ofCA during bacterial fermentation reduces its concentration in the broth, resulting in low yields. Thisstudy investigated the use of temperature reduction to obtain high CA production during fermentationemploying cultures of S. clavuligerus. Simple batch cultures, and batch cultures with glycerol pulses, wereperformed at different temperatures in shake flasks at 250 rpm, and pH 6.8. Firstly, three batch culturesusing glycerol as carbon source were carried out at temperatures of 30 (control), 25, and 20 ◦C. Next,
three batch cultures were carried out with temperature reductions from 30 to 25 C, 30 to 20 C, and25 to 20 ◦C, after glycerol exhaustion. CA production generally increased in cultures with temperaturereduction and glycerol feeding. Maximum CA concentration of 1534.3 mg L−1 was achieved in a culturemaintained at a constant temperature of 20 ◦C, with one pulse of glycerol. Under these conditions, bothglycerol uptake rate and CA degradation were low. The results obtained demonstrate the potential of
the o
temperature reduction in
. Introduction
The production of beta-lactamase enzymes is the most commonechanism of bacterial resistance to beta-lactam antibiotics such
s penicillins and cephalosporins. These enzymes are secreted by wide range of important pathogenic Gram-positive and Gram-egative bacteria. Beta-lactamases catalyze hydrolysis of the beta-
actam ring, splitting the amide bond. As a result, the antibioticsecome ineffective against bacterial growth. Clavulanic acid (CA)
s a secondary beta-lactam metabolite produced by Streptomyceslavuligerus that has a potent beta-lactamase inhibitory activity [1].A has been used clinically in conjunction with beta-lactamase-ensitive and beta-lactam antibiotics to treat diseases caused byeveral pathogenic bacteria.
Like all beta-lactam compounds, in its crude form CA is chem-cally unstable at acidic and basic pH, and at high temperatures.revious studies have demonstrated that temperature has a majorffect on CA degradation [2–4].
CA is traditionally produced by Streptomyces clavuligerus using aomplex culture medium containing soybean derivatives, and glyc-
rol or lipid, as sources of nitrogen, carbon, and energy [5–9]. Glyc-rol is the most commonly used carbon source, and in the biosyn-hetic route of CA is converted to d-glyceraldehyde-3-phosphate,
the primary metabolic precursor of CA [10]. Chen et al. [5] observedthat the biosynthesis of CA was inhibited by glycerol concentra-tions higher than 15 g L−1, while Rius and Demain [11] concludedthat concentrations of glycerol above 2% (w/v) suppressed produc-tion of the compound. Fed-batch culture using glycerol has beenused to increase the productivity of microbial processes, and feed-ing strategies have been employed to control substrate inhibitionand metabolite repression in order to improve CA production [7–9].This is based on the fact that substrate inhibition is caused by highsubstrate concentrations, with a high substrate uptake rate causingaccumulation of metabolic intermediates that inhibit or repress theproduction of secondary metabolites such as CA. However, the sub-strate uptake rate can be controlled to a low level using a reducedtemperature, even when the available substrate concentration ishigh.
It has been reported in the literature that lower operational tem-peratures result in reduced growth, decreased metabolism, andimprovement in cell viability, with variable effects on productiv-ity in batch cultures [12]. In animal cell cultures, an increase inrecombinant protein production has been achieved by lowering thecell culture temperature [13]. Constantinides et al. [14] reported a15% increase in penicillin yield at a reduced temperature in batchfermentation.
Many studies have been undertaken concerning the nutrientrequirements of S. clavuligerus cultures, and the influences of pHand temperature on CA degradation [2–4]. However, the influenceof temperature on cell growth and substrate consumption during
A production processes has received much less attention, despitehe importance of temperature in optimization of biomolecule pro-uction using cell cultures. Many experiments have suggested that
reduction of the culture temperature results in higher viabilitynd shear resistance, with a decrease in the specific growth rate anduppression of the release of waste products [15,16]. The influencef temperature on the oxygen transfer rate has been demonstrated,nd low temperatures are known to decrease product degradation.n important positive factor is the increase in oxygen solubility in
he aqueous phase at lower temperatures.CA is traditionally produced in cultures performed at 28 or 30 ◦C
6]. Although these temperatures favor S. clavuligerus growth, theyre not appropriate for a slow consumption of the carbon source,hich could minimize the inhibition effects, while CA degradation
s more pronounced than at 20 ◦C [2].Considering the important effect of temperature on cellular
rowth, substrate consumption, and CA degradation, the presentork investigated CA production by S. clavuligerus in cultureserformed under different temperature conditions. Simple batchultures and batch cultures with pulses of glycerol were performedt constant temperatures and with temperature reduction after thenitial cellular growth phase, in order to evaluate the effects on thelycerol uptake rate, and on the production and degradation of CA.
. Materials and methods
.1. Microorganism
The microorganism used in this work was Streptomyceslavuligerus ATCC 27064, stored as vegetative cells (5 g L−1 dryeight) at −70 ◦C in 4 mL cryotubes containing glycerol (10%, v/v).
.2. Culture media
The seed medium [17] had the following composition (g L−1):lycerol 15.0; bacto peptone, 10.0; malt extract, 1.0; K2HPO4,.8; MgSO4·7H2O, 0.75; MnCl2·4H2O, 0.0001; FeSO4·7H2O, 0.001;nSO4·7H2O, 0.001; and 3-(N-morpholino) propanesulfonic acidMOPS) buffer, 21.0 (100 mM). The pH of the medium was 6.8.
The composition of the inoculum medium [9] was as followsg L−1): glycerol, 15.0; soybean protein isolate, 25.0; K2HPO4, 0.8;
gSO4·7H2O, 0.75; and 3-(N-morpholino) propanesulfonic acidMOPS) buffer, 21.0 (100 mM). The pH of the medium was 6.8.
The production medium had the same composition as the inocu-um medium. All media were autoclaved at 121 ◦C for 15 min.
.3. CA production assays
Vegetative cell suspensions were transferred from the cryotubeso 500 mL Erlenmeyer flasks containing 50 mL of seed medium, andncubated in a rotary shaker at 250 rpm for 24 h at 30 ◦C. Erlen-
eyer flasks (500 mL) containing 45 mL of the inoculum mediumere inoculated with 5 mL of cultivated seed broth, and incubated
t 250 rpm for 24 h at 30 ◦C. In the production stage, the inocu-um suspension (in a proportion of 10%, v/v) was transferred undergitation to a 3 L flask containing the production medium. 50 mLolumes of this inoculated medium were individually pumped into00 mL Erlenmeyer flasks, and incubated at 250 rpm under the fol-
owing conditions:Batch cultures at constant temperature: three batch cultures were
erformed at constant temperatures of 30 (B30-30), 25 (B25-25),nd 20 ◦C (B20-20).
Batch cultures at constant temperature and with glycerol pulses:welve batch cultures with 1, 2, 3, or 4 pulses of glycerol (BP) wereonducted at constant temperatures of 30, 25, and 20 ◦C. These cul-ures were denoted BP30-30-1, BP30-30-2, BP30-30-3, BP30-30-4,
Engineering Journal 69 (2012) 1– 7
BP25-25-1, BP25-25-2, BP25-25-3, BP25-25-4, BP20-20-1, BP20-20-2, BP20-20-3, and BP20-20-4, respectively.
Batch cultures with temperature reduction: three batch cultureswere conducted at either 30 or 25 ◦C during the phase of cellulargrowth and glycerol consumption, after which the culture tem-perature was reduced to either 25 (B30-25) or 20 ◦C (B30-20 andB25-20).
Batch cultures with temperature reduction and glycerol pulses:twelve batch cultures were performed with temperature reduc-tion (30 to 25, 30 to 20, and 25 to 20 ◦C) and with 1, 2, 3, or 4pulses of glycerol (BP). The cultures were denoted BP30-25-1, BP30-25-2, BP30-25-3, BP30-25-4, BP30-20-1, BP30-20-2, BP30-20-3,BP30-20-4, BP25-20-1, BP25-20-2, BP25-20-3, and BP25-20-4,respectively.
For the cultivations with glycerol pulses, a pre-established vol-ume of glycerol solution (100 g L−1) was added every time itsconcentration in the broth fell below 2.0 g L−1, in order to main-tain the concentration between 9.0 and 12.0 g L−1 after the pulse.The culture conditions are summarized in Table 1. During the cul-tures, samples (1 mL) were withdrawn approximately every 12 h,and centrifuged at 3720 × g for 15 min. The supernatants were usedfor analyses of CA and glycerol. All experiments were performed intriplicate, giving a total of 90 cultures. The batch culture performedat 30 ◦C (B30-30) was used as the control.
2.4. CA degradation assays
In parallel with the CA production assays, degradation of CAin the fermentation broths was evaluated at constant tempera-ture during batch cultures with four pulses of glycerol (BP30-30-4,BP25-25-4, and BP20-20-4). Samples of the culture broth (in 500 mLErlenmeyer flasks with 50 mL of broth in simultaneous culture)were withdrawn immediately before the glycerol pulses and afterglycerol exhaustion in the end of the culture, totaling five sam-ples for each culture. The samples were centrifuged, sterilized byfiltering through a sterile 0.2 �m membrane, and used in the CAdegradation assays. Kinetic studies of CA hydrolysis were carriedout using 100 mL Erlenmeyer flasks containing 25 mL of fermentedbroth at constant temperatures of 20, 25, and 30 ◦C. Samples(0.5 mL) of culture broth were periodically withdrawn, and the con-centrations of CA that had not been degraded (CCA, mg L−1) weremeasured.
2.5. Determination of CA production and degradation rates
A pseudo-first order kinetic model (Eq. (1)) was used to describethe CA degradation rate (rPd, mg L−1 h−1) in the fermentation broth:
CCA = CCA0 · e−kPd·t (1)
where, CCA0 is the initial CA concentration, and kPd (h−1) is theproduct degradation constant.
The kPd values were estimated from the fitting of Eq. (1) to theCA experimental data. Five samples of the fermentation broths fromcultures BP20-20-4, BP25-25-4, and BP30-30-4 were used, and anaverage value of kPd was calculated for each temperature. Substi-tuting values of kPd and CCA in Eq. (2) enabled calculation of theCA degradation rate (rPd, mg L−1 h−1). Values of the CA productionrate (rP, mg L−1 h−1) could then be obtained from the mass bal-ance for CA during the cultures, using the experimental data for CAaccumulation (dCCA/dt, mg L−1 h−1) and rPd (Eq. (3)).
i: initial temperature; TG: temperature after initial glycerol exhaustion.
he dCCA/dt values were obtained as the simple derivative of CCAith respect to time.
.6. Analytical methods
The CA concentration (CCA) in the fermentation broth was mea-ured using the classical analytical methods proposed by Foulstonend Reading [18] (HPLC with UV detector) and by Bird et al. [19]UV spectrophotometry), and the results were compared. Since theA concentrations obtained using both methods were very similar,ith a difference of less than 7%, in all cultures the CA concentrationas determined by the spectrophotometric technique, employing
derivative produced by the reaction of CA with imidazole, asroposed by Bird et al. [19]. CA contained in the pharmaceuticalroduct Clavulin (Glaxo-SmithKline Farmacêutica, Rio de Janeiro,razil) was used as a standard.
The glycerol concentration (CG) in the supernatants was deter-ined by an enzymatic method using a triglycerides GPO-PAP test
it (Laborlab, Brazil).The method proposed by Mayer and Deckwer [4] was used to
etermine the cell concentration in the presence of solid parti-les. Firstly, the cell dry weight concentration (CX) was determinednd correlated with the optical density at 600 nm (OD600) in cul-ures employing solid-free media, which generated a linear modelescribing the relationship between cell dry weight and OD600. Inhe cultures with media containing insoluble particles (soybeanrotein isolate, SPI), the broth was decanted for 45 s (the timeecessary to precipitate the SPI) and the OD600 was measured.
he value of CX was obtained from the linear regression equationescribed above.
There are many reviews of techniques used to measureell concentrations in media where solid particles are absent;
0.156 0.34 26.5 108.1
however, little information is available concerning measurementsof cell concentrations in the presence of solid particles. In thepresent work, other techniques for the determination of cell con-centrations in the presence of solid particles were also tested. Theseincluded measurements of total protein [20,21], as well as colonycounting on plates [22]. However, protein extraction by wash-ing did not efficiently eliminate all the protein of the medium; itwas not possible to measure the DNA content, since the lipopro-tein layer formed made it difficult to extract the total DNA fromsmall samples; and colony counting on plates gave false resultsbecause Streptomyces sp. form pellets in liquid media. The mostreliable measurements of cell concentrations in culture media con-taining solid particles were achieved using the method describedabove.
3. Results and discussion
3.1. CA production assays
Table 1 summarizes the main results obtained for the differentcultures.
3.1.1. Batch cultures at constant temperatureThree batch cultures of S. clavuligerus ATCC 27064 were per-
formed in triplicate using the production medium containing15 g L−1 of glycerol, at temperatures of 30 (B30-30), 25 (B25-25),and 20 ◦C (B20-20).
The maximum CA concentration (CCAmax) was increased by
decreasing the temperature (Fig. 1). For the batch culture per-formed at 30 ◦C (the control culture, B30-30), the CCAmax in thebroth was 168.7 mg L−1. At 25 ◦C (B25-25), the yield was 3.8-foldhigher than that of the control culture. The highest CA production
Fig. 1. Time courses of concentrations in batch cultures conducted at 30 ◦C (B30-30, square), 25 ◦C (B25-25, circle), and 20 ◦C (B20-20, triangle): (a) CA concentration(CCA); (b) cellular concentration (CX) (empty symbols) and glycerol concentration(CG) (half-filled symbols).
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3.1.3. Batch cultures with temperature reduction and glycerolpulses
◦
as observed for the culture grown at 20 ◦C (B20-20), which yielded266.2 mg L−1, almost 7.5-fold higher than that of the control cul-ure. The glycerol uptake rates (rG) were similar at 25 and 30 ◦C,t about 0.25 g L−1 h−1, 2.5 times higher than the value obtained at0 ◦C. It has been reported that when the concentration of glycerol
s higher than 20 g L−1, CA production may decrease due to sub-trate inhibition [5,7]. It was also found that CA production mighte hindered when rG is high.
There was a significant effect of fermentation temperature onellular growth. Cell growth was highest when incubated at 30 ◦C,nd was very similar to the results observed at 25 ◦C, while at 20 ◦Coth the cell growth rate and the cell concentration during theeriod of fermentation were lower. Although growth was reducedt 20 ◦C, the cell death rate was delayed at the lower temperature,hich probably extended cellular viability and contributed to max-
mum CA accumulation in the broth. The cell yield coefficient inelation to glycerol consumption (YX/G) decreased by about 33% at0 ◦C, compared to 30 ◦C. On the other hand, the CA yield coef-cient in relation to glycerol consumption (YCA/G) was favored byhe reduced temperature of 20 ◦C, with a value of 58.5 mg g−1, about.2-fold greater than the value obtained at 30 ◦C. These results indi-ate that the temperatures favoring either S. clavuligerus cell growth
r CA accumulation in the fermentation broth were quite different,nd should be considered.
Fig. 2. Time courses of CA concentrations (CCA) in the control culture (B30-30,squares) and in batch cultures with temperature reductions of 30 to 25 ◦C (B30-25,circles), 30 to 20 ◦C (B30-20, triangles), and 25 to 20 ◦C (B25-20, stars).
3.1.2. Batch cultures with temperature reductionSince the optimal temperatures for cell growth and CA forma-
tion are different, the use of different temperatures during thecourse of the culture should be able to benefit net CA production.The effect of temperature reduction on CA production was there-fore investigated by changing the temperature after sufficient cellgrowth had been achieved to minimize the effect of total cell massunder the different temperature conditions. Fig. 2 shows the evo-lution of CA concentrations during culture B30-30 and where thetemperature was reduced during the production phase (culturesB30-25, B30-20, and B25-20).
Firstly, cultures were maintained at 30 and 25 ◦C until theglycerol concentration was depleted. The rG values for culturesperformed at these temperatures were very similar, with glyc-erol exhaustion occurring after approximately 50 h of cultivation.In a second stage, the temperature was reduced to values of 25and 20 ◦C. In these cultures, net CA production showed a suddenincrease (in all cultures) when the temperature was reduced andglycerol was depleted in the broth, probably due to removal of theinhibition effect as well as reduced degradation of CA at lowertemperatures. For cultures B30-25 and B30-20, CCAmax were 2.9and 2.7-fold higher than for culture B30-30, respectively. Althoughthese CCAmax values were lower than those obtained in the constanttemperature batch cultures (B25-25 and B20-20), temperaturereduction increased CCAmax by about 2.2-fold in the B30-20 cul-ture and 2.9-fold in the B30-25 culture (relative to B30-30). In theB25-20 culture, CCAmax was 4-fold greater than for the control cul-ture.
Cell concentrations in the B30-30, B30-25, and B30-20 cultureswere very similar, although the cells died more slowly after thetemperature change to 20 ◦C (Fig. 1), as discussed above. In cultureswith temperature reduction, all parameters related to CA produc-tion (CCAmax, PCAmax, YCA/G and YCA/X) were improved if comparedto B30-30, but not if compared to the cultures performed at con-stant reduced temperature (B25-25 and B20-20). The higher netCA production of 683.4 mg L−1 in culture B25-20 was due mainlyto reduced degradation at low temperature, since a high rG in theearly stage is detrimental to CA production by S. clavuligerus.
At 30 C, the initial amount of glycerol was rapidly consumedin primary metabolism during the trophophase, which resulted
Fig. 3. Time courses of concentrations in the best batch cultures with temperaturereduction and pulses of glycerol: (a) CA concentration (CCA), and (b) cell concen-t(
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CG (
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together with low CA degradation, provided the best conditions
ration (CX). Cultures: BP30-25-3 (circles), BP30-20-2 (triangles), and BP25-20-1squares).
n lower CA production. Cultures were therefore performed usingupplementation with pulses of glycerol and with temperatureeduction. Fig. 3 illustrates the time courses of CA and cell concen-rations for cultures BP30-25-3, BP30-20-2, and BP25-20-1, whichrovided the best results in terms of CA production.
For the batch cultures performed with temperature reductionrom 30 to 25 ◦C, three pulses of glycerol (BP30-25-3) gave the bestesults in terms of CA production. The maximum CA concentrationas 6.6-fold higher than the value obtained for B30-30. The best
esults for the batch cultures with pulses of glycerol and reductionf temperature from 30 to 20 ◦C were obtained using two pulsesBP30-20-2). The maximum CA concentration was 6.1-fold higherhan the value for (B30-30). At the lower temperatures, the CA con-entration was maintained at high levels over longer periods (from50 to 260 h). The average rG was 17% lower than for B30-30. Forhe cultures with reduction of temperature from 25 to 20 ◦C, a sin-le glycerol pulse (BP25-20-1) provided the highest CA productivity
PCAmax), of 10.5 mg L−1 h−1, almost 3.9-fold higher than the control.he yield coefficients, YX/G and YCA/X, were about half and 11-foldigher, respectively, compared to the control culture, supporting
Fig. 4. Time courses of glycerol (CG, squares), cell (CX, circles), and CA (CCA, triangles)concentrations in batch cultures at 20 ◦C with one pulse of glycerol (BP20-20-1).
the notion that lower temperature favors the use of glycerol forincorporation in secondary metabolism biosynthesis of CA.
3.1.4. Batch cultures at constant temperature and with glycerolpulses
The influence of glycerol feeding was also evaluated using cul-tures performed at constant temperatures of 30, 25, and 20 ◦C.For the batch cultures performed at 30 ◦C with pulses of glycerol,the best results in terms of CA production were achieved usingfour glycerol pulses (BP30-30-4). The CCAmax was 2.6-fold higherthan that obtained for B30-30. After pulse initiation, the aver-age was 38% higher than the value obtained for B30-30. At 25 ◦C,the values of CCAmax fell within a narrow range for all cultures.In these cultures, the highest CA volumetric productivity (PCAmax)values ranged around 9.7 mg L−1 h−1. CCAmax of 1184.6 mg L−1 wasachieved using two pulses of glycerol (BP25-25-2). Considering allcultures, the best results in terms of CCAmax were obtained for thecultures at 20 ◦C, with values in a narrow range from 1500 mg L−1.The PCAmax values obtained for the cultures at 20 ◦C (Table 1) weremuch lower than obtained for the cultures performed at 25 ◦C;however, they were nearly twice that obtained for B30-30. Thetime courses of the CA, cell, and glycerol concentrations for thebest culture (BP20-20-1) are illustrated in Fig. 4.
Glycerol is essential for the biosynthesis of CA, and is one of thebest carbon sources for CA fermentation [23]. In the present work,the effect of feeding using pulses of glycerol on CA production byS. clavuligerus was evaluated at constant temperature, as well asin cultures with temperature reduction after initial glycerol con-sumption. In general, CA production increased with increase in thenumber of pulses of glycerol, as reported by Chen et al. [5]. Thisindicates that S. clavuligerus can continuously utilize glycerol forthe biosynthesis of CA, and that temperature control of glycerolconsumption and CA degradation can effectively enhance net CAproduction. As the temperature decreased, the CCAmax occurred incultures given a smaller number of glycerol pulses.
The CCAmax of 1534.3 mg L−1, achieved for the culture at 20 ◦Cgiven a single pulse of glycerol (BP20-20-1), was 9.3-fold higherthan that obtained for B30-30 and about 7.6-fold higher than thatobtained for culture BP30-30-1, corroborating the beneficial effectof low temperature during the fermentation. In culture BP20-20-1,the value of rG reflected slower cellular metabolism of glycerol,which can reduce the effect of carbon source inhibition. This,
for CA accumulation in the fermentation broth. The higher CA pro-duction could also be explained by maintenance of cell viability forlonger periods at lower temperature, as observed previously for
6 C.L.L. Costa, A.C. Badino / Biochemical
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ig. 5. Profiles of CA production rate (rP) and CA degradation rate (rPd) during cul-ures B30-30 (full line), B20-20 (dashed line), and BP20-20-1 (dotted line).
nimal cell lines [13]. The value of YX/G was similar to that obtainedor B30-30, while the value of YCA/X was 15.6-fold higher. Althoughhe CCAmax obtained in cultures at 20 ◦C occurred during a latereriod of fermentation, the PCAmax of the process was still higherhan that of the control culture.
The CCAmax value found in the present study was similar to thealue of 1.6 g L−1 recently reported by Teodoro et al. [9] for fed-atch cultures using bench-scale bioreactors (5 and 10 L workingolumes), with glycerol and ornithine feeding. Teodoro et al. [9]alue was described as being the highest value ever reported in theiterature for cultures utilizing a wild strain of S. clavuligerus andimilar complex media with soybean derivatives and glycerol oroybean oil as nitrogen and carbon sources. This demonstrates theuccess of the strategy employing low temperature cultures withlycerol feeding for CA production by S. clavuligerus.
.2. CA degradation assays
The average values of the degradation constant (kPd) obtainedt 30, 25, and 20 ◦C were 0.00536 ± 0.00072, 0.00304 ± 0.00047,nd 0.00173 ± 0.00046 h−1, respectively. There was a clear decreasen the value of kPd with decreasing temperature. The valuesbtained in the present work were much smaller than the valuesf 0.0415 h−1 (30 ◦C) and 0.0240 h−1 (20 ◦C) reported by Bersanettit al. [2]. However, the culture media used in this work and byersanetti et al. [2] had different compositions, while in the earlierork the degradation assays employed frozen fermentation broths.
It was therefore possible to obtain the profiles of rPd and rP (Eqs.2) and (3)) during the course of the cultures, and evaluate the indi-idual effects of temperature on rP and rPd. This procedure waspplied to the control culture (B30-30), and to the best cultures inerms of CA production (B20-20 and B20-20-1) (Fig. 5).
The maximum CA degradation rates (rPdmax) were similar for allultures. For the low temperature (20 ◦C) fermentations, the low-st value of kPd was accompanied by the highest concentrationf CA, and vice versa for the B30-30 culture. However, the ratiosbtained between the maximum rates of CA degradation and pro-uction (rPdmax/rPmax) were much lower for the cultures performedt low temperature (around 0.2 for the cultures at 20 ◦C), comparedo the value of 0.38 obtained for the control culture. Comparison of
ultures B20-20 and BP20-20-1 showed that there was a decreasen the value of rP after the pulse of glycerol in culture BP20-20-1at 125 h). However, rP still remained high relative to rPd during the
[
Engineering Journal 69 (2012) 1– 7
subsequent 100 h of cultivation, resulting in a high accumulationof CA in the broth.
4. Conclusions
Clavulanic acid production by S. clavuligerus in shake flask cul-tures was enhanced by manipulating the culture temperature. Themaximum net CA production was obtained for culture BP20-20-1,which yielded 1534.3 mg L−1, with a productivity of 5.3 mg L−1 h−1.This could be attributed to low rates of glycerol uptake and CAdegradation.
Comparison of the rates of CA production (rp) and degradation(rPd) showed that low temperature had a greater influence on CAproduction than on reduction of CA degradation.
The results demonstrated that S. clavuligerus can grow wellat low temperature, with extended duration of cell viability, andreduced CA degradation. Cellular metabolism appeared to be betterdirected towards CA production, and there was a high accumulationof CA in the broth. The beneficial effect of lower culture temperaturefor CA production by S. clavuligerus may be particularly attractivefor industrial applications, because temperature is a convenientcontrol variable that is easy to manipulate.
Acknowledgements
The authors gratefully acknowledge FAPESP (Proc. 2011/23807-1) and Coordenac ão de Aperfeic oamento de Pessoal de NívelSuperior (CAPES) for financial support of this work.
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