## Brazilian Journal of Chemical Engineering

##
*Print version* ISSN 0104-6632*On-line version* ISSN 1678-4383

### Braz. J. Chem. Eng. vol.17 n.4-7 São Paulo Dec. 2000

#### http://dx.doi.org/10.1590/S0104-66322000000400045

**STUDIES ON THE RHEOLOGY AND OXYGEN MASS TRANSFER IN THE CLAVULANIC ACID PRODUCTION BY STREPTOMYCES CLAVULIGERUS**

**E. R. Gouveia, A.Baptista-Neto, C.O. Hokka, and A.C.Badino Jr.* **Departamento de Engenharia Química – Universidade Federal de São Carlos

C.P. 676, 13565-905, São Carlos, SP, Brazil

Phone: (016) 260-8264, Fax: (016) 260-8266

E-mail: badinojr@deq.ufscar.br

*(Received: November 3, 1999 ; Accepted: April 18, 2000)*

Abstract- In the present work rheological characteristics and volumetric oxygen transfer coefficient (k_{L}a) were investigated during batch cultivations ofStreptomyces clavuligerusNRRL 3585 for production of clavulanic acid. The experimental rheological data could be adequately described in terms of the power law model and logistic equation. Significant changes in the rheological parameters consistency index (K) and flow behavior index (n) were observed with the fermentation evolution. Interesting correlations between the consistency index (K)/biomass concentration (C_{X}) and the flow behavior index (n)/biomass concentration were proposed. Volumetric oxygen mass transfer coefficient (k_{L}a) was determined by the gas balance method. Classical correlation relating the volumetric oxygen mass transfer coefficient to the operating conditions, physical and to transport properties, including apparent viscosity (m_{ap}), could be applied to the experimental results.

Keywords: rheology, oxygen transfer, clavulanic acid,Streptomyces clavuligerus.

**INTRODUCTION**

The streptomycetes constitute the most important group of microorganism for the commercial production of antibiotics and exhibit two extreme types of morphology in submerged culture: pellet and free filamentous form (Packer and Thomas, 1990). The pellet growth form consists of branched hyphae intertwined to make as a stable aggregate, producing therefore less viscous fluids than the filamentous form. The pellet growth form may, however, impose other problems such as nutrient limitation in the pellets. As the viscosity in streptomycetes fermentations may strongly increase during the course of a batch process, the mass and heat transfer rates and the degree of mixing are remarkably affected. These facts, in turn, affect growth, morphology and product formation (Olsvik and Kristiansen, 1994).

It has been well established that the biomass concentration, the specific growth rate and the dissolved oxygen concentration influence the rheological properties of filamentous fermentation broth. Decrease in oxygen transfer rate with increasing mycelial cell concentration has also been shown elsewhere (Ghildyal *et al*., 1987; Warren *et al*., 1995).

Metz *et al*. (1979) presented correlations relating the biomass concentration during mycelial growth of the fungi to the rheological properties of the broth. Fatile (1985) correlated each one of the power-law constants with the microbial concentration and the shape of the mycelial aggregate. Queiroz *et al*. (1997) proposed mathematical relationships between the rheological properties and the microorganism physiological state, expressed by both specific growth and enzyme production rates and Badino Jr. *et al*. (1999) described the rheological experimental data in terms of the power law model, during *Aspergillus awamori* cultivation for glucoamylase production. The consistency index was conveniently related to the biomass concentration and to the shear conditions represented by the impeller speed and airflow rate*.*

In the present work mathematical correlations between biomass concentration and rheological properties, during clavulanic acid production process utilizing *Streptomyces clavuligerus *are proposed. Also, values of the volumetric oxygen transfer coefficient, obtained experimentally by gas balance method, were related to physical properties and operating conditions, including stirrer speed (N) and a transport property, the apparent viscosity (m_{ap}).

**MATERIALS AND METHODS**

**Microorganism**

*Streptomyces clavuligerus* NRRL 3585, kindly provided by the Institute of Antibiotics of the Universidade Federal de Pernambuco, Recife, PE, Brazil, was utilized throughout. The spores were obtained by cultivation in the sporulation medium proposed by Butterworth (1984) and suspensions containing approximately 10^{8 }spores per millilitre were maintained in cryovials at -50ºC in freezer using glycerol (20% w/w) as cryoprotector.

**Culture Media**

The germination media contained (in g/l): glycerol, 20.0 and protein extract of soybean (or peptone), 32.0; initial pH was 6.7. Two fermentation culture media with two different nitrogen sources were utilized. They contained (in g/l): glycerol, 15.0, Samprosoy 90 NB (or peptone), 47.0 and K_{2}HPO_{4}, 0.8, initial pH was 6.5. Distilled water was used throughout.

**Cultivation Conditions**

Initially, 1 ml of a cell suspension was inoculated in 50 ml of germination medium in a 500 ml Erlenmeyer flask. This flask was incubated at 27ºC, 250 rev/min for 24 hours and then 10% was used to inoculate 3600 ml of the production medium. The production stage continued for about 36 hours cultivation time and was performed in a New Brunswick 5 liters BIOFLO II bioreactor with automatic temperature and dissolved oxygen control. The stirrer speed changed from 300 to 1000 rpm, and aeration varied from 0.5 to 1.0 vvm. Dissolved oxygen was kept at 40% and temperature at 28^{0}C. Samples were withdrawn each 3 hours until the end of clavulanic acid production. Portions of 20 ml of fermentation broth were centrifuged at 3720×g for 20 minutes.

**Analytical Methods**

Biomass concentration was determined as dry weight of solids at 105ºC for 24 hours.

Clavulanic acid was determined by High Performance Liquid Chromatography (HPLC) as proposed by Foulstone and Reading (1982) with modifications. A C-18 m-Bondapak column (3.9 x 300mm) was used and the equipment was operated under the following conditions: temperature, 28ºC; flow rate, 1.5 ml/min and the UV detector model 486 set to 227 nm. The standardisation was carried out with 0-0.2 mM solutions of potassium clavulanate starting from the commercially available pharmaceutical product Clavulin (125 mg of potassium clavulanate and 325 mg of amoxicillin), produced by SmithKline Beecham Laboratórios Ltda., Rio de Janeiro, Brazil. The mobile phase was composed of KH_{2}PO_{4} (50 mM) at pH 4.5.

Glycerol was determined by High Performance Liquid Chromatography (HPLC), calibrated with glycerol solutions of 0.125, 0.25, 0.375 and 0.5 g.L^{-1}. NaOH (1 mM) solution was used as the mobile phase. The equipment was operated at 38ºC with 1ml/min flowrate. A Shodex KS 802 (Lonpak - division of Millipore) column was utilized.

Rheological measurements were made using BROOKFIELD viscometers, models LVT and RVT, with concentric cylinders. Shear stress (t) and shear rate () were calculated using equations (1) and (2). The rheological parameters consistency index, K and flow behavior index n, from the power law model were calculated by non-linear regression of experimental values of t and.

(1) |

(2) |

(3) |

The volumetric oxygen transfer coefficient (k_{L}a) was calculated by the gas balance method considering that the reactor is completely mixed. Such procedure requires the accurate measurements of oxygen concentrations in all gas exit streams and a reliable measurement of the dissolved oxygen concentration (C). A molar balance on O_{2} in the gas allows the determination global oxygen uptake rate () and the volumetric oxygen transfer coefficient (k_{L}a) was calculated as follows:

(4) |

**RESULTS AND DISCUSSION**

**Fermentation Characteristics**

Time course of process variables such as biomass concentration, concentration of glycerol, clavulanic acid concentration, dissolved oxygen levels, and the stirrer speed changes are shown in Figures 1 and 2 for both culture media. The biomass concentrations increased rapidly reaching a maximum value of 9.5 g.L^{-1} (dry weight) after 21 h, which was accompanied by the depletion of glycerol. Clavulanic acid concentrations subsequently increased during trophophase and reached a value of 0.8 g.L^{-1} for culture medium with Samprosoy 90 NB, approximately twice as much clavulanic acid as that obtained with peptone. Moreover, Samprosoy 90 NB is an excellent source of nutrients for this bioprocess (Gouveia et al., 1999). The dissolved oxygen levels fell during the first 5 hours of fermentation and afterwards the stirrer speed was increased in order to maintain dissolved oxygen concentration at 40% of the saturation with air in the broth.

**Rheological Parameters**

** **In this work, studies on rheological characteristic and the oxygen transfer capacity during clavulanic acid production process by *Streptomyces clavuligerus* in submerged culture was performed. For the two culture media the broth apparent viscosity (m_{ap}) rapidly increased, showing non-Newtonian pseudoplastic behavior. Rheological data could be described in terms of power law model. Figure 3 shows K and n for both culture media utilized.** **Consistency index (K) significantly increased when Samprosoy 90 NB medium was used. It is probably due to the presence of insoluble solids in this medium. On the other hand, n decreased rapidly from 1 to 0.5 during the first 9 hours of fermentation in both media. The flow index reported by others workers for *Streptomyces *strains were in the range 0.20-0.25 (Warren et al., 1995).

According to Olsvik and Kristiansen (1994), expressions like the equation (5) have been widely used to describe an power relationship between K and C_{x}. However, in batch fermentation systems, it is likely that the relation between consistency index, K, and biomass concentration, C_{x}, is sigmoidal (Goudar et al., 1999).

(5) |

Figures 4a and 4b show the inadequacy of equations (6) and (7) to describe experimental data K versus C_{x} in the culture media containing Samprosoy 90NB or peptone, respectively.

r | (6) |

r | (7) |

Goudar et al. (1999) propose a more accurate description of the relationship between K and C_{x} provided by a three parameter logistic equation:

(8) |

where K_{0} and K_{f }represent initial and final values of K, and c is a constant. Table 1 summarizes this parameters estimated by nonlinear least-squares analysis using experimental K and C_{x} values*.* Moreover, Figures 4a and 4b show that the equation (8) was able to describe the relationship between K and C_{x} accurately, not being the case of equations (6) and (7).

Given the similarity in the relationships between pseudoplastic fluid viscosity/shear rate and n/biomass concentration, the following equation describes adequately the relationship between n and C_{x} (Goudar et al., 1999):

(9) |

where n_{f} is final value of n and d and e are constants. Figures 5a and 5b show the correlation between experimental n and cell concentration (C_{x}) for *Streptomyces clavuligerus* broths obtained in both culture media. Also, the relationship predicted by equation (9) is shown. It can be observed that it describes well the relation of n with the biomass concentration. Table 1 also shows the estimates of n_{f}, d and e. Equations (8) and (9) adequately describe the relationships of the power law parameters K and n, with the biomass concentration. The applicability of these equations was examined for other non-Newtonian fermentation systems, using rheological data from *Aspergillus awamori*, *Aspergillus niger *and *Penicillium chrysogenum* fermentation broths. In all cases, the relationships between K/C_{x} and n/C_{x} were adequately described by the equations (8) and (9) (Goudar et al., 1999).

**Characteristics of Oxygen Transfer**

The stirrer speed, N, and apparent viscosity, m_{ap}, influence the values of volumetric oxygen transfer coefficient, k_{L}a, during fermentations of filamentous organisms such as *Streptomyces clavuligerus*. As oxygen transfer is limited by diffusion into flocs, an increase of N results in smaller flocs and so in an increased oxygen uptake rate (Metz et al., 1979). Higher stirrer speeds also acts decreasing the

apparent viscosity of fluid. The k_{L}a values obtained in this study were correlated to N and m_{ap}, according to the relationship proposed by Li et al. (1995) as:

(10) |

where,

(11) |

and k=11.5 is a constant for a given experimental system.

The data were correlated by non linear regression analysis, and values of C_{1}, a and b were found to be 2.4× 10^{5}, 1.042 and –0.037, respectively for both culture media:

(12) |

The correlation coefficient between the transformed data of k_{L}a and the remaining of variables was r^{2}=0.98. Experimental values of k_{L}a were plotted against the k_{L}a values calculated by equation (12). As shown in Figure 6, good agreement are observed, indicating that the equation (12) can be applied to predict this important transport coefficient, in non-Newtonian fermentation broths such as produced by *Streptomyces clavuligerus* cultivation.

**ACKNOWLEDGEMENTS**

The authors would like to thank financial support from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil), Proc. 98/12.271-7.

**NOMENCLATURE**

a | constant defined by Eq. 5 |

b | Empirical exponent to Eq. 5 |

c | Empirical exponent to Eq. 8 |

C | Dissolved oxygen concentration, mmol.L^{-1} |

C* | Dissolved oxygen saturation concentration, mmol.L^{-1} |

C_{1} | Proportionality constant to Eq. 10 |

d | Constant defined by Eq. 9 |

D | Diffusivity of oxygen in water, m^{2}.s^{-1} |

d_{i} | Impeller diameter, m |

e | Empirical exponent for Eq. 9 |

g | Gravitational acceleration, m.s^{-2} |

k_{L}a | Volumetric oxygen transfer coefficient, s^{-1} |

K | Consistency index, Pa.s^{n} |

n | Flow behavior index, - |

N | Impeller speed, s^{-1} |

Global oxygen uptake rate, mmol.L^{-1}.h^{-1} | |

r^{2} | correlation coefficient, - |

T | Impeller torque, N.m |

*Greek letters:*

a | Empirical exponent for Eq. 10 | |

b | Empirical exponent for Eq. 10 | |

Shear rate, s^{-1} | ||

m | Viscosity, Pa.s | |

t | Shear stress, Pa |

*Subscripts*

ap | Apparent condition |

0 | Initial condition |

f | Final condition |

x | Biomass |

w | Water |

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*To whom correspondence should be addressed