Fluidized bed adsorption for whole broth extraction.

Fluidized bed adsorption using a high-density synthetic resin has proven to be an invaluable technique for separating novel compounds from unfiltered fermentation broths during the very early stages of fermentation development, where product concentrations are typically in the parts per million range. Previous initial downstream processing strategies consisted of cell separation from whole broth or direct extraction with water-immiscible solvents, both of which resulted in lengthy time cycles, conflicts with existing operations requiring the use of high-cost centrifugal separators, and environmental/solvent recovery concerns. Laboratory and subsequent pilot plant process development work along with concomitant improvements in yield, quality, and time cycles are presented for one of several fluidized bed processes piloted in Merck's Natural Product Isolation facility.     

Investigations on cephalosporin C adsorption kinetics and equilibria

Summary The kinetics and equilibria of cephalosporin C adsorption on different commercial adsorbents were investigated. Adsorption isotherms could be analysed according to the Brunauer, Emmett and Teller theory. For the interpretation of adsorption kinetics it was necessary to develop a more complex model comprising both a rapid and a slower step. An integrated approach combining kinetics and equilibria allowed for simulation of the experimental data and can be used as a basis for predicting technical approaches such as fluidized-bed technology.Dedicated to Professor F. Wagner on the occasion of his 60th birthdayOffprint requests to: K. Buchholz     

Fluidized bed biomethanation of acetic acid

Summary The kinetics of acetate biomethanation was studied in a high recycle ratio biological fluidized bed reactor behaving in practice as a completely mixed reactor. The active biofilm consisted of bacteria from a methane fermenter that after spontaneous immobilization on the bed particles (sand) were adapted to acetate as the only carbon source. The effects of temperature (13°, 20°, 25° and 35°C), substrate concentration (500, 1000 and 1500 mg chemical oxygen demand (COD) l^-1) and hydraulic retention time (1 to 8 h) on substrate consumption were studied. Maximum substrate consumption (as % COD reduction) amounted from 25% (13°C, 1500 mg COD l^-1) to 93% (35°C, 500 mg COD l^-1). At 35°C the concentration of attached biomass presented a weakly increase with reactor substrate concentration (from 3.10 g VS l^-1 to 4.54 g VS l^-1 for 32 and 1150 mg COD l^-1 respectively). On the other hand when reducing , a sharp incrase in biomass loss coefficient was observed showing that excess biofilm growth was continuously removed by shearing forces. Thus in the assayed conditions the attached biomass concentration was basically determined by the bed superficial velocity. Result show that diffusional resistances are negligible. Data are fairly well correlated by a variable order kinetic model. The apparent reaction order is a function of temperature and increases from 0.27 to 0.7 when temperature decreases from 35° C to 13°C.Nomenclature b Total biomass loss coefficient (T^-1) - J Flux of substrate removal into the biofilm surface (ML^-2 T^-1) - J _d Flux of substrate removed into the biofilm surface in deep conditions (ML^-2 T^-1) - k Maximum specific rate of substrate utilization (T^-1) - K Variable order kinetic constant (T^-1 M^n-1 L^3n-3) - K _s9 Hall saturation constant (ML^-3) - n Reaction order - q Feed flow rate (L³ T^-1) - S Substrate concentration (ML^-3) - Se Effluent substrate concentration (ML^-3) - So Influent substrate concentration (ML^-3) - Se_min Minimum substrate concentration able to sustain a steady-state biofilm (ML^-3) - T Temperature - t Time(T) - V Bed volume (L³) - VS Volatile solids (M) - VSS Volatile suspended solids - X Attached biomass concentration (ML^-3) - X _c Effluent volatile suspended solids (ML^-3) - Y Yield coefficient - Hydraulic retention time (T) This work forms part of a Doctoral Thesis of senior author     

Development of the cephalosporin C fermentation taking into account the instability of cephalosporin C

Summary Cephalosporin C undergoes chemical hydrolysis during fermentation at 25°C at a rate of 0.48% per hour to give increasing amounts of 2-(D-4-amino-4-carboxybutyl)thiazole-4-carboxylic acid (compound X). Although the cephalosporin C titer in a fermentation levels off at 160–170h, the total of C+X rises almost linearly up to 200h. Cells, therefore, biosynthesize cephalosporin C at an almost constant rate up to 200h. This instability greatly influences attempts to improve the cephalosporin strain by genetic manipulation, or the process by alterations in technology.     

Factors affecting cephalosporin C biosynthesis

Summary Caprolactam, butan-2-one, butan-2-ol, and butan-1, 3-diol were found to have a stimulating effect on the biosynthesis of cephalosporin C by a mutant strain ofCephalosporium. Caprolactam was shown to have a synergistic effect, when added to the fermentation medium together with each of the other three compounds.     

Fluidized bed design parameters affecting novel lactic Acid downstream processing.

Lactic acid purification was directly done from fermentation utilizing a fluidized bed column refilled with a strong anionic exchange resin. The purpose of this work was to study the influence of two important design parameters, bed-diameter (D) and bed-height (H), in the lactic acid binding and elution capacity of the matrix. By changing the settled bed height from 2.5 to 5 cm for each diameter of column analyzed it was possible to obtain an 50% increase in the binding capacity of the resin in all experiments. This fact was attributed to a higher contact time between the culture broth and the anionic resin produced by the increase of back mixing and lactic acid residence time.     

Methionine control of cephalosporin C formation

DL -Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.     

Extraction of cephalosporin C from whole broth and separation of desacetyl cephalosporin C by aqueous two-phase partition

Cephalosporin C was extracted from diluted or whole broth by PEG/salt aqueous two-phase systems. Parameters such as PEG molecular weight, salt type, pH, and salt concentration were investigated for finding a suitable extraction system. In PEG 600/ammonium sulfate or phosphate systems, K(c) (partition coefficienct of cephalosporin C) was observed to be larger than 1, with K(d) (partition coefficient of desacetyl cephalosporin C) being smaller than 1. The particular values of these coefficients would imply that the difficult separation of cephalosporin C and desacetyl cephalosporin C could possibly be achieved via the aqueous two-phase extraction. The addition of surfactants, water-miscible solvents, and neutral salts for enhancement of the separation efficiency was also investigated. The addition of surfactants to the system did not affect the separation efficiency substantially. K(c) would increase whereas K(d) decreased as a result of the addition of acetone, MeOH, EtOH, IPA, and n-BuOH. Meanwhile both K(c) and K(d) would decrease whenever neutral salts, NaCl, KCl, Kl, or KSCN, were added. The partitioning behavior of cephalosporin C and desacetyl cephalosporin C in filtered, whole, and different batches of broth was notably quite similar to that of diluted broth. The recovery yield of cephalosporin C in whole broth extraction was observed to be a function of centrifugal force used in phase separation. (c) 1994 John Wiley & Sons, Inc.     

Expanded bed adsorption: elution in expanded bed mode

Elution in expanded bed mode has been investigated in the expanded bed adsorption process. Elution was performed at different sample loads and at different liquid velocities using bovine serum albumin as a model. The effect on mixing in the liquid phase and on the volume of the eluted peak were determined. Mixing in the liquid phase was almost unaffected when elution was performed at 100 cm/h, regardless of sample load. However, mixing increased significantly when elution was carried out at high liquid velocities (300 cm/h) at high sample loads. The eluted peak volume increased with liquid velocity and increased sample load. It was approx. 80% higher in expanded bed mode than in packed bed from an adsorbent completely saturated with protein eluted at 300 cm/h.     

Separation of cephalosporin C and desacetyl cephalosporin C by high speed counter-current chromatography in aqueous two-phase systems

Summary In the recovery of cephalosporin C (CPC) from fermentation broth, the separation of desacetyl cephalosporin C (DAC) is a major concern. Multistage extraction in aqueous two-phase systems, mainly PEG/ammonium sulfate systems, proved to be promising. In preparative scale operation, high speed counter-current chromatography (HSCCC) with eccentric columns was used with aqueous two-phase systems to obtain baseline resolution of CPC and DAC. Solvents (e.g. 5% acetone) or neutral salts (e.g. 1.45% KSCN) added into aqueous two-phase systems enhanced the separation efficiency. Operation parameters of HSCCC such as rotational speed and mobile phase flow rate can affect the retention of the stationary phase and HETP.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of an acylase active on cephalosporin C

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.