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Production of cephalosporin C by Acremonium chrysogenum in semisynthetic medium
Process Biochemistry 38 (2002) 263 /272 www.elsevier.com/locate/procbio
Production of cephalosporin C by Acremonium chrysogenum in semisynthetic medium M. Ju¨rgens a, G. Seidel b, K. Schu¨gerl a,* a
b
Institute for Technical Chemistry, University Hanover, Callinstrasse 3, D-30167 Hannover, Germany Aventis Pharma, Deutschland GmbH, Wirkstoffe, Wirkstoffproduktion Biotechnik Industriepark Hoechst, D-65926 Frankfurt am Main, Germany Received 12 February 2002; accepted 19 March 2002
Abstract The production of Cephalosporin C by Acremonium chrysogenum 3/2 (C3) strain was performed in shake flasks with semisynthetic medium and corn steep liquor (CSL) supplement, after removal of the sediment and the sediment and proteins from the medium, respectively. Product formation was also carried out in shake flasks with semisynthetic media without buffer and with different type of buffers, to avoid the strong fall in pH during cultivation. In addition, cultivations were carried out in 10 and 30 l bioreactors with semisynthetic medium and pH control in batch operation without and with oxygen supplement. The performances of these cultivations were compared with each other and with cultivations in 30 l reactors with complex media in batch and fed-batch operation. Fed-batch cultivations with complex media had the highest performance. Batch cultivations with semisynthetic media and pH control have nearly the same performance as batch cultivation with complex media. Cultivations with semisynthetic media with pH control had a better production than those with buffers. Complex media after removal of the sediment of CSL and after separation of sediments and proteins of CSL had low performances. Semisynthetic media without buffer had the lowest performance. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acremonium chrysogenum ; Cephalosporin C; Complex media; Semisynthetic media; Osmotic pressure; Shake flask cultivation; Controlled cultivation
1. Introduction Production of antibiotics on complex medium has the advantages of low raw material cost and high productivity, but the disadvantages of high energy cost, because of high viscosity and formidable process monitoring. The determination of the biomass concentration is especially laborious, because of the solid content of the cultivation medium. The recovery and purification of the product is difficult because of the great number of (non well defined) medium components. The evaluation of the influence of the medium composition on the cell growth and product formation is demanding because of the complex interaction of not well defined medium components. Process engineering aspects of the produc-
* Corresponding author. Tel./fax: /49-511-762-2253. E-mail address: [email protected] Schu¨gerl).
(K.
tion of cephalosporin C (CPC) by Acremonium chrysogenum by using complex media was considered earlier [1,2]. In the present paper the production of CPC by the same strain, but in semisynthetic medium is investigated. The development of cultivation medium is usually performed in shake flasks, in which the control of process variables is not possible. In particular, the uncontrolled variations of the pH-value and the dissolved oxygen concentration (pO2) can influence the results of the investigations. In complex media the strong variation of the pH-value is partly suppressed by the high buffer capacity of the medium. In semisynthetic media no such effects exist. Therefore, it is necessary to supplement the medium with buffers to avoid the excessive drop in pH. In the first part of this paper the influence of various buffers is investigated in shake flasks on the biomass and product formation. In the second part cultivations are performed in a bioreactor under well controlled conditions.
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 8 0 - 8
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Table 1 Composition of the semisynthetic cultivation medium Compound
Concentration (g l 1)
Compound
Concentration (g l 1)
Glucose NH4SO4 Na2SO4 KH2 PO4 L-valine DL-alanine L-asparagin L-methionine L-arginine Urea
35 4.2 5 3.1 1 2 3.33 14.67 3.33 2.1
L-serine
1 20a 1 ml 6.67 0.09 0.03 0.02 0.02
a
Soy oil Desmophen (antifoam agent) MgSO4 FeSO4 MnSO4 ZnSO4 CuSO4
40 g l 1 soy oil concentration in cultivations in the 10 l bioreactor.
Most investigations of the production of CPC were performed in synthetic medium (e.g. [3 /10]). The authors often used a synthetic medium in shake flasks with 2-morpholinoethansulphanic acid (MES) and 3morpholinopropansulphonic acid (MOPS) buffers, respectively, which were recommended by Aharonowitz and Demain, because, they do not influence growth and Cephalosporin production by Streptomyces clavuligerus [11]. The same buffers are used in the present paper in various concentrations and in addition an inorganic phosphate buffer. Cultivations in 10 and 30 l bioreactors were performed by close process monitoring and control and compared with cultivations on complex media.
2. Materials and methods 2.1. Strain and pre- and main cultures The A. chrysogenum 3/2(C3) strain was donated by HMR Deutschland. Each of the ampoules with 2.2 ml spore suspension were stored in liquid nitrogen. Agar slant cultures of the strain were used to inoculate a 1 l shake flask with 160 ml malt and yeast containing medium which was sterilised before at 121 8C for 20 min. This was cultivated on a rotary shaker with 280 rpm at 25 8C for 72 h. This was the preculture for 10 and 30 l bioreactors. 2.1.1. Shake culture runs Thirty ml medium in a 500 ml shake flask was autoclaved at 121 8C for 20 min and after cooling 3 ml sterilised concentrated sugar solution was added to the flasks. A 3 ml inoculum was added to it and cultivated at 25 8C on rotary shaker at 280 rpm for 160 /225 h. 2.1.2. 10 l Bioreractor runs In the 10 l volume bioreactor (BIOSTAT C, Braun Melsungen) the semisynthetic cultivation medium (Ta-
ble 1) autoclaved at 121 8C for 30 min and inoculated 160 ml preculture, which was prepared in a 1 l shake flask and cultivated at 25 8C for 72 h on a rotary shaker with 280 rpm. 2.1.3. 30 l Bioreactor runs A 30 l volume bioreactor (BIOSTAT UD Braun Melsungen) was used for these runs. The reactor was filled with 25 l semisynthetic cultivation medium (Table 1), sterilised at 121 8C for 30 min cooled to 25 8C and stored for 12 h. The amount of inoculum was 19% of the medium volume. 2.2. Process monitoring For determination of the biomass concentration 10 ml sample was centrifuged at 3800 rpm for 10 min (Laborfuge GL, Heaeus Crist), the sediment was dried at 110 8C for 72 h and weighed. An Olympus BH2microscope was used for the observation of the morphology of the fungus. Phosphate concentration was determined by the phosphate test (BMB) of Merck. Total sugar content was determined by a p -hydroxybenzoic-acid-hydrazide (pHBAH)-method. Glucose and lactate concentrations were determined by the equipment of Yellow Springs Instruments. Protein concentration was evaluated by the method of Bradford. Ammonia concentration was measured by a German standard method (DIN 38 406). Amino acid concentrations were determined by HPLC consisting of an autosampler (Pharmacia), degasser (Irica), precolumn and analytical column (ResovedTM C18, Waters), thermostate (Julabo), fluorescence detector (Shimadzu RS535), control unit (Autochrom CS12 Interface), gradient control (Autochrom CIM interface) and data processing (APEX chromatography workstation). Samples were treated with methanol and centrifuged at 0 8C to separate proteins. The protein free probes were diluted with borate buffer pH 10 to 1/20 (in general) and 1/2000 (for methionine determination), respectively. Ten ml sample is mixed with 10 ml OPA-reagent and
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
injected after 2 min reaction time into the column. The analysis is performed at 25 8C. A mixture of 96% buffer solution (33 mM Na-acetate, 38 mM Na phosphate, pH 7), 2% methanol and 2% tetrahydrofurane was used as eluent A. Methanol (54%) in bidistilled water was applied as eluent B. Within 5 min the eluent was changed from 0 to 30% B, and within 35 min it was rised to 100%. The duration of the analysis was 55 min. The thiole containing amino acids (L-cystein) and peptides (ACV) cannot be analysed with this method and they need a sample preconditioning with OPA / MCE (mercaptoethanol) according to Holzhauer/Rieger [12]. Samples were injected to the column after derivatisation with OPA /MCE. Analysis of the product and precursors were performed by HPLC consisting of a pump (Irica), injection valve (Valco). degasser, precolumn, column (Macherey and Nagel), thermostate (Julabo), two channel UVdetector (Gynotek). On-line analysis was performed by a cooled autosampler (Gilson /1222, abimed) and a controller (CSI2 interface). Data logging and evaluation were carried out on a PC (486 DX66) by an APEX program. A packed column with Nucleosil 5† with reverse phase mode was operated at 30 8C with a mixture of 5% methanol, 95% 14 mM phosphate buffer and 10.2 mM tetrabutylammoniumhydrohensulphate (TBAHS) pH 6.5 as eluent, which was degassed in an ultrasonic bath for 20 min at a wavelength of 2260 nm. The standards for CPC, DAC, DAOC and PENN were donated by HMR Deutschland. For more details see [13]. The reactors were provided with thermometer (Pt100), pH-meter (Pa 12/120, Ingold), pO2-electrode (Ingold) and off gas analyser (EGAS1 (Braun) with O2 and CO2 detectors. The reactors were equipped with measuring and control system (DCU, RS 422) and a PC as supervisor.
3. Results 3.1. Investigations of shake flask cultivations One hundred and ten grams per liter corn steep liquor (CSL) was added to the basic cultivation media (Table 1). CSL has a solid sediment content of 40 /50%. By centrifugation at 4500 rpm the sediment was removed and the solid particle free liquor was used for cultivation The medium contained 46 g l 1 dry glucose syrup (TGS) instead of glucose and had an initial pH 6.44 (run 1). Another cultivation was performed with the same basic cultivation media (Table 1) and with 110 g l 1 CSL. The sediment was removed by centrifugation at 4500 rpm and the sediment free liquor was treated with ZEN-medium at 100 8C for 60 min [14]. The precipitate
265
was removed by centrifugation at 4500 rpm and the liquor was used for cultivation. The medium contained 46 g l 1 TGS instead of glucose. The initial pH was 6.44 (run 2). The semisynthetic media did not contain CSL and TGS (Table 1). Their initial pH was 6.44. These cultivations were performed / / / / /
without buffer (run 3), with 50 mM MES-buffer (run 4), with 100 mM MES buffer (run 5), with 50 mM phosphate buffer (run 6), with 200 mM MOPS buffer (run 7).
In all of these cultivations the following process variables were monitored: pH-value, (dry) biomass, total sugar, phosphate, lactate, ammonia, asparagine, arginine, alanine, methionine, a-amino adipinic acid, cystein, valine, penicillin N (PENN), deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC), cephalosporin C (CPC). 3.1.1. Run 1 Cell growth started without lag phase. The maximum (dry) biomass concentration was 45 g l 1. The concentrations of phosphate, sugars, lactate, asparagine, arginine, alanine, methionine and valine were consumed and exhausted upto about 100 h. pH drops to 5.5 at 70 h and than increased to 8.5. Ammonia passed a minimum at 87 h, the concentrations of cystein and a-aminoadipinic acid increased with the time. Precursor concentrations were below 1.5 g l 1. The concentration of CPC attained a maximum (6.5 g l 1) at 112 h. 3.1.2. Run 2 Cell growth had no lag phase. The maximum (dry) biomass concentration was 35 g l 1. The consumption rates of sugars, lactate, amino acids were lower than those in run 1. The pH value passed a minimum (5.4) at 120 h and increased to 7.0. Ammonia concentration passed a minimum at the same time and increased only slightly. The DAOC and DAC concentrations were low (below 0.5 g l 1) PENN concentration below 1.0 g l 1 and the CPC concentration increased and attained 3.5 g l 1 at the end of the cultivation (175 h). 3.1.3. Run 3 The cultivation started with a long lag phase. The maximum (dry) biomass concentration was 25 g l 1. Glucose was consumed slowly and exhausted at 165 h. The pH value dropped to 4.25 at 120 h and remained low. The ammonia concentration diminished parallel to pH. The concentrations of asparagine, arginine and valine varied only slightly. Alanine was exhausted at 120 h. The concentration of methionine gradually dropped to half of its original value. Precusor concentrations
266
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were below 0.1 g l 1. The maximum CPC concentration (1.0 g l 1) was attained at 120 h and then diminished slowly. 3.1.4. Run 4 No lag phase was observed. Maximum (dry) biomass concentration (35 g l 1) was obtained at 112 h and it remained constant. The pH gradually dropped to 4.75. The ammonia concentration diminished parallel with pH. Glucose was exhausted at 110 h. The amino acid concentrations gradually diminished to about 0.3 of their initial value. Alanine was exhausted at 130 h. Valine was exhausted at 210 h. PENN concentration attained 1.9 g l 1 at the end of the cultivation. CPC concentration had its maximum (3.4 g l 1) at 175 h. 3.1.5. Run 5 No lag phase was observed. The (dry) biomass concentration gradually increased to 40 g l 1. Glucose was exhausted at 95 h. pH-value passed a minimum ((5.0) at 120 h and increased to 5.45. The course of ammonia ran parallel to pH. Valine and alanine were exhausted 187 h, asparagine at 212 h. The other amino acids were nearly exhausted at the end of the cultivation at 215 h. PENN concentration increased to 2.5 g l 1 and CPC concentration attained a maximum (4.0 g l 1) at 137 h. After that it varied only slightly. 3.1.6. Run 6 A lag phase existed. The (dry) biomass concentration attained a maximum (23 g l 1) at 130 h and remained constant. Glucose was exhausted at 112 h. The pH-value dropped to 4.75 at 187 h and than changed only slightly. Ammonia concentration run parallel to pH-value. Phosphate concentration passed a maximum (6.45) at 85 h and after that it diminished to 4.75. Valine was exhausted at 112 h. The concentrations of the other amino acids gradually decreased to about half of their initial values. PENN concentration attained a maximum (1.75 g l 1) at 137 h, and CPC concentration reached a maximum (3.2 g l 1) at 162 h. 3.1.7. Run 7 There was a short lag phase. The (dry) biomass concentration attained a maximum (21 g l 1) at 140 h. Glucose was exhausted at the same time. The pH-value passed a minimum (5.45) at 124 h. After that it increased to 6.7. Ammonia concentration run parallel to pHvalue. Alanine and valine were exhausted at 187 h. The other amino acid concentrations diminished to about 50% of their initial value at the same time. PENN concentration had a maximum value (1.0 g l 1) and the CPC concentration attained its maximum value (3.0 g l 1) at 190 h.
3.2. Investigations of cultivations in the 10 l bioreactors with semisynthetic medium 3.2.1. Run 8 In bioreactors the pH-value was controlled by 8 N NaOH and conc. H2SO4 solutions, respectively, and it was not necessary to use buffer supplements. The semisynthetic medium of the buffer free runs (Table 1) was used with 40 g l 1 soy oil concentration. CO2 production rate (CPR) and oxygen transfer rate (OTR) and optical density (OD) were determined in addition to the process variables of shake flask cultivations. The concentrations of the precursors and CPC were monitored on-line. The reactor was aerated with 0.5 vvm min 1. The dissolved oxygen concentration diminished during the batch cultivation from 100 to 0% at 110 h. Therefore, after 80 h oxygen limitation prevailed. The pH dropped between 20 and 40 h from 6.4 to 5.8. The pH was maintained at this value. The ammonia concentration followed this course and diminished from 1.75 to 0.5 g l 1. The concentration of the (dry) biomass gradually increased and attained 42 g l 1 at 120 h. The OD followed the same course. OTR increased to 29 mM l 1 h 1, because of the increasing biomass concentration. Glucose was exhausted at 79 h. CPR increased with the cultivation time, but it had two minima caused by the pH-shift at 40 h and caused by oxygen limitation at 80 h. Asparagine, alanine and valine were exhausted between 130 and 140 h. The concentration of arginine dropped to the half of its initial value and that of methionine to 0.3 of its initial value at 140 h. The concentrations of DAOC and DAC remained below 0.5 g l 1, PENN concentration increased to 2.5 g l 1 and that of CPC to 4.4 g l 1. CPC formation rate gradually increased to 0.07 g l 1 h 1 and methionin consumption rate attained two maxima: 0.06 g l 1 h 1 at 20 h and 0.175 g l 1 h 1 at 90 h. The steep reduction of the methionine consumption rate after these two maxima is caused by the drop of pH and the oxygen limitation, respectively. 3.2.2. Run 9 In order to avoid the oxygen limitation, in the second run the aeration rate was increased to 1 vvm and after pO2 dropped to 70%, oxygen was added to the pressurised air to keep pO2 at 100%. After a lag phase the biomass concentration increased to 47.5 g l 1 and after this maximum it slightly diminished. Glucose was exhausted at 75 h. This caused a minimum of CPR. The second minimum of CPR was caused by diauxie at 125 h. After 150 h CPR dropped, because, exhaustion of the soy oil. The pH-value diminished from 6.5 to 5.75 between 50 and 75 h and was maintained at this value. Ammonia followed the pH-change and dropped from 1.8 to 0.75 g l 1 during this time period and later to 0.25 g l 1. Valine was exhausted at 125 h, asparagin at 137 h
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
267
Fig. 1. pO2, pH and CPR in the 30 l bioreactor as a function of cultivation time.
Fig. 2. Concentrations of (wet) biomass (BFM) and (dry) biomasss (BMT) and OD in the 30 l reactor as a function of cultivation time.
and alanine, arginine and methionine at 160 h. The concentrations of DAOC and DAC remained below 0.7 g l 1. PENN concentration increased to 4 g l 1 and CPC concentration to 7.7 g l 1. The CPC formation rate gradually increased and attained a maximum of 0.11 g l 1 h 1 at 124 h. Methionine consumption rate passed two maxima: 0.175 g l 1 h 1 at 95 h and 0.30 g l 1 h 1 at 137 h. By abolishing the oxygen limitation the (dry) biomass concentration was increased from 42 to 47.5 g l 1, the CPC concentration from 4.4 to 7.7 g l 1 and the PENN concentration from 2.5 to 4 g l 1. Cell growth was less
influenced by oxygen limitation than the biosynthesis of CPC. 3.3. Investigation of cultivations in 30 l bioreactor on semisynthetic medium 3.3.1. Run 10 Batch cultivations in 10 l bioreactor were repeated in a 30 l reactor. The medium composition was the same (Table 1). The pO2 value was controlled and kept at 60% of the saturation value by addition of oxygen to the pressurised air. The pH-value was controlled by 8 N
268
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
Fig. 3. Concentrations of ammonia, phosphate and total sugar in the 30 l reactor as a function of cultivation time.
Fig. 4. Concentrations of methionine, alanine and asparagin in the 30 l reactor as a function of cultivation time.
NaOH and conc. H2SO4, respectively, and pH 5.8 was maintained during the cultivation. In Fig. 1 the courses of pO2, the pH value and CPR are shown as a function of the cultivation time. At 140 h the pO2 dropped to 60% and was maintained at this value. The pH value diminished from 6.3 to 5.8 during the first 60 h and kept at this value. CPR increased exponentially in the first 90 h, passed a minimum, because, at this time glucose was exhausted and the fungus had to change from glucose substrate to soy oil. In Fig. 2 the concentrations of (wet) biomass ( BFM) and (dry) biomass (BTM) are shown together with OD. The diauxic between 90 and 140 h can be clearly recognised.
The (dry) biomass concentration increases after the diauxic phase again and attained a final concentration of 40 g l 1. The difference between BFM, BTM and OD disappeared after 100 h. The concentrations of ammonia, phosphate and sugar are shown in Fig. 3. Sugar content was exhausted at 80 h. Phosphate concentration passed a maximum at 40 h, which is unexpected, and ammonia concentration gradually diminished from 1.8 to 0.5 g l 1. Alanine, asparagine and methionine were consumed at a nearly constant rate (Fig. 4), similar to serine, valine and glutamate (Fig. 5). Glutamine and cystein concentrations were very low. The concentration of a-aminoadipinic acid increased from 90 h nearly exponentially, which was unexpected. The concentrations of PENN, DAOC, DAC and CPC increased with time (Fig. 6). The final concentrations of b-lactam compounds were 0.13 g l 1 DAOC and DAC, 4.5 g l 1 PENN, and 7.7 g l 1 CPC. The CPC production rate had two minima: The first was caused by exhaustion of glucose at 90 h and the second appeared at 139 h. To obtain more information on the biosynthesis, the intracellular concentrations of ACV and proteins and ACV-synthetase (ACVS) activity were also determined. All three values passed a steep maximum at 90 h and slowly decreased further (Fig. 7). The concentration of PENN and the activities of isopenicillin N-synthetase (IPNS) are shown in Fig. 8. PENN formation started at 50 h, passed a maximum at 65 h and after 75 h increased to a maximum concentration of 1.5 g l 1. IPNS activity started to increase also at 50 h. After a steep increase it stagnated from 75 h and at 120 h further increased to a maximum activity of 20 U g 1. The courses of ACV and
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
269
Fig. 5. Concentrations of some extracellular aminoacids in the 30 l bioreactor as a function of cultivation time.
Fig. 6. Concentrations of b-lactam precursors: PENN, deacetoxycephalosrotin C (DAOC), deacetyl cephalosporin C (DAC) and the product: CPC as well as the CPC-formation rate in the 30 l bioreactor as a function of cultivaton time.
PENN as well as ACVS and IPNS activities are similar to those observed in cultivations with complex medium [1].
4. Discussion 4.1. Comparison of the shake flask cultivations By the removal of the sediment of CSL in run 1, a maximum concentrations of CPC of 6.5 g l 1 and (dry) biomass of 45 g l 1 were obtained. After the precipita-
tion of the dissolved proteins in run 2 the maximum concentration of CPC was reduced to 3.5 g l 1 and that of (dry) biomass diminished to 35 g l 1. In Table 2 the osmotic pressure, buffer capacity and maximum CPC concentrations of runs 3 /7 with semisynthetic medium were compiled. Without buffer only 1 g l 1 CPC and 25 g l 1 (dry) biomass were obtained. With increasing buffer capacity the osmotic pressure enlarged, but the biomass and CPC production was not influenced. In run 5 with an osmotic pressure of 16.10 bar the highest CPC concentration of 4.0 g l 1 and (dry) biomass concentration of 40 g l 1 were obtained.
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
270
Fig. 7. Intracellular concentration of ACV and proteins and specific activity of ACVS in the 30 l bioreactor as a function of cultivation time.
Fig. 8. Intracellular concentration of isopenicillin N (IPN) and proteins and specific activity of IPNS in the 30 l bioreactor as a function of cultivation time.
Table 2 Osmotic pressures, buffer capacities and maximum CPC concentrations of the semisynthetic media (runs 3 /7) Run
3 4 5 6 7
Buffer
No buffer 50 mM MES 100 mM MES 50 mM PO3 4 200 mM MOPS
Osmotic pressure (bar)
13.623 14.86 16.10 14.43 18.58
CPC concentration (g l 1)
Buffer capacity (mM) pH 5.5
pH 6.0
pH 6.5
1.94 19.09 36.66 5.38 13.26
4.57 25.0 56.25 11.80 47.22
10.08 21.91 45.45 20.0 100.0
1.0 3.4 4.0 3.2 3.0
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Table 3 Production of CPC by A. chrysogenum 3/(C3) strain in semisynthetic and complex media under various cultivation conditions Run Cultivation conditions
Biomass concentration PENN concentration (g l 1) (g l 1)
1 2
45 35
2.5 1.0
6.5 3.5
25 40
B 0.1 2.5
1.0 4.0
42 47.5
2.5 4.0
4.4 7.7
40
4.5
7.7 9.5
[1]
23.0
[1]
3 5 8 9 10
Complex with sediment separation shake flask culture Complex with sediment and protein separation shake flask culture Semisynthetic without buffer shake flask culture Semisynthetic with 100 mM MES buffer shake flask culture Semisynthetic with pH control, batch 10 l reactor Semisynthetic pH control, batch Oxygen supplement 10 l reactor Semisynthetic with pH control, batch 30 l reactor Complex medium pH control, batch 30 l reactor oxygen supplement Complex medium pH control, fed-batch 30 l reactor oxygen supplement
98
3.2
CPC concentration (g l 1)
Reference
Comparison of maximum concentrations of biomass, CPC and PENN.
Obviously at too high osmotic pressure (18.58 bar) the biomass and product formation were impaired and the maximum CPC concentration reduced to 3.0 g l 1 and the maximum (dry) biomass concentration diminished to 21 g l 1. Phosphate buffer was less suitable for suppression of the pH-change. A comparison of runs 4 and 6 indicate, that at about the same osmotic pressures (14.8 and 14.4. bar) with MES buffer a somewhat higher CPC concentrations (3.4 vs. 3.2 g l 1) and higher biomass concentrations (25 vs. 23 g l 1) was obtained than with phosphate buffer. However, the difference was dramatic. 4.2. Comparison of shake flask and reactor cultivations A comparison of the cultivations with buffer with those of pH-control indicates, that pH control was superior to buffering of the medium. The best cultivation with buffer resulted 40 g l 1 (dry) biomass concentration and 4 g l 1 CPC concentration. The pH-control for non-oxygen limited cultivation resulted 47.5 g l 1 (dry) biomass concentration and 7.7 g l 1 CPC concentration. CPC formation was more sensitive to osmotic pressure than the cell growth. A comparison of cultivations in 10 l reactor without and with oxygen supplement shows than oxygen limitation reduced the biomass from 47.5 to 42 g l 1, and the CPC concentration from 7.7 to 4.4 g l 1. The biosynthesis of CPC is more sensitive to oxygen limitation than the cell growth [15]. In the 10 and 30 l reactors under the same cultivation conditions similar results were obtained. A comparison of batch cultivation in semisynthetic and complex media in 30 l reactor shows that with suitable synthetic medium composition (7.7 g l 1 CPC)
similar results can be obtained than with complex medium (9.5 g l 1 CPC) (Table 3). A comparison of batch and fed-batch cultivations in 30 l bioreactor with the same medium composition indicates that with fed-batch operation much better results were obtained (23 g l 1 CPC) than with the batch operation (9.5 g l 1 CPC).
Appendix A: Nomenclature ACV ACVS Arg Asp BFM BMT CPC CPR CSL Cys DAC DAOC Glu Gln IPN IPNS MCE MES MOPS Met OD OPA OTR PHBAH
a-L-amimnoadipyl-L-cyteinyl-D-valine ACV synthetase arginine aspartate (wet) biomass (dry) biomass cephalosporin C CO2 formation rate corn steep liquor cystein deacetyl cephalosporin C deacetoxy cephalosporin C glutamate glutamin isopenicillin IPN synthetase mecaptoethanol 2-morpholinoethansulphanic acid 3-morpholinopropansulphonic acid methionine optical density o -phtalaldehyd oxygen transfer rate p -hydroxybenzoic acid hydrazide
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272
pO2 PENN Ser TGS Val
dissolved oxygen concentration with regard to its saturation penicillin N serine dry glucose syrup valine
Acknowledgements This work was financed by HMR Deutschland, Hoechst. The authors thank Dr B. Ko¨nig, HMR Deutschland and Biochemie Kundl, for his support.
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Production of cephalosporin C by Acremonium chrysogenum in semisynthetic medium M. Ju¨rgens a, G. Seidel b, K. Schu¨gerl a,* a
b
Institute for Technical Chemistry, University Hanover, Callinstrasse 3, D-30167 Hannover, Germany Aventis Pharma, Deutschland GmbH, Wirkstoffe, Wirkstoffproduktion Biotechnik Industriepark Hoechst, D-65926 Frankfurt am Main, Germany Received 12 February 2002; accepted 19 March 2002
Abstract The production of Cephalosporin C by Acremonium chrysogenum 3/2 (C3) strain was performed in shake flasks with semisynthetic medium and corn steep liquor (CSL) supplement, after removal of the sediment and the sediment and proteins from the medium, respectively. Product formation was also carried out in shake flasks with semisynthetic media without buffer and with different type of buffers, to avoid the strong fall in pH during cultivation. In addition, cultivations were carried out in 10 and 30 l bioreactors with semisynthetic medium and pH control in batch operation without and with oxygen supplement. The performances of these cultivations were compared with each other and with cultivations in 30 l reactors with complex media in batch and fed-batch operation. Fed-batch cultivations with complex media had the highest performance. Batch cultivations with semisynthetic media and pH control have nearly the same performance as batch cultivation with complex media. Cultivations with semisynthetic media with pH control had a better production than those with buffers. Complex media after removal of the sediment of CSL and after separation of sediments and proteins of CSL had low performances. Semisynthetic media without buffer had the lowest performance. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acremonium chrysogenum ; Cephalosporin C; Complex media; Semisynthetic media; Osmotic pressure; Shake flask cultivation; Controlled cultivation
1. Introduction Production of antibiotics on complex medium has the advantages of low raw material cost and high productivity, but the disadvantages of high energy cost, because of high viscosity and formidable process monitoring. The determination of the biomass concentration is especially laborious, because of the solid content of the cultivation medium. The recovery and purification of the product is difficult because of the great number of (non well defined) medium components. The evaluation of the influence of the medium composition on the cell growth and product formation is demanding because of the complex interaction of not well defined medium components. Process engineering aspects of the produc-
* Corresponding author. Tel./fax: /49-511-762-2253. E-mail address: [email protected] Schu¨gerl).
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tion of cephalosporin C (CPC) by Acremonium chrysogenum by using complex media was considered earlier [1,2]. In the present paper the production of CPC by the same strain, but in semisynthetic medium is investigated. The development of cultivation medium is usually performed in shake flasks, in which the control of process variables is not possible. In particular, the uncontrolled variations of the pH-value and the dissolved oxygen concentration (pO2) can influence the results of the investigations. In complex media the strong variation of the pH-value is partly suppressed by the high buffer capacity of the medium. In semisynthetic media no such effects exist. Therefore, it is necessary to supplement the medium with buffers to avoid the excessive drop in pH. In the first part of this paper the influence of various buffers is investigated in shake flasks on the biomass and product formation. In the second part cultivations are performed in a bioreactor under well controlled conditions.
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 8 0 - 8
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Table 1 Composition of the semisynthetic cultivation medium Compound
Concentration (g l 1)
Compound
Concentration (g l 1)
Glucose NH4SO4 Na2SO4 KH2 PO4 L-valine DL-alanine L-asparagin L-methionine L-arginine Urea
35 4.2 5 3.1 1 2 3.33 14.67 3.33 2.1
L-serine
1 20a 1 ml 6.67 0.09 0.03 0.02 0.02
a
Soy oil Desmophen (antifoam agent) MgSO4 FeSO4 MnSO4 ZnSO4 CuSO4
40 g l 1 soy oil concentration in cultivations in the 10 l bioreactor.
Most investigations of the production of CPC were performed in synthetic medium (e.g. [3 /10]). The authors often used a synthetic medium in shake flasks with 2-morpholinoethansulphanic acid (MES) and 3morpholinopropansulphonic acid (MOPS) buffers, respectively, which were recommended by Aharonowitz and Demain, because, they do not influence growth and Cephalosporin production by Streptomyces clavuligerus [11]. The same buffers are used in the present paper in various concentrations and in addition an inorganic phosphate buffer. Cultivations in 10 and 30 l bioreactors were performed by close process monitoring and control and compared with cultivations on complex media.
2. Materials and methods 2.1. Strain and pre- and main cultures The A. chrysogenum 3/2(C3) strain was donated by HMR Deutschland. Each of the ampoules with 2.2 ml spore suspension were stored in liquid nitrogen. Agar slant cultures of the strain were used to inoculate a 1 l shake flask with 160 ml malt and yeast containing medium which was sterilised before at 121 8C for 20 min. This was cultivated on a rotary shaker with 280 rpm at 25 8C for 72 h. This was the preculture for 10 and 30 l bioreactors. 2.1.1. Shake culture runs Thirty ml medium in a 500 ml shake flask was autoclaved at 121 8C for 20 min and after cooling 3 ml sterilised concentrated sugar solution was added to the flasks. A 3 ml inoculum was added to it and cultivated at 25 8C on rotary shaker at 280 rpm for 160 /225 h. 2.1.2. 10 l Bioreractor runs In the 10 l volume bioreactor (BIOSTAT C, Braun Melsungen) the semisynthetic cultivation medium (Ta-
ble 1) autoclaved at 121 8C for 30 min and inoculated 160 ml preculture, which was prepared in a 1 l shake flask and cultivated at 25 8C for 72 h on a rotary shaker with 280 rpm. 2.1.3. 30 l Bioreactor runs A 30 l volume bioreactor (BIOSTAT UD Braun Melsungen) was used for these runs. The reactor was filled with 25 l semisynthetic cultivation medium (Table 1), sterilised at 121 8C for 30 min cooled to 25 8C and stored for 12 h. The amount of inoculum was 19% of the medium volume. 2.2. Process monitoring For determination of the biomass concentration 10 ml sample was centrifuged at 3800 rpm for 10 min (Laborfuge GL, Heaeus Crist), the sediment was dried at 110 8C for 72 h and weighed. An Olympus BH2microscope was used for the observation of the morphology of the fungus. Phosphate concentration was determined by the phosphate test (BMB) of Merck. Total sugar content was determined by a p -hydroxybenzoic-acid-hydrazide (pHBAH)-method. Glucose and lactate concentrations were determined by the equipment of Yellow Springs Instruments. Protein concentration was evaluated by the method of Bradford. Ammonia concentration was measured by a German standard method (DIN 38 406). Amino acid concentrations were determined by HPLC consisting of an autosampler (Pharmacia), degasser (Irica), precolumn and analytical column (ResovedTM C18, Waters), thermostate (Julabo), fluorescence detector (Shimadzu RS535), control unit (Autochrom CS12 Interface), gradient control (Autochrom CIM interface) and data processing (APEX chromatography workstation). Samples were treated with methanol and centrifuged at 0 8C to separate proteins. The protein free probes were diluted with borate buffer pH 10 to 1/20 (in general) and 1/2000 (for methionine determination), respectively. Ten ml sample is mixed with 10 ml OPA-reagent and
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
injected after 2 min reaction time into the column. The analysis is performed at 25 8C. A mixture of 96% buffer solution (33 mM Na-acetate, 38 mM Na phosphate, pH 7), 2% methanol and 2% tetrahydrofurane was used as eluent A. Methanol (54%) in bidistilled water was applied as eluent B. Within 5 min the eluent was changed from 0 to 30% B, and within 35 min it was rised to 100%. The duration of the analysis was 55 min. The thiole containing amino acids (L-cystein) and peptides (ACV) cannot be analysed with this method and they need a sample preconditioning with OPA / MCE (mercaptoethanol) according to Holzhauer/Rieger [12]. Samples were injected to the column after derivatisation with OPA /MCE. Analysis of the product and precursors were performed by HPLC consisting of a pump (Irica), injection valve (Valco). degasser, precolumn, column (Macherey and Nagel), thermostate (Julabo), two channel UVdetector (Gynotek). On-line analysis was performed by a cooled autosampler (Gilson /1222, abimed) and a controller (CSI2 interface). Data logging and evaluation were carried out on a PC (486 DX66) by an APEX program. A packed column with Nucleosil 5† with reverse phase mode was operated at 30 8C with a mixture of 5% methanol, 95% 14 mM phosphate buffer and 10.2 mM tetrabutylammoniumhydrohensulphate (TBAHS) pH 6.5 as eluent, which was degassed in an ultrasonic bath for 20 min at a wavelength of 2260 nm. The standards for CPC, DAC, DAOC and PENN were donated by HMR Deutschland. For more details see [13]. The reactors were provided with thermometer (Pt100), pH-meter (Pa 12/120, Ingold), pO2-electrode (Ingold) and off gas analyser (EGAS1 (Braun) with O2 and CO2 detectors. The reactors were equipped with measuring and control system (DCU, RS 422) and a PC as supervisor.
3. Results 3.1. Investigations of shake flask cultivations One hundred and ten grams per liter corn steep liquor (CSL) was added to the basic cultivation media (Table 1). CSL has a solid sediment content of 40 /50%. By centrifugation at 4500 rpm the sediment was removed and the solid particle free liquor was used for cultivation The medium contained 46 g l 1 dry glucose syrup (TGS) instead of glucose and had an initial pH 6.44 (run 1). Another cultivation was performed with the same basic cultivation media (Table 1) and with 110 g l 1 CSL. The sediment was removed by centrifugation at 4500 rpm and the sediment free liquor was treated with ZEN-medium at 100 8C for 60 min [14]. The precipitate
265
was removed by centrifugation at 4500 rpm and the liquor was used for cultivation. The medium contained 46 g l 1 TGS instead of glucose. The initial pH was 6.44 (run 2). The semisynthetic media did not contain CSL and TGS (Table 1). Their initial pH was 6.44. These cultivations were performed / / / / /
without buffer (run 3), with 50 mM MES-buffer (run 4), with 100 mM MES buffer (run 5), with 50 mM phosphate buffer (run 6), with 200 mM MOPS buffer (run 7).
In all of these cultivations the following process variables were monitored: pH-value, (dry) biomass, total sugar, phosphate, lactate, ammonia, asparagine, arginine, alanine, methionine, a-amino adipinic acid, cystein, valine, penicillin N (PENN), deacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC), cephalosporin C (CPC). 3.1.1. Run 1 Cell growth started without lag phase. The maximum (dry) biomass concentration was 45 g l 1. The concentrations of phosphate, sugars, lactate, asparagine, arginine, alanine, methionine and valine were consumed and exhausted upto about 100 h. pH drops to 5.5 at 70 h and than increased to 8.5. Ammonia passed a minimum at 87 h, the concentrations of cystein and a-aminoadipinic acid increased with the time. Precursor concentrations were below 1.5 g l 1. The concentration of CPC attained a maximum (6.5 g l 1) at 112 h. 3.1.2. Run 2 Cell growth had no lag phase. The maximum (dry) biomass concentration was 35 g l 1. The consumption rates of sugars, lactate, amino acids were lower than those in run 1. The pH value passed a minimum (5.4) at 120 h and increased to 7.0. Ammonia concentration passed a minimum at the same time and increased only slightly. The DAOC and DAC concentrations were low (below 0.5 g l 1) PENN concentration below 1.0 g l 1 and the CPC concentration increased and attained 3.5 g l 1 at the end of the cultivation (175 h). 3.1.3. Run 3 The cultivation started with a long lag phase. The maximum (dry) biomass concentration was 25 g l 1. Glucose was consumed slowly and exhausted at 165 h. The pH value dropped to 4.25 at 120 h and remained low. The ammonia concentration diminished parallel to pH. The concentrations of asparagine, arginine and valine varied only slightly. Alanine was exhausted at 120 h. The concentration of methionine gradually dropped to half of its original value. Precusor concentrations
266
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were below 0.1 g l 1. The maximum CPC concentration (1.0 g l 1) was attained at 120 h and then diminished slowly. 3.1.4. Run 4 No lag phase was observed. Maximum (dry) biomass concentration (35 g l 1) was obtained at 112 h and it remained constant. The pH gradually dropped to 4.75. The ammonia concentration diminished parallel with pH. Glucose was exhausted at 110 h. The amino acid concentrations gradually diminished to about 0.3 of their initial value. Alanine was exhausted at 130 h. Valine was exhausted at 210 h. PENN concentration attained 1.9 g l 1 at the end of the cultivation. CPC concentration had its maximum (3.4 g l 1) at 175 h. 3.1.5. Run 5 No lag phase was observed. The (dry) biomass concentration gradually increased to 40 g l 1. Glucose was exhausted at 95 h. pH-value passed a minimum ((5.0) at 120 h and increased to 5.45. The course of ammonia ran parallel to pH. Valine and alanine were exhausted 187 h, asparagine at 212 h. The other amino acids were nearly exhausted at the end of the cultivation at 215 h. PENN concentration increased to 2.5 g l 1 and CPC concentration attained a maximum (4.0 g l 1) at 137 h. After that it varied only slightly. 3.1.6. Run 6 A lag phase existed. The (dry) biomass concentration attained a maximum (23 g l 1) at 130 h and remained constant. Glucose was exhausted at 112 h. The pH-value dropped to 4.75 at 187 h and than changed only slightly. Ammonia concentration run parallel to pH-value. Phosphate concentration passed a maximum (6.45) at 85 h and after that it diminished to 4.75. Valine was exhausted at 112 h. The concentrations of the other amino acids gradually decreased to about half of their initial values. PENN concentration attained a maximum (1.75 g l 1) at 137 h, and CPC concentration reached a maximum (3.2 g l 1) at 162 h. 3.1.7. Run 7 There was a short lag phase. The (dry) biomass concentration attained a maximum (21 g l 1) at 140 h. Glucose was exhausted at the same time. The pH-value passed a minimum (5.45) at 124 h. After that it increased to 6.7. Ammonia concentration run parallel to pHvalue. Alanine and valine were exhausted at 187 h. The other amino acid concentrations diminished to about 50% of their initial value at the same time. PENN concentration had a maximum value (1.0 g l 1) and the CPC concentration attained its maximum value (3.0 g l 1) at 190 h.
3.2. Investigations of cultivations in the 10 l bioreactors with semisynthetic medium 3.2.1. Run 8 In bioreactors the pH-value was controlled by 8 N NaOH and conc. H2SO4 solutions, respectively, and it was not necessary to use buffer supplements. The semisynthetic medium of the buffer free runs (Table 1) was used with 40 g l 1 soy oil concentration. CO2 production rate (CPR) and oxygen transfer rate (OTR) and optical density (OD) were determined in addition to the process variables of shake flask cultivations. The concentrations of the precursors and CPC were monitored on-line. The reactor was aerated with 0.5 vvm min 1. The dissolved oxygen concentration diminished during the batch cultivation from 100 to 0% at 110 h. Therefore, after 80 h oxygen limitation prevailed. The pH dropped between 20 and 40 h from 6.4 to 5.8. The pH was maintained at this value. The ammonia concentration followed this course and diminished from 1.75 to 0.5 g l 1. The concentration of the (dry) biomass gradually increased and attained 42 g l 1 at 120 h. The OD followed the same course. OTR increased to 29 mM l 1 h 1, because of the increasing biomass concentration. Glucose was exhausted at 79 h. CPR increased with the cultivation time, but it had two minima caused by the pH-shift at 40 h and caused by oxygen limitation at 80 h. Asparagine, alanine and valine were exhausted between 130 and 140 h. The concentration of arginine dropped to the half of its initial value and that of methionine to 0.3 of its initial value at 140 h. The concentrations of DAOC and DAC remained below 0.5 g l 1, PENN concentration increased to 2.5 g l 1 and that of CPC to 4.4 g l 1. CPC formation rate gradually increased to 0.07 g l 1 h 1 and methionin consumption rate attained two maxima: 0.06 g l 1 h 1 at 20 h and 0.175 g l 1 h 1 at 90 h. The steep reduction of the methionine consumption rate after these two maxima is caused by the drop of pH and the oxygen limitation, respectively. 3.2.2. Run 9 In order to avoid the oxygen limitation, in the second run the aeration rate was increased to 1 vvm and after pO2 dropped to 70%, oxygen was added to the pressurised air to keep pO2 at 100%. After a lag phase the biomass concentration increased to 47.5 g l 1 and after this maximum it slightly diminished. Glucose was exhausted at 75 h. This caused a minimum of CPR. The second minimum of CPR was caused by diauxie at 125 h. After 150 h CPR dropped, because, exhaustion of the soy oil. The pH-value diminished from 6.5 to 5.75 between 50 and 75 h and was maintained at this value. Ammonia followed the pH-change and dropped from 1.8 to 0.75 g l 1 during this time period and later to 0.25 g l 1. Valine was exhausted at 125 h, asparagin at 137 h
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
267
Fig. 1. pO2, pH and CPR in the 30 l bioreactor as a function of cultivation time.
Fig. 2. Concentrations of (wet) biomass (BFM) and (dry) biomasss (BMT) and OD in the 30 l reactor as a function of cultivation time.
and alanine, arginine and methionine at 160 h. The concentrations of DAOC and DAC remained below 0.7 g l 1. PENN concentration increased to 4 g l 1 and CPC concentration to 7.7 g l 1. The CPC formation rate gradually increased and attained a maximum of 0.11 g l 1 h 1 at 124 h. Methionine consumption rate passed two maxima: 0.175 g l 1 h 1 at 95 h and 0.30 g l 1 h 1 at 137 h. By abolishing the oxygen limitation the (dry) biomass concentration was increased from 42 to 47.5 g l 1, the CPC concentration from 4.4 to 7.7 g l 1 and the PENN concentration from 2.5 to 4 g l 1. Cell growth was less
influenced by oxygen limitation than the biosynthesis of CPC. 3.3. Investigation of cultivations in 30 l bioreactor on semisynthetic medium 3.3.1. Run 10 Batch cultivations in 10 l bioreactor were repeated in a 30 l reactor. The medium composition was the same (Table 1). The pO2 value was controlled and kept at 60% of the saturation value by addition of oxygen to the pressurised air. The pH-value was controlled by 8 N
268
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
Fig. 3. Concentrations of ammonia, phosphate and total sugar in the 30 l reactor as a function of cultivation time.
Fig. 4. Concentrations of methionine, alanine and asparagin in the 30 l reactor as a function of cultivation time.
NaOH and conc. H2SO4, respectively, and pH 5.8 was maintained during the cultivation. In Fig. 1 the courses of pO2, the pH value and CPR are shown as a function of the cultivation time. At 140 h the pO2 dropped to 60% and was maintained at this value. The pH value diminished from 6.3 to 5.8 during the first 60 h and kept at this value. CPR increased exponentially in the first 90 h, passed a minimum, because, at this time glucose was exhausted and the fungus had to change from glucose substrate to soy oil. In Fig. 2 the concentrations of (wet) biomass ( BFM) and (dry) biomass (BTM) are shown together with OD. The diauxic between 90 and 140 h can be clearly recognised.
The (dry) biomass concentration increases after the diauxic phase again and attained a final concentration of 40 g l 1. The difference between BFM, BTM and OD disappeared after 100 h. The concentrations of ammonia, phosphate and sugar are shown in Fig. 3. Sugar content was exhausted at 80 h. Phosphate concentration passed a maximum at 40 h, which is unexpected, and ammonia concentration gradually diminished from 1.8 to 0.5 g l 1. Alanine, asparagine and methionine were consumed at a nearly constant rate (Fig. 4), similar to serine, valine and glutamate (Fig. 5). Glutamine and cystein concentrations were very low. The concentration of a-aminoadipinic acid increased from 90 h nearly exponentially, which was unexpected. The concentrations of PENN, DAOC, DAC and CPC increased with time (Fig. 6). The final concentrations of b-lactam compounds were 0.13 g l 1 DAOC and DAC, 4.5 g l 1 PENN, and 7.7 g l 1 CPC. The CPC production rate had two minima: The first was caused by exhaustion of glucose at 90 h and the second appeared at 139 h. To obtain more information on the biosynthesis, the intracellular concentrations of ACV and proteins and ACV-synthetase (ACVS) activity were also determined. All three values passed a steep maximum at 90 h and slowly decreased further (Fig. 7). The concentration of PENN and the activities of isopenicillin N-synthetase (IPNS) are shown in Fig. 8. PENN formation started at 50 h, passed a maximum at 65 h and after 75 h increased to a maximum concentration of 1.5 g l 1. IPNS activity started to increase also at 50 h. After a steep increase it stagnated from 75 h and at 120 h further increased to a maximum activity of 20 U g 1. The courses of ACV and
M. Ju¨rgens et al. / Process Biochemistry 38 (2002) 263 /272
269
Fig. 5. Concentrations of some extracellular aminoacids in the 30 l bioreactor as a function of cultivation time.
Fig. 6. Concentrations of b-lactam precursors: PENN, deacetoxycephalosrotin C (DAOC), deacetyl cephalosporin C (DAC) and the product: CPC as well as the CPC-formation rate in the 30 l bioreactor as a function of cultivaton time.
PENN as well as ACVS and IPNS activities are similar to those observed in cultivations with complex medium [1].
4. Discussion 4.1. Comparison of the shake flask cultivations By the removal of the sediment of CSL in run 1, a maximum concentrations of CPC of 6.5 g l 1 and (dry) biomass of 45 g l 1 were obtained. After the precipita-
tion of the dissolved proteins in run 2 the maximum concentration of CPC was reduced to 3.5 g l 1 and that of (dry) biomass diminished to 35 g l 1. In Table 2 the osmotic pressure, buffer capacity and maximum CPC concentrations of runs 3 /7 with semisynthetic medium were compiled. Without buffer only 1 g l 1 CPC and 25 g l 1 (dry) biomass were obtained. With increasing buffer capacity the osmotic pressure enlarged, but the biomass and CPC production was not influenced. In run 5 with an osmotic pressure of 16.10 bar the highest CPC concentration of 4.0 g l 1 and (dry) biomass concentration of 40 g l 1 were obtained.
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270
Fig. 7. Intracellular concentration of ACV and proteins and specific activity of ACVS in the 30 l bioreactor as a function of cultivation time.
Fig. 8. Intracellular concentration of isopenicillin N (IPN) and proteins and specific activity of IPNS in the 30 l bioreactor as a function of cultivation time.
Table 2 Osmotic pressures, buffer capacities and maximum CPC concentrations of the semisynthetic media (runs 3 /7) Run
3 4 5 6 7
Buffer
No buffer 50 mM MES 100 mM MES 50 mM PO3 4 200 mM MOPS
Osmotic pressure (bar)
13.623 14.86 16.10 14.43 18.58
CPC concentration (g l 1)
Buffer capacity (mM) pH 5.5
pH 6.0
pH 6.5
1.94 19.09 36.66 5.38 13.26
4.57 25.0 56.25 11.80 47.22
10.08 21.91 45.45 20.0 100.0
1.0 3.4 4.0 3.2 3.0
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271
Table 3 Production of CPC by A. chrysogenum 3/(C3) strain in semisynthetic and complex media under various cultivation conditions Run Cultivation conditions
Biomass concentration PENN concentration (g l 1) (g l 1)
1 2
45 35
2.5 1.0
6.5 3.5
25 40
B 0.1 2.5
1.0 4.0
42 47.5
2.5 4.0
4.4 7.7
40
4.5
7.7 9.5
[1]
23.0
[1]
3 5 8 9 10
Complex with sediment separation shake flask culture Complex with sediment and protein separation shake flask culture Semisynthetic without buffer shake flask culture Semisynthetic with 100 mM MES buffer shake flask culture Semisynthetic with pH control, batch 10 l reactor Semisynthetic pH control, batch Oxygen supplement 10 l reactor Semisynthetic with pH control, batch 30 l reactor Complex medium pH control, batch 30 l reactor oxygen supplement Complex medium pH control, fed-batch 30 l reactor oxygen supplement
98
3.2
CPC concentration (g l 1)
Reference
Comparison of maximum concentrations of biomass, CPC and PENN.
Obviously at too high osmotic pressure (18.58 bar) the biomass and product formation were impaired and the maximum CPC concentration reduced to 3.0 g l 1 and the maximum (dry) biomass concentration diminished to 21 g l 1. Phosphate buffer was less suitable for suppression of the pH-change. A comparison of runs 4 and 6 indicate, that at about the same osmotic pressures (14.8 and 14.4. bar) with MES buffer a somewhat higher CPC concentrations (3.4 vs. 3.2 g l 1) and higher biomass concentrations (25 vs. 23 g l 1) was obtained than with phosphate buffer. However, the difference was dramatic. 4.2. Comparison of shake flask and reactor cultivations A comparison of the cultivations with buffer with those of pH-control indicates, that pH control was superior to buffering of the medium. The best cultivation with buffer resulted 40 g l 1 (dry) biomass concentration and 4 g l 1 CPC concentration. The pH-control for non-oxygen limited cultivation resulted 47.5 g l 1 (dry) biomass concentration and 7.7 g l 1 CPC concentration. CPC formation was more sensitive to osmotic pressure than the cell growth. A comparison of cultivations in 10 l reactor without and with oxygen supplement shows than oxygen limitation reduced the biomass from 47.5 to 42 g l 1, and the CPC concentration from 7.7 to 4.4 g l 1. The biosynthesis of CPC is more sensitive to oxygen limitation than the cell growth [15]. In the 10 and 30 l reactors under the same cultivation conditions similar results were obtained. A comparison of batch cultivation in semisynthetic and complex media in 30 l reactor shows that with suitable synthetic medium composition (7.7 g l 1 CPC)
similar results can be obtained than with complex medium (9.5 g l 1 CPC) (Table 3). A comparison of batch and fed-batch cultivations in 30 l bioreactor with the same medium composition indicates that with fed-batch operation much better results were obtained (23 g l 1 CPC) than with the batch operation (9.5 g l 1 CPC).
Appendix A: Nomenclature ACV ACVS Arg Asp BFM BMT CPC CPR CSL Cys DAC DAOC Glu Gln IPN IPNS MCE MES MOPS Met OD OPA OTR PHBAH
a-L-amimnoadipyl-L-cyteinyl-D-valine ACV synthetase arginine aspartate (wet) biomass (dry) biomass cephalosporin C CO2 formation rate corn steep liquor cystein deacetyl cephalosporin C deacetoxy cephalosporin C glutamate glutamin isopenicillin IPN synthetase mecaptoethanol 2-morpholinoethansulphanic acid 3-morpholinopropansulphonic acid methionine optical density o -phtalaldehyd oxygen transfer rate p -hydroxybenzoic acid hydrazide
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272
pO2 PENN Ser TGS Val
dissolved oxygen concentration with regard to its saturation penicillin N serine dry glucose syrup valine
Acknowledgements This work was financed by HMR Deutschland, Hoechst. The authors thank Dr B. Ko¨nig, HMR Deutschland and Biochemie Kundl, for his support.
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