Measurement of penicillin acylase and cephalosporin acylase in fermentation broths

Summary The p-dimethylamino-benzaldehyde method of Bomstein and Evans, the ninhydrin method of Marrelli and the new p-dimethyl-amino-benzaldehyde method of Kornfeld were evaluated for analysis of penicillin-V acylase and cephalosporin-V acylase in fermentation broths. The ninhydrin method was the method of choice for cephalosporin-V acylase, whereas the Kornfeld method had certain advantages with respect to penicillin-V acylase.     

Oxygen-stabilized enzyme electrode for d-glucose analysis in fermentation broths

A new principle for the construction of oxygen-dependent enzyme electrodes is presented. The enzyme electrode is based on a galvanic oxygen electrode which is furnished with an electrolysis anode covered by immobilized enzyme and placed close to the oxygen-sensing surface. An ordinary oxygen electrode is used as a reference electrode. The enzymatic consumption of oxygen in the enzyme electrode generates a potential difference between the electrodes which is utilized to control electrolytic generation of oxygen from water in such a way that zero differential potential is maintained. Thus, the enzyme electrode operates under ambient oxygen tension and does not suffer from oxygen limitation. The electrolytic current in this system gives a measure of the concentration of substrate surrounding the enzyme electrode. The electrode has been applied for continuous d-glucose analysis in situ during batch cultures of Candida utilis.     

Parallel capillary electrophoresis for the quantitative screening of fermentation broths containing natural products

Directed molecular evolution is a recursive process of controlled genetic diversification and functional screening. The success of this approach is dependent on both the quality of the genetic diversity and the ability to accurately screen a large population of individual genetic variants for those having improved function. In this paper, the application of parallel capillary electrophoresis to rapidly quantitate lovastatin production levels by Aspergillus terreus mutants is described. A parallel 96 capillary instrument analyzed 900 samples in 8 h. with a 100 mM MES at pH 5.2 running buffer. In this manner, the fermentation broths of thousands of mutated strains were efficiently and inexpensively screened for increased lovastatin production. The ability to develop high-throughput methods to both separate and quantitate the components of complex mixtures greatly facilitates the ability to apply evolutionary engineering methods to complex biological systems.     

Cultivation of the gonococcus in nutrient broths

The dynamics of the multiplication of gonococci and the parameters of their growth have been studied in the process of batch cultivation in liquid culture media with different content of bovine blood serum. The protective and stimulating action of the serum on the growth of gonococci has been shown. The optimization of the process of cultivation has been carried out; as a result, the growth of test strains in a culture medium containing no serum has been achieved. Phasic changes in the ultrastructure of gonococci in the process of their cultivation in liquid media have been followed. 9- to 12-hour cultures of gonococci grown in liquid culture media have been found the most valuable and physiologically active.     

Procedure for isolation of Thienamycin from fermentation broths

An improved process for the isolation of thienamycin, produced by the actinomycete Streptomyces cattleya, has been developed. The isolation procedure consists of three chromatographic steps, volume reduction by reverse osmosis between the steps, and freezedrying for obtaining the final product. The chromatographic steps are as follows: (1) ion exchange chromatography on Dowex 1 × 2 resin in the bicarbonate cycle, (2) gel chromatography on Dowex 1 × 2 resin in the chloride cycle, (3) reverse phase chromatography on XAD-2 resin. This procedure is useful for processing large volumes of fermentation broth.     

Recovery of ethanol from fermentation broths using selective sorption-desorption

Industrialized nations face a critical problem in replacing the sources of liquid fuels that traditionally have been supplied by petroleum. One solution that has gained increasing support in this country is the use of ethanol produced by fermentation of renewable biomass as an extender in, or supplement to, gasoline for transportation fuel. Distillation, the present method of separating ethanol from the fermentation broth, is an energy-intensive one and frequently uses more energy than is available from the ethanol recovered. There are many investigations under way to find alternative, less energy-intensive techniques for the ethanol-water separation. The separations method described in this article involves the use of solid materials to preferentially remove ethanol from fermentation broths. Subsequent stripping of the ethanol from the sorbent with a dry gas reduces dramatically the energy required for the separation. Three solid sorbents have been investigated experimentally. Their sorption/desorption characteristics are described, and their incorporation in an ethanol recovery process is evaluated. Three sorbents were investigated: two commercially available divinylbenzene crosslinked polystyrene resins in bead form (one with a nominal surface area of 300 m(2)/g, the other with 750 m(2)/g) and an experimental proprietary molecular sieve with hydrophobic properties. Equilibrium adsorption isotherms for two of the sorbents were obtained at ambient temperature (21 degrees C) for ethanol-water solutions containing up to 12 wt. % ethanol. In addition, 40 degrees C isotherms were obtained for the polystyrene sorbents. Although different, the equilibrium isotherms for the sorbents indicated that ethanol could be preferentially sorbed from a dilute solution. Column breakthrough curves indicated very favorable kinetics. Desorption of the ethanol was readily effected with warm (60-80 degrees C), dry nitrogen.     

Sensitivity analysis of fed-batch fermentation for the production of penicillin G

Summary The mixed metabolism model of Nicolaiet al. (1991) has been used to analyse the sensitivity of fed-batch fermentation for penicillin G with respect to four parameters. The cases of fully endogenous, fully maintenance and mixed metabolisms have been studied. Sensitivities with respect to two parameters,K _x andK _i, were much larger than for the other two. Differences between the three kinds of metabolism were sharpest forK _p. The implications of both observations are discussed.     

Effect of complex nitrogen sources on the production of penicillin acylase byBacillus megaterium

The following complex nitrogen sources were evaluated for the production of penicillin acylase byBacillus megaterium: casein hydrolysate, corn steep liquor, stick water concentrate, blood meal and defatted sunflower meal. Experiments were run in shake flasks at 30‡C and pH 7.0. Best results were obtained with casein hydrolysate: 244 IU/I were produced with a productivity of 20.3 IU/l/h and yield of 717.6 IU/g of nitrogen. The lowest results correspond to sunflower meal with 39 IU/1.     

Reverse osmosis processing of organic model compounds and fermentation broths

Post-treatment of an anaerobic fermentation broth was evaluated using a 150 gal/day, single cartridge prototype reverse osmosis (RO) system. Baseline tests were conducted at 25 degrees C using six organic model compounds representing key species found in the fermentation broth: ethanol, butanol, acetic acid, oxalic acid, lactic acid, and butyric acid. Correlations of the rejection and recovery efficiencies for these organic species, individually and in simulated mixtures, were obtained as a function of feed pressure with and without recirculation of the retentate. The actual fermentation broth obtained from a continuous-flow biohydrogen process was treated by the RO system under the operating conditions similar to those used in the baseline tests, resulting in greater than 95% removal of total organic carbon. These results are encouraging and useful for further studies on the feasibility of incorporating the RO technology into an integrated and field deployable wastewater management and water recovery system.     

Influence of microbial concentration on the rheology of non-Newtonian fermentation broths

The objective of this study was to quantify the effect of fungal biomass concentration on the rheology of non-Newtonian fermentation systems. Batch fermentations of Penicillium chrysogenum were carried out with glucose as the sole carbon source. The flow behavior of the system was characterized at various fermentation times and was adequately described by the power-law model. The apparent viscosity of the fermentation broth was significantly affected by biomass concentrations in the fermenter. Fermentation broths containing 17.71 g/l biomass as dry weight were characterized by an apparent viscosity of 0.25 Pa s at a shear rate of 50 s⁻¹. Microbial concentration also affected the power-law flow-behavior index and the consistency index. The value of the consistency index ranged from 0.002 Pa s ⁿ at a biomass concentration of 0.1 g/l to 6.14 Pa s ⁿ at a biomass concentration of 17.71 g/l. The flow-behavior index decreased from an initial value of 1 to a final value of 0.17. Simple empirical correlations have been proposed to quantify the dependence of the power-law parameters on fungal biomass concentration. Experimental data obtained in this study were accurately described by these correlations. The general applicability of these relationships was tested, using previously published rheological data on Aspergillus awamori and Aspergillus niger fermentation broths, and good agreement was seen between experimental data and the predictions from the empirical correlations. Received: 24 March 1998 / Received revision: 10 September 1998 / Accepted: 16 October 1998     

Separation of L-valine from fermentation broths using a supported liquid membrane

A carrier-mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L-valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days.     

Antibiotic purification from fermentation broths by counter-current chromatography: analysis of product purity and yield trade-offs

Counter-current chromatography (CCC) is a low pressure, liquid–liquid chromatographic technique which has proven to be a powerful purification tool for the high-resolution fractionation of a variety of active pharmaceutical compounds. The successful integration of CCC into either existing or new manufacturing processes requires the predictable purification of target compounds from crude, fermentation-derived, feed streams. This work examines the feasibility of CCC for the purification of fermentation-derived erythromycin A (EA) from its structurally and chemically similar analogues. At the laboratory scale, the effect of feed pre-treatment using either clarified, forward extracted (butyl acetate) or back extracted broth on EA separation was investigated. This defined the degree of impurity removal required, i.e. back extracted broth, to ensure a reproducible elution profile of EA during CCC. Optimisation and scale-up of the separation studied the effects of mobile phase flow (2–40 mlmin^–1) and solute loading (0.1–10 g) on the attainable EA purity and yield. The results in all cases demonstrated a high attainable EA purity (>97% w/w) with throughputs up to 0.33 kgday^–1. Secondly, a predictive scale-up model was applied demonstrating, that from knowledge of the solute distribution ratio of EA (K_EA) at the laboratory scale, the EA elution time at the pilot scale could be predicted to within 3–10%, depending upon the solute injection volume. In addition, this study has evaluated a fractionation diagram approach to visually determine the effects of key operational variables on separation performance. This resulted in accurate fraction cut-point determination for a required degree of product purity and yield. Overall, the results show CCC to be a predictable and scaleable separation technique capable of handling real feed streams.     

Comparative analysis of oxygen transfer rate distribution in stirred bioreactor for simulated and real fermentation broths

Study of the distribution of the oxygen mass transfer coefficient, k _l a, for a stirred bioreactor and simulated (pseudoplastic solutions of carboxymethylcellulose sodium salt) bacterial (P. shermanii), yeast (S. cerevisiae), and fungal (P. chrysogenum free mycelia) broths indicated significant variation of transfer rate with bioreactor height. The magnitude of the influence of the considered factors differed from one region to another. As a consequence of cell adsorption to bubble surface, the results indicated the impossibility of achieving a uniform oxygen transfer rate throughout the whole bulk of the microbial broth, even when respecting the conditions for uniform mixing. Owing to the different affinity of biomass for bubble surface, the positive influence of power input on k _l a is more important for fungal broths, while increasing aeration is favorable only for simulated, bacterial and yeast broths. The influence of the considered factors on k _l a were included in mathematical correlations established based on experimental data. For all considered positions, the proposed equations for real broths have the general expression k_l a = aC_X^b ( \fracP_a V )^g v_S^d , k_{\rm l} a = \alpha C_{\rm X}^{\beta } \left( {{\frac{{P_{\rm a} }}{V}}} \right)^{\gamma } v_{\rm S}^{\delta } , exhibiting good agreement with experimental results (with maximum deviations of ±10.7% for simulated broths, ±8.4% for P. shermanii, ±9.3% for S. cerevisiae, and ±6.6% for P. chrysogenum).     

The foams of fermentation broths. I. Some parameters of the foaming of fermentation media

The stability of foam formed during fermentation is decisively affect ed by the nature of the nutrient media used. In froth-flotation models, (a) the foam formation time, characteristic of the tendency to foam, and (b) foam subsistence time, characteristic of the stability of foams formed, have been studied. With the utilization of these two parameters, the stability of foam from aqueous solution of several surface active components of nutrient media has been noted as a function of concentrations. Further, but, without attempting completeness, the viscosity enhancing effect of carbohydrate components, and the effect of the subsistence time of their foam, upon the stability of foam have been studied together with the correlation between “standing” time after sterilization and tendency to foam. Taking soy-bean meal as a model, the stability of foam films in function of pi I, at constant concentration, has been studied. It seems that though a proper control of the factors mentioned, nutrient media with a low tendency of foaming can be formulated.