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Autolysis in Batch Cultures of Penicillium Chrysogenum at Varying Agitation Rates
Autolysis in batch cultures of Penicillium chrysogenum at varying agitation rates L. M. Harvey, B. McNeil, D. R. Berry, and S. White Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, United Kingdom The process of autolysis in batch cultures of an industrial strain of Penicillium chrysogenum was investigated at a range of stirrer speeds. Autolysis in the cultures was monitored by conventional analysis (biomass decline, NH1 4 release), direct measurement of autolysing regions (image analysis), and enzyme activity assays (proteases and b-glucanases). Image analysis measurements provided a sensitive indicator of the onset and progress of autolysis in the culture. Autolysis could be brought about by C or N limitation. Culture history could have a marked effect on the process. Autolysis resulted in degradation of penicillin V and problems with broth filterability. It was closely associated with increased intracellular proteolytic and b-1-3-glucanolytic activities. Although stirrer speed affected both growth and penicillin V production, final mean main hyphal lengths were similar in autolysing cultures. This indicated a possible role of an intrinsic characteristic in fragmentation. © 1998 Elsevier Science Inc. Keywords: Autolysis; impeller speed; Penicillium chrysogenum; image analysis; proteases; b 1-3 glucanases
Introduction The process by which microbial cells cease growing and commence to breakdown by the action of their own enzymes is generally referred to as autolysis. This process has received a great deal of attention in some bacterial species.1 Much research has also focused on autolysis in the commercially important yeast species.2 In contrast, studies of autolysis in the filamentous fungi have been far fewer,3 which given the biotechnological importance of these organisms, is rather surprising. Those studies which have in the past considered fungal autolysis have often had very lengthy experimental duration (10 – 60 days),4,5 the relevance of which to the current industrial process involving fungi, is questionable. Similarly, most previous studies have concentrated on various starvation regimes in terms of macronutrients (particularly carbon and nitrogen) to ‘‘induce’’ autolysis.5 Among the extrinsic factors which have been demonstrated to bring about the process of autolysis in fungi as C-source starvation (in fact, in most cases this is more correctly
Address reprint requests to Dr. L. M. Harvey, University of Strathclyde, Dept. of Bioscience and Biotechnology, 204 George Street, Glasgow G1 1XW, UK Received 12 November 1996; revised 10 February 1997; accepted 22 October 1997
Enzyme and Microbial Technology 22:446 – 458, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
termed carbon and energy source starvation), nitrogen starvation,6 increased temperature,2,7 and the presence of toxic compounds or metabolites including, e.g., ethanol.2 Regardless of the extrinsic factors which bring about autolysis, there is evidence that the process may take place in a number of stages8 involving an initial disruption of normal cellular metabolism. This leads to progressive loss of membrane function, failure of compartmentalization within the cell, allowing release/activation of the enzymes stored in compartments such as the vacuole.8,9 Release of these vacuolar enzymes and their activation leads rapidly to widespread proteolysis10 and to a rather slower and less complete degradation of the polymeric cell wall materials matrix polymers11 and chitin.5,11,12 Most authors emphasize the essential role of proteases in the process.6,10 With regard to the methods used to assess the extent of autolysis, in mold cultures conventionally, these have involved mean (i.e., whole culture measurements) of the decline in biomass4 or of cellular breakdown products (e.g., 5 NH1 4 release). While these may be useful overall, biomass decline itself is not always necessarily indicative of the process of autolysis and can result from ordered degradation of cellular reserves (e.g., glycogen).13 Changes in cellular composition can also lead to a decline in biomass levels. The process of autolysis in multicellular non-unitary systems such as filamentous fungi is complicated by both morphological/physiological differentiation and nutrient
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Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al. translocation throughout the mycelial network;14 thus, while some hyphal elements may be exhibiting signs of growth, others at the same process time may exhibit autolytic signs.5,14,15 In examining the process of autolysis in an industrial strain of Penicillium chrsyogenum, we adopted three methods of analysis:
Table 1 Seed flask (inoculum) medium in g dm23
1. Conventional analyses of changes in biomass, nutrient levels, and release of NH1 4, 2. Enzyme assays: Measuring the activity of classes of enzymes which might be of key importance in cellular breakdown, particularly intracellular proteases and b 1,3 glucanases as indicators of degradation of intracellular material and cell wall, respectively, 3. Image analysis: To visualize and quantify the actual extent of autolysis in the fungal culture. Image analysis techniques have been widely used to study bioprocesses involving morphological differentiation within the biocatalyst.16 The studies of Nielsen et al.17,18 and Paul et al.19,20 on differentiation and degradation in P. chrysogenum were particularly useful in the selection of criteria for definition of autolytic regions as was the very detailed description of the cytological changes during yeast autolysis by Hernawan and Fleet.2 This allowed direct quantification of the extent of autolysing regions within the culture samples.
the flask. Spore counts were performed by means of serial dilution (if required) and use of a Neubauer counting chamber.
A number of studies in P. chrysogenum have examined the process of mycelial fragmentation17,18 often exclusively from the viewpoint of extrinsic factors (power input, shearing forces) while, with the notable exception of Nielsen and Krabben18 and Paul et al.20 omitting any mention of the intrinsic factors. Paul et al.,20 however, noted a link between the process of hyphal degeneration in P. chyrsogenum (as measured by an increase in vacuolation in older hyphal elements), and fragmentation, clarifying the findings of an earlier study by Righelato et al13. which had indicated the possibility of such a link. In view of these findings and the end point of the processes of cellular degeneration, it seemed worthwhile to investigate the effects of processes occurring during or immediately before autolysis, such as wall weakening, loss of cell rigidity, and decreased turgor pressure, on fragmentation. We therefore examined autolysis in an industrial strain of P. chrysogenum (SKB) in a complex medium in submerged batch cultures at a range of impeller speeds. Culture D.O.T. was maintained at a minimum of 40% saturation at all times both to exclude this source of variability and to avoid effects of O2 limitation with subsequent deleterious influence on penicillin V synthesis.21,22
Corn steep liquor Glucose (NH4)2SO4 Rape seed oil
70.0 15.0 4.0 0.5(ml)
Inoculum production Spore suspension (1 ml) was added to 0.2 dm3 of seed medium in 0.5 dm3 Erlenmeyer flasks. Flasks were incubated at 25°C and 200 rpm for 48 h. This vegetative culture was used as the inoculum for the bioreactor.
Media For media preparation, see Tables 1 and 2.
Bioreactor A fully instrumented BIOSTAT ED10 (B. Braun Biotech. Ltd, Melfungen, Germany) with an operating volume of 9 dm3 was employed in this study. Dissolved oxygen tension was maintained at or above 40% of air saturation (unless otherwise stated) using a Uniprobe dissolved oxygen controller linked to a gas mixing unit (B. Braun Biotech. Ltd) which supplied air or an air/O2 mix. Culture conditions were as follows: initial pH, 4.9; agitation rate variable, aeration rate 1 vvm; and temperature, 25°C. All fermentations were performed in batch mode. Sodium phenoxyacetate was added to each process continuously from 68 h at a rate of 8 ml h21 of a 0.3% solution. The agitation rate was constant throughout each batch process and ranged from 200 – 800 rpm. The bioreactor was equipped with three six-bladed Rushton turbine impellers (each 0.5 vessel diameter).
Biomass determination Biomass was measured by means of dry weight measurement. Whole broth (10 ml) was filtered through predried and weighed Whatman GF/C filter papers. The filter and biomass were then dried in a microwave oven on the defrost setting for 20 min or until a constant dry weight was obtained. The sample was then placed in a desiccator for a further 20 min until cool and then was weighed. All samples were analyzed in triplicate. The filtrate, which was required for further analyses, was immediately frozen at 220°C to prevent deterioration of the sample. All further analyses were performed as rapidly as possible.
Exit gas analysis The concentration of oxygen and carbon dioxide in the inlet and outlet gas streams were measured using a paramagnetic oxygen
Materials and methods Microorganism and bioreactor
Table 2 g dm23
Batch fermenter medium in
An industrial strain of P. chrysogenum was supplied by SmithKline Beecham Pharmaceuticals (Irvine, UK) in the form of liquid spore suspensions contained in sealed 10-ml glass bottles. These were stored at 220°C and brought to room temperature before use as the inoculum for seed flasks. Approximately 1 ml of spore suspension was added to each seed flask (fermenter inoculum). This was equivalent to a spore concentration of 2.1 3 105 ml21 in
Corn steep liquor Lactose KH2PO4 K2HPO4 MgSO4 z 7H2O Rape seed oil
75.0 70.0 2.5 2.5 0.4 0.5(ml)
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Papers analyzer (Model 540A, Servomex Ltd., Sussex, UK) and an infrared CO2 analyzer (Model 7000, Analytical Development Co. Ltd., Cambridge, UK), respectively. Gases were passed over a drying column of medium grade silica gel before measurements were taken.
Viscosity measurements Apparent viscosity of the whole broth was measured using a Ferranti concentric cylinder viscometer, model VL. Readings were taken using 0.1 dm3 broth samples at 25°C and a shear rate of 68 s21.
digitized on the basis of each pixel greyscale level (where 0 is black and 255 is white) and then converted to a binary two-color image. Objects within the image were then analyzed; however, objects shown in the binary image were determined by the greyscale threshold level set by the operator. Whole broth samples (2 ml) were taken from the fermenter at regular intervals and added to 1 ml of lactophenol blue and then made up to a 20 ml volume using a fixative. This was comprised of 5.6% formaldehyde and 2.5% glacial acetic acid made up to a 200 ml volume with 50% ethanol. Samples were stored at 4°C before analysis.25 For each sample, 50 free mycelial elements (organisms) were analyzed although it has been proposed that 40 would be adequate.26
Total carbohydrate assay The total amount of sugars present in the fermentation broth was determined using the method of Dubois et al.23 Standards were prepared using a concentration range between 10 –100 mg dm23. All measurements were made in triplicate.
Ammonium ion assay The ammonium ion concentration was measured using an assay kit by Sigma (No. 640, St. Louis, MO). Standards of ammonium chloride were prepared in the range 3.125–100 mg dm23. Samples were diluted 1:10 before use.
Penicillin V analysis Penicillin V concentration was assayed by HPLC analysis. The following method (modified from that of Deo and Gaucher)24 was employed. Apparatus. A Perkin-Elmer 250LC pump with a LC90J detector connected to a Perkin-Elmer Nelson 900 series interface with PC Integrator (software version 5.1.5., Perkin-Elmer Nelson Systems Inc.) was used. The column was a Nucleosil C18, 250 mm 3 4.6 mm with 5 mm particle packing (Burke Analytical, Glasgow, UK). The guard column was 20 mm 3 2 mm (internal diameter) packed with Spherisorb (3 g) Reverse Phase 18 (Anachem). The operating pressure was between 1,500 –2,500 psi. Mobile phase. This was an isocratic system maintaining constant eluent composition using a mobile phase of acetonitrile:sodium phosphate buffer (25:75 v/v) at room temperature. The mobile phase was filtered through a 0.22 mm Whatman filter paper and was degassed daily with helium before use. Sample preparation. All samples and standards were filtered through 0.2 mm Whatman nylon filter papers (diameter 13 mm). Samples were diluted 1 in 20 with mobile phase prior to filtration and injection. Absorbance was measured at 220 nm and the range setting was 0.5. The sample/standard was injected using a 20 ml injection loop and a flow rate of 1 ml min21. The internal standard used was penicillin G since this had a distinct retention time from that of penicillin V.
Image analysis Morphological characteristics of the culture were examined using a Seescan image analysis system. This produced digitized monochrome images via a Nikon Optiphot-2 microscope on which was mounted a Sony CCD video camera (Model XC-77CE). The magnifications used were 3100 and 3200 depending on the dimensions of the mycelial elements. The images of dispersed mycelial elements were then analyzed manually. The image obtained on the monochrome video camera was
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Measurements. Mean main hyphal length, number and length of branches, and autolytic lengths were measured. The mean hyphal growth unit was calculated for each hyphal tree (organism) by dividing the total hyphal length by the number of branches.
Enzymology Approximately 50 g of whole broth sample was weighed (noting volume) and centrifuged at 20,000 rpm for 10 min at 4°C. From this, 20 ml of supernatant was removed and stored for further analyses. The pellet obtained was transferred to a preweighed centrifuge tube, washed with 50 ml of 50 mm phosphate buffer pH 6.2, vortex mixed, and then re-centrifuged as above. This process was repeated a further four times. The pellet obtained was weighed and then transferred to a cell disrupter. A high pressure cell disrupter (Model 4000, Constant Systems Ltd., Warwick, UK) which gave very rapid sample disruption was used to disrupt mycelial samples before further sample treatment and analysis. After centrifugation (as above), the supernatant was then transferred to dialysis tubing (M. W. cut-off, 12,000 –14,000; pore size, 2.4 nm) and dialyzed for 3 3 8 h in 10 mm phosphate buffer pH 6.2 at 4°C. After dialysis, samples were stored at 220°C until required for analysis.10
Total proteolytic activity Determination of total proteolytic activity. The method of Santamaria and Reyes10 was employed. Casein (0.1% w/v) was dissolved in 0.25 m phosphate buffer pH 6.0, made up to 0.1 dm23, and used as the substrate. The solution was stored at 4°C. To 0.5 ml of substrate solution, 0.5 ml of suitably diluted enzyme (sample) solution was added. This was then incubated for 30 min at 30°C. The reaction was terminated by boiling for 5 min. The a-amino nitrogen released was determined using the ninhydrin method. The activity of the samples is expressed in terms of mg amino acid equivalents released min21 of the reaction (the conversion rate). Activity is also expressed in terms of specific activity where the conversion rate is divided by the total protein or biomass. This is then expressed as mg amino acid equivalents released min21 unit21 protein or unit21 biomass. Determination of b-glucanase activity. The method of Mulenga and Berry27 was used. Activity is expressed as mg glucose equivalents released min21 (conversion rate). Specific activity is in terms of mg glucose equivalents released min21 unit21 total protein or unit21 biomass in the sample.
Results and discussion Many studies have examined mycelial fragmentation in P. chrysogenum17,18,20,28 –31 and although it has been shown that changes in morphological parameters of the dispersed
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al. form are generally related to stirrer speed, Nielsen et al.17 have shown that a steady reduction in mean main hyphal length, taken as an indicator of mycelial fragmentation, occurs even at low impeller speeds. Few of these studies consider the physiology of the microorganism itself as being the factor likely to contribute to the frequency/location of fracture points. Given recent studies relating to differences in fungal cell wall structure along the hyphal length, this could be a significant oversight.32 Our initial view was that the process of autolysis might well influence that of mycelial fragmentation with areas undergoing autolysis being more likely to fragment. We thus examined, at a fixed minimum dissolved oxygen level of 40%, a range of impeller speeds using a complex medium. (Since dissolved oxygen (DO) is critically important in P. chrysogenum in terms of growth and penicillin synthesis,21,33 DO was controlled at 40% in all processes). Without medium supplementation, it was expected that this medium would be depleted of the readily available C and/or n source between 5–7 days, which based on literature reports on other organisms, should lead to autolysis in this culture.2– 6 This should permit examination of effects of impeller speed on autolysis and the effect of exhaustion of a major nutrient independently of dissolved oxygen tension.
800 rpm Process, pelleted inoculum The time course of this process is shown in Figures 1a–1c and 2a and 2b (enzymology). In this process the lag, rapid growth phase, stationary phase, and decline (autolytic) phase are clearly apparent in Figure 1a. NH1 4 was absent from the filtrate from 48 h onwards. Penicillin V synthesis and the commencement of ‘‘stationary’’ phase (a much reduced growth rate) begin around 72 h. At this stage, some hyphal segments were beginning to show signs of autolysis despite the bulk of the culture having a healthy appearance (Figure 1b). Trinci and Righelato5 noted that such autolytic segments may well occur in the middle of otherwise healthy hyphae. This was a point also emphasised by Fencl.14 Cellular breakdown was thus occurring while culture cell mass was still increasing. Around 164 h the available C source was exhausted. From this point, NH1 4 reappeared in the filtrate presumably from deamination of proteins.5,34,35 Biomass declined sharply (Figure 1a) and autolysis (as measured by image analysis) in the culture continued (Figure 1b). These events coincided with a peak in intracellular protease activity and specific intracellular protease activity at 213 h (Figure 2a) and peaks in b-1,3 glucanase activity and specific activity at 190 h (Figure 2b). Image analysis measurements (Figure 1b) showed significant signs of autolysis in the culture before other indicators such as dry weight decline and NH1 4 release; thus, these measurements of micromorphology may well be a more sensitive indicator of the onset of autolysis than biomass decline throughout the culture as a whole. The exhaustion of NH1 4 did not, in this instance, lead to rapid autolysis despite other studies showing this to be the case.14,34,36 This may be due to uptake of other n sources (e.g., amino acids) by the cells from the complex medium or, more probably, from the results obtained, autolysis may
be related primarily to the disappearance of the energy source. A number of authors have indicated that exhaustion of an exogenous energy source in fungi rapidly leads to a depletion in cellular storage compounds (e.g., glycogen) and then to a suspension of normal metabolism.2,5 These results show that when the C source is exhausted, a series of events occurs involving morphological change, proteolysis/ release of NH1 4 and increased b-glucanase activity. Despite the latter, no free C source appeared in the filtrates. This confirmed the results of Cohen36 and Pitson et al.34 who indicated that b-glucanase activity in autolysis generally did not lead to release of glucose but did lead either to partial wall restructuring (loosening) or the observation that any monosaccharides generated were utilized for cryptic growth. In this study, intrahyphal growth was noted in the autolytic phase as was the appearance of apparently healthy hyphal tips from degraded hyphal masses. This is strong evidence for the occurrence of cryptic growth in certain culture elements. Comparing C consumption (Figure 1a) with C.E.R. (Figure 1c), it can be seen that for most of the decline phase (164 h), C.E.R. remained steady. This indicated that energy generation/consumption were probably still proceeding in a fraction of the culture at this stage. The collapse in C.E.R. after 218 h is due to the fact that the great bulk of the culture is completely degraded by this stage as confirmed by microscopic and IA examination. As can be seen in Figure 2a, specific intracellular protease activity (g21 dry weight and g protein) is maximal near the end of the rapid growth phase (;80 h). It thereafter falls sharply. This may well reflect the rapid protein turnover in the growing culture at this point. Later peaks in specific intracellular proteolytic activity and specific activity (occuring between 180 –214 h) are almost certainly related to the processes of autolysis which, as a number of authors have indicated, is predominantly a proteolytic event.21 It is possible that assaying for total proteolytic activity may be obscuring our view of autolysis somewhat in that the high proteolytic activity seen in the growth phase may represent different types of protease from those in the autolytic phase. In the latter case, vacuolar proteases have been implicated9 and, in general, this fits well with the observed increase in culture vacuolation noted in this study and in yeast;2 however, another group of proteases has been shown to be especially important to the cell during transitional periods, e.g., the transition from exponential to stationary phase when nutritional status rapidly changes.37 Figures 1a and 2a show that the early specific protease peak occurs very close to the entry into ‘‘stationary’’ phase (based on the biomass curve). These proteases are generally agreed to be largely concerned with stress responses in cells while the vacuolar hydrolases seem largely to be involved during major changes in nutritional status. Based on our results, such a pattern is plausible with the release/activation of vacuolar hydrolases (especially proteolytic enzymes) being associated with C exhaustion leading to the syndrome noted in the decline/autolysis phase in this culture. In this process, the sequence of events in late stationary/ early decline phase may thus be exhaustion of energy source, depletion of endogenous reserves, failure to maintain membrane function leading to release of compartmentalized hydrolases (largely proteases), activation of hydroEnzyme Microb. Technol., 1998, vol. 22, May 1
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Figure 1 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (pelleted inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (pelleted inoculum) (B). Carbon dioxide evolution rate and dissolved oxygen concentration vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (pelleted inoculum) (C)
lytic enzymes (based on the literature, this may be about the pH shift on release from the storage compartment),2,9 proteolysis, deamination of amino acids, partial glucan hydrolysis, wall weakening, loss of structural integrity, and 450
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hyphal disintegration. The results of this fermentation process seem to point toward such a sequence. The IA results (Figure 1b) generally indicate the trend in morphology noted in P. chrysogenum processes by oth-
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al.
Figure 2 Intracellular protease activity found in disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm with a pelleted inoculum. Activity is expressed as mg amino acid equivalent released min21 and specific activity mg21 protein or g21 dry cell weight (A). Intracellular b-1,3 glucanase activity found in the disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm with a pelleted inoculum. Activity is expressed as mg of reducing sugars released min21 and specific activity mg21 protein or g21 dry cell weight (B)
ers18,28 –30 with a steady decline in the value of key parameters including mean main hyphal length with time due to mycelial fragmentation and possibly also to the increased tendency of the longer dispersed hyphal elements to form aggregates.30 The general engineering view of this process of fragmentation is that it is an event which occurs randomly and is related solely to power input. In our view, it is wrong not to consider the physiological condition of the microorganism with respect to mycelial fragmentation, and Neilsen35 indicates that it may be wise to do so. The earlier suggestion by Righelato et al.13 that there could be a link between vacuolation and fragmentation (which was confirmed by Paul et al.20) also indicates the relevance of considering the physiological aspects of fragmentation. If fragmentation increased in highly autolytic cultures, it might be expected to be reflected in a sharp decrease in,
e.g., mean main hyphal length and branch length during the autolytic phase. From Figure 1b, this did not appear to happen with the decline in both values being approximately linear from the time of entry to the stationary phase (76 – 80 h) onward; however, visual examination showed a high proportion of cellular debris (particles , 30 mm in length) especially after 28 h. The values of morphological parameters measured were based on freely dispersed organisms which became far less common in the autolytic phase. One of the major drawbacks to IA measurements in the late stages of the process was the lengthy time spent locating 50 well-dispersed ‘‘organisms’’ upon which to make measurements. Even those few ‘‘organisms’’ which remained intact by late autolytic stage showed very extensive signs of autolysis. IA techniques are useful in measurement of regions which are autolysing, but since the end process of
Table 3 Time taken to filter a given volume (10 ml) of whole broth and to wash filter cake 800 800 400 200
rpm pellet inoculum rpm rpm rpm
Up Up Up Up
to to to to
190 190 163 162
h–immediate h–immediate h–immediate h–immediate
194 214 186 186
h–56 h–20 h–60 h–90
min s s s
236 h–56 min 236 h–60 s 210 h–60 s –
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Figure 3 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (dispersed mycelial inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (dispersed mycelial inoculum) (B). Carbon dioxide evolution rate and dissolved oxygen concentration vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (dispersed mycelial inoculum) (C)
autolysis is cellular disintegration, IA clearly has practical limits. Consideration of micromorphology is very valuable especially as a very early and sensitive indicator of the process 452
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of autolysis in cultures which, based on conventional analysis (dry weight and NH1 4 release) and enzymology are not yet clearly in the decline or autolytic phases, but quantitative information in micromorphology must be inter-
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al.
Figure 4 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (B). Carbon dioxide evolution rate, dissolved oxygen concentration, and apparent viscosity vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (C)
preted very carefully and, if possible, integrated with some consideration of macromorphology. The onset of autolysis in culture elements coincides closely with that of penicillin synthesis (Figures 1a and 1b) and entry to the stationary phase. A striking feature of the autolytic phase (from 164 h on) is the marked decline in penicillin V concentration. This analysis was repeated on three separate occasions to confirm it. Nielsen35 indicates that the degradation of penicillin V in a complex medium is
largely to penicilloic acid (40%); the remainder is to compounds as yet unknown. The striking decrease in penicillin V titer may represent not so much an increased degradation rate as a cessation of synthesis, since the penicillin V to penicilloic acid reaction proceeds even during the penicillin synthetic period with a first-order degradation rate based on penicillin concentration;38 however, the rate of degradation reactions are heavily dependent on operational conditions. It is possible that degradation Enzyme Microb. Technol., 1998, vol. 22, May 1
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Figure 5 Intracellular protease activity found in disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm with a dispersed mycelial inoculum. Activity is expressed as mg amino acid equivalent released min21 and specific activity mg21 protein or g21 dry cell weight (A). Intracellular b-1,3 glucanase activity found in the disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm with a dispersed mycelial inoculum. Activity is expressed as mg reducing sugars released min21 and specific activity mg21 protein or g21 dry cell weight (B)
during the autolytic phase may be due to release of peptidases or amidohydrolases39 which are normally physically separated from the penicillin V, but may come into contact on membrane breakdown. Another aspect of autolysis in P. chrysogenum which may have considerable economic impact is the increase in filtration time (Table 3), i.e., the time taken to separate the cells/cellular debris from the filtrate. This increased very substantially, thus any recovery process involving initial solids separation would be lengthened and suffer a further decrease in penicillin V titer. Based on morphological examination, the decrease in filtrability may be due to the disintegration of the ordered mycelial network which would tend to maintain a relatively open fibrous structure on filtering into a largely degraded material with large quantities of cellular debris which forms a densely packed filter cake.
800 rpm Dispersed mycelial sample A younger vegetative inoculum was used in this and subsequent processes. The inoculum was of a dispersed mycelial nature as opposed to the pellet inoculum of the first process. The results of the processes are shown in Figures 3a–3c. 454
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In this process, the rate of biomass increase was lower and the growth phase more prolonged. Free NH1 4 did not disappear from the filtrate until 120 h; some of the C source was present until the end of the process. The decline in the biomass concentration (past 180 h) was again accompanied by the reappearance of free NH1 4 ions in the culture fluid. Signs of autolysis were apparent in the culture in the growth phase. The percentage of total hyphal length showing signs of autolysis rose linearly from 90 –213 h. This again emphasizes that autolysis of some hyphal regions occurs even during the growth processes. As with the previous process, widespread mycelial disintegration occurred from 180 h onward. It became time consuming to find organisms for measurement of morphological indices; however, those organisms examined were generally healthier with less apparent autolytic regions than in the previous process. This may well have been due to the presence of the available C source in this phase. Overall, this phase makes it clear that the previous history of the culture has an influence on subsequent culture behavior and the process of autolysis, and that NH1 4 limitation/exhaustion does not increase the occurrence of autolysis in hyphae in this batch process. The presence of an available energy source late in the process
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al.
Figure 6 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm (dispersed mycelial inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (B). Carbon dioxide evolution rate, dissolved oxygen concentration, and apparent viscosity vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm (dispersed mycelial inoculum) (C)
did not prevent widespread culture disintegration, but those organisms present were maintained in a healthier condition than when no C source was available (process 1). A number of reports have indicated the key role of the C/energy source in the maintenance of cell viability and the process of autolysis.2,5,13
It is possible that autolysis may be an age-related phenomenon and that cells of a particular age within a hyphal network may become metabolically inactive leading to release of vacuolar hydrolases and autolysis. Many authors refer to age and age distribution in fungal cultures usually in a rather fleeting fashion.2,30,33 This cursory Enzyme Microb. Technol., 1998, vol. 22, May 1
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Figure 7 Intracellular protease activity of a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm with a dispersed mycelial inoculum without dissolved oxygen control. Activity is expressed as mg amino acid equivalent released min21 and specific activity mg21 protein or g21 dry cell weight (A). Intracellular b-1,3 glucanase activity found in the disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm with a dispersed mycelial inoculum without dissolved oxygen control Activity is expressed as mg reducing sugars released min21 and specific activity mg21 protein or g21 dry cell weight (B)
treatment is understandable in dealing with the filamentous fungi since physiological (culture) age and the age of individual compartments within the hyphae may differ.14 The distinction between the age of individual hyphae and culture age must be borne in mind when considering age. Nutritional status, the process of aging, and autolysis may well be linked in the fungi. As our results show, in young growing cultures, some hyphal regions showing autolysis are present, and as might be expected, as the mean age of cells in the culture increases, autolysis becomes more extensive regardless of whether excess C substrate is available or not. Similarly, cultures may be subject to some form of nutrient exhaustion. Both these effects may contribute to the extent of autolysis during the rapid growth phase. It therefore is clearly important to establish which forms of nutrient limit autolysis in a system where culture age is more readily controlled. The final values of mean main hyphal length in processes 1 and 3 are comparable (56 – 62 mm). This may be the effective equilibrium length of active hyphae for this strain18 below which further fragmentation due to mechanical damage does not occur; however, our results indicate that autolysis does lead to increased particle breakdown leading to generation of much cellular debris. 456
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400 rpm The time course of this process is shown in Figures 4a– 4c and 5a and 5b. Comparing the results in Figures 4a and 4a, it can be seen that there is again a close correlation between the early peak in specific intracellular proteolytic activity and the onset of the stationary phase. As with process one (800 rpm), a later peak in the mid-decline phase was apparent in protease and specific protease activity with the usual increase in broth NH1 4 ion concentration. In this process, there was a very large increase in intracellular b-1,3-glucanase activity between 182–213 h (Figure 5b). This occurred in the presence of the available C source. Values of morphological parameters in this process fell sharply in the phase 24 –72 h, which may have been due to entanglement of the longer mycelial particles to form pellets or aggregates30 while the proportion of the culture showing signs of autolysis rose until 185 h when it fell sharply. Two processes may have contributed to this: firstly, new or cryptic growth5 at the expense of autolysing regions, and secondly, the disintegration of autolysed regions to generate cellular debris. The latter process was particularly marked on microscopic examination of late process samples. From Figure 4b, it can be seen that signs of autolysis were apparent in some culture elements during the stationary
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al. phase (96 –164 h); thus, IA techniques can be used to detect and measure autolysis in advance of other methods (biomass decline, NH1 4 release). Some penicillin degradation occurred very late in the process associated with widespread disintegration of culture. Figure 4c indicates that C.E.R. declined only slightly at 132 and 186 h, a period when biomass was sharply declining and autolysis was proceeding; however, this CO2 evolution was not associated with C source consumption after 164 h. Autolysis is clearly an active (energy-consuming) process. The final mean main hyphal lengths are very similar to the processes at 800 rpm at around 60 mm.
perhaps indicating a ‘‘threshold’’ effect or an intrinsic characteristic of this strain.
Acknowledgments The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council, Chemicals and Pharmaceuticals Directorate.
References 1. 2.
200 rpm Process In this process, an early rapid growth phase (up to 64 h) was associated with NH1 4 decrease and rapid C utilization. As NH1 4 concentration reached a minimum (64 h), the rate of biomass increase slowly and penicillin V titer rose (Figure 6a). Morphologically, the early rapid growth phase coincided with pronounced decreases in the value of the key morphological indices (Figure 6b) and maximum apparent viscosity occurred at 64 h. Until 92 h, autolysis was scarcely present, but increased from this point onwards (Figure 6b). The onset of autolysis as measured by IA was associated with a sharp decrease in the penicillin V titer, and preceded biomass decline (post 120 h) and NH1 4 release (164 h). After 164 h, the culture extensively disintegrated into cellular debris, leading to the decrease in IA-measured autolysis. Final mean main hyphal length in these cultures was similar to those at higher impeller speeds, namely around 60 mm. The close similarity of the final mean main hyphal lengths in processes ranging from 800 rpm down to 200 rpm may indicate that this may be the result of an interaction between an innate characteristic of this particular strain and its physical environment. The difference between the findings of this study and others demonstrating a fall in morphological indices with increased stirring may be due to the fact that in the present study, all cultures were permitted to extensively autolyse; thus, the mean main hyphal length reported may be a consequence of that process and even modest agitation. Figure 7a and 7b again show two specific protease peaks— one early in the rapid growth phase and the other in the mid-autolytic phase as before while b-glucanase activity peaks very late in the process.
3.
4. 5.
6.
7. 8.
9. 10. 11. 12. 13.
14. 15.
Conclusions IA techniques can provide a sensitive indicator of the onset and progress of autolysis in fungal cultures. Autolysis may be effected by C and n limitation and/or culture aging. Culture history and inoculum type can have marked effects on the process. Autolysis P. chrysogenum may lead to loss of penicillin V and problems in broth filtration, and is associated with pronounced increases in the activity of both proteases and b-1,3 glucanases. Regardless of impeller speed, final mean main hyphal lengths in these autolysing cultures were very similar,
16. 17.
18. 19. 20.
de Pedro, M. A., Ho¨ltje, J. V. and Lo¨ffelhardt, W. (Eds.). Bacterial Cell Growth and Lysis. Plenum Press, New York, 1– end. 1993 Hernawan, T. and Fleet, G. Chemical and cytological changes during the autolysis of yeasts. J. Ind. Microbiol. 1995, 14, 440 – 450 Nombela, C., Molero, G., Martin, H., Cenamor, R., Molina, M., and Sanchez, M. Genetic control of fungal cell wall autolysis. In: Bacterial Cell Growth and Lysis (de Pedro, M. A., Ho¨ltje, J. V. and Lo¨ffelhardt, W., Eds.). Plenum Press, New York, 285–294. 1993 Lahoz, R., Reyes, F., and Perez Leblic, M. I. Lytic enzymes in the autolysis of filamentous fungi. Mycopathologia 1976, 60, 45– 49 Trinci, A. P. J. and Righelato, R. C. Changes in constituents and ultrastructure of hyphal compartments during autolysis of glucosestarved Penicillium chrysogenum. J. Gen. Microbiol. 1970, 60, 239 –249 Gordon, L. J. and Lilly, W. W. Quantitative analysis of Schizophyllum commune metalloprotease ScPrB activity in sds-gelatin PAGE reveals differential mycelial localization of nitrogen limitationinduced autolysis. Curr. Microbiol. 1995, 30, 337–343 Lahoz, R., Ballesteros, A. M., and Jimeno, L. Influence of temperature on the autolytic phase of growth of Aspergillus niger. Ann. Bot. 1974, 38, 661 Rober, B., Riemay, K. H., and Hilpert, A. Growth and autolysis of the ascomycete Hypomyces ochraceus. Metabolism of U-14c-lleucine, 2-14c-dl-threonine and U-14c-l-aspartic acid. J. Basic Microbiol. 1986, 26(8), 475– 482 Klionsky, D. J., Herman, P. K., and Emr, S. D. The fungal vacuole: Composition, function, and biogenesis. Microbiol. Rev. 1990, Sept, 266 –292 Santamaria, F. and Reyes, F. Proteases produced during the autolysis of filamentous fungi. Trans. Br. Mycol. Soc. 1988, 91(2), 217–220 Pitson, S. M., Seviour, R. J., and McDougall, B. M. Noncellulolytic fungal b-glucanases: Their physiology and regulation. Enzyme Microb. Technol. 1993, 15, 178 –192 Perez-Leblic, M. I., Reyes, F., and Lahoz, R. Autolysis of Penicillium oxalicum with special reference to its cell walls. Can. J. Microbiol. 1982, 28, 1289 –1295 Righelato, R. C., Trinci, A. P. J., and Pirt, S. J. The influence of maintainance energy and growth rate on the metabolic activity, morphology, and conidiation of Penicillium chrysogenum. J. Gen. Microbiol. 1968, 50, 399 – 412 Fencl, Z. Cell ageing and autolysis. In: The Filamentous Fungi III, Developmental Mycology. Edward Arnold, London, 389 – 405, 1974 (Smith, J. E. and Berry, D. R., (Eds.). Bilbrey, R. E., Penheiter, A. R., Gathman, A. C., and Lilly, W. W. Characterization of a novel phenylalanine-specific aminopeptidase from Schizophyllum commune. Mycol. Res. 1996, 100(4), 462– 466 Paul, G. C. and Thomas, C. R. Applications of image analysis in cell technology. Curr. Opinion Biotechnol. 1996, 7, 35– 45; Penicillium chrysogenum. Trans. I. Chem. E. 1994, 72C, 95–105 Nielsen, J., Johansen, C. L., Jacobsen, M., Krabben, P., and Villadsen, J. Pellet formation and fragmentation in submerged cultures of Penicillium chrysogenum and its relation to penicillin production. Biotechnol. Prog. 1995, 11, 93–98 Nielsen, J. and Krabben, P. Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures. Biotechnol. Bioeng. 1995, 46, 588 –598 Paul, G. C., Kent, C. A., and Thomas, C. R. Image analysis for characterizing differentiation of Penicillium chrysogenum. Trans. I. Chem. E. 1994, 72C, 95–105 Paul, G. C., Kent, C. A., and Thomas, C. R. Hyphal vacuolation and
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Papers
21. 22. 23. 24.
25.
26.
27.
28. 29.
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fragmentation in Penicillium chrysogenum. Biotechnol. Bioeng. 1994, 44, 655– 660 Vardaar, F. and Lilly, M. D. Effect of cycling dissolved oxygen concentrations on product formation in penicillin production. Eur. J. Appl. Microbiol. Biotechnol. 1982, 14, 203–211 Jorgenesen, H., Nielsen, J., and Villadsen, J. Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations. Biotechnol. Bioeng. 1995, 46, 117–131 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 1956, 28, 350 –356 Deo, Y. M. and Gaucher, G. M. Semicontinous and continous production of penicillin G by Penicillium chrysogenum cells immobilized in K-carrageenan beads. Biotechnol. Bioeng. 1983, 26, 285–295 Tucker, K. G. and Thomas, C. R. Effect of biomass concentration and morphology on the rheological parameters of Penicillium chrysogenum fermentation broths. Trans. I. Chem. E. 1993, 71, 111–117 Vanhoutte, B., Pens, M. N., Thomas, C. R., Louvel, L., and Vivier, H. Characterization of Penicillium chrysogenum physiology in submerged cultures by color and monochrome image analysis. Biotechnol. Bioeng. 1995, 48, 1–11 Mulenga, D. K. and Berry, D. R. Characteristics and role of b-glucanase enzymes associated with blastospore formation in Saccharomycopsis fibuligera NCYC 451. Microbios 1995, 82, 75– 86 Packer, H. L. and Thomas, C. R. Morphological measurements on filamentous microorganisms by fully automatic image analysis. Biotechnol. Bioeng. 1990, 35, 870 – 881 Tucker, K. G., Kelly, T., Delgrazia, P., and Thomas, C. R. Fully
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30.
31.
32. 33. 34. 35. 36. 37. 38. 39.
automatic measurement of mycelial morphology by image analysis. Biotechnol. Prog. 1992, 8, 353–359 Makagiansar, H. Y., Ayazi Shamlou, P., Thomas, C. R., and Lilly, M. D. The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum. Bioproc. Eng. 1993, 9, 83–90 Cronenberg, C. C. H., Ottengraf, S. P. P., van den Heuvel, J. C., Pottel, F., Sziele, D., Schugerl, K., and Bellgardt, K. H. Influence of age and structure of Penicillium chrysogenum pellets on the internal concentration profiles. Bioproc. Eng. 1994, 10, 209 –216 Wessels, J. G. H. Gene expression during fruiting of Schizophyllum commune. Mycol. Res. 1992, 18, 609 – 620 Nielsen, J. and Jorgensen, H. S. Metabolic control analysis of the penicillin biosynthetic pathway in a high-yielding strain of Penicillium chrysogenum. Biotechnol. Prog. 1995, 11, 299 –305 Pitson, S., Sevoiur, R. J., Bott, R., and Stasinopoulos, S. J. Production and regulation of b-glucanases in Acremonium and cephalosporin isolates. Mycol. Res. 1991, 95, 352–356 Nielsen, J. Physiological engineering aspects of Penicillium chrysogenum. D.Sc. thesis, Danish Technical University, Lyngby, 1994 Cohen, B. L. Regulation of protease production in Aspergillus. Trans. Br. Mycol. Soc. 1981, 76, 447– 450 Gottesman, S. and Maurizi, M. R. Regulation of proteolysis: Energy-dependent proteases and their targets. Microbiol. Rev. 1992, 56, 592– 621 Christensen, L., Nielsen, J., and Villadsen, J. Degradation of penicillin V in fermentation media. Biotechnol. Bioeng. 1994, 44, 165–169 Bilbery, R. E., Penheiter, A. R., Gathman, A. C., and Lilly, W. W. Characterisation of a novel phenylalanine-specific aminopeptidase from Schizophyllum commune. Mycol. Res. 1996, 100, 462– 466
Introduction The process by which microbial cells cease growing and commence to breakdown by the action of their own enzymes is generally referred to as autolysis. This process has received a great deal of attention in some bacterial species.1 Much research has also focused on autolysis in the commercially important yeast species.2 In contrast, studies of autolysis in the filamentous fungi have been far fewer,3 which given the biotechnological importance of these organisms, is rather surprising. Those studies which have in the past considered fungal autolysis have often had very lengthy experimental duration (10 – 60 days),4,5 the relevance of which to the current industrial process involving fungi, is questionable. Similarly, most previous studies have concentrated on various starvation regimes in terms of macronutrients (particularly carbon and nitrogen) to ‘‘induce’’ autolysis.5 Among the extrinsic factors which have been demonstrated to bring about the process of autolysis in fungi as C-source starvation (in fact, in most cases this is more correctly
Address reprint requests to Dr. L. M. Harvey, University of Strathclyde, Dept. of Bioscience and Biotechnology, 204 George Street, Glasgow G1 1XW, UK Received 12 November 1996; revised 10 February 1997; accepted 22 October 1997
Enzyme and Microbial Technology 22:446 – 458, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
termed carbon and energy source starvation), nitrogen starvation,6 increased temperature,2,7 and the presence of toxic compounds or metabolites including, e.g., ethanol.2 Regardless of the extrinsic factors which bring about autolysis, there is evidence that the process may take place in a number of stages8 involving an initial disruption of normal cellular metabolism. This leads to progressive loss of membrane function, failure of compartmentalization within the cell, allowing release/activation of the enzymes stored in compartments such as the vacuole.8,9 Release of these vacuolar enzymes and their activation leads rapidly to widespread proteolysis10 and to a rather slower and less complete degradation of the polymeric cell wall materials matrix polymers11 and chitin.5,11,12 Most authors emphasize the essential role of proteases in the process.6,10 With regard to the methods used to assess the extent of autolysis, in mold cultures conventionally, these have involved mean (i.e., whole culture measurements) of the decline in biomass4 or of cellular breakdown products (e.g., 5 NH1 4 release). While these may be useful overall, biomass decline itself is not always necessarily indicative of the process of autolysis and can result from ordered degradation of cellular reserves (e.g., glycogen).13 Changes in cellular composition can also lead to a decline in biomass levels. The process of autolysis in multicellular non-unitary systems such as filamentous fungi is complicated by both morphological/physiological differentiation and nutrient
0141-0229/98/$19.00 PII S0141-0229(97)00234-2
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al. translocation throughout the mycelial network;14 thus, while some hyphal elements may be exhibiting signs of growth, others at the same process time may exhibit autolytic signs.5,14,15 In examining the process of autolysis in an industrial strain of Penicillium chrsyogenum, we adopted three methods of analysis:
Table 1 Seed flask (inoculum) medium in g dm23
1. Conventional analyses of changes in biomass, nutrient levels, and release of NH1 4, 2. Enzyme assays: Measuring the activity of classes of enzymes which might be of key importance in cellular breakdown, particularly intracellular proteases and b 1,3 glucanases as indicators of degradation of intracellular material and cell wall, respectively, 3. Image analysis: To visualize and quantify the actual extent of autolysis in the fungal culture. Image analysis techniques have been widely used to study bioprocesses involving morphological differentiation within the biocatalyst.16 The studies of Nielsen et al.17,18 and Paul et al.19,20 on differentiation and degradation in P. chrysogenum were particularly useful in the selection of criteria for definition of autolytic regions as was the very detailed description of the cytological changes during yeast autolysis by Hernawan and Fleet.2 This allowed direct quantification of the extent of autolysing regions within the culture samples.
the flask. Spore counts were performed by means of serial dilution (if required) and use of a Neubauer counting chamber.
A number of studies in P. chrysogenum have examined the process of mycelial fragmentation17,18 often exclusively from the viewpoint of extrinsic factors (power input, shearing forces) while, with the notable exception of Nielsen and Krabben18 and Paul et al.20 omitting any mention of the intrinsic factors. Paul et al.,20 however, noted a link between the process of hyphal degeneration in P. chyrsogenum (as measured by an increase in vacuolation in older hyphal elements), and fragmentation, clarifying the findings of an earlier study by Righelato et al13. which had indicated the possibility of such a link. In view of these findings and the end point of the processes of cellular degeneration, it seemed worthwhile to investigate the effects of processes occurring during or immediately before autolysis, such as wall weakening, loss of cell rigidity, and decreased turgor pressure, on fragmentation. We therefore examined autolysis in an industrial strain of P. chrysogenum (SKB) in a complex medium in submerged batch cultures at a range of impeller speeds. Culture D.O.T. was maintained at a minimum of 40% saturation at all times both to exclude this source of variability and to avoid effects of O2 limitation with subsequent deleterious influence on penicillin V synthesis.21,22
Corn steep liquor Glucose (NH4)2SO4 Rape seed oil
70.0 15.0 4.0 0.5(ml)
Inoculum production Spore suspension (1 ml) was added to 0.2 dm3 of seed medium in 0.5 dm3 Erlenmeyer flasks. Flasks were incubated at 25°C and 200 rpm for 48 h. This vegetative culture was used as the inoculum for the bioreactor.
Media For media preparation, see Tables 1 and 2.
Bioreactor A fully instrumented BIOSTAT ED10 (B. Braun Biotech. Ltd, Melfungen, Germany) with an operating volume of 9 dm3 was employed in this study. Dissolved oxygen tension was maintained at or above 40% of air saturation (unless otherwise stated) using a Uniprobe dissolved oxygen controller linked to a gas mixing unit (B. Braun Biotech. Ltd) which supplied air or an air/O2 mix. Culture conditions were as follows: initial pH, 4.9; agitation rate variable, aeration rate 1 vvm; and temperature, 25°C. All fermentations were performed in batch mode. Sodium phenoxyacetate was added to each process continuously from 68 h at a rate of 8 ml h21 of a 0.3% solution. The agitation rate was constant throughout each batch process and ranged from 200 – 800 rpm. The bioreactor was equipped with three six-bladed Rushton turbine impellers (each 0.5 vessel diameter).
Biomass determination Biomass was measured by means of dry weight measurement. Whole broth (10 ml) was filtered through predried and weighed Whatman GF/C filter papers. The filter and biomass were then dried in a microwave oven on the defrost setting for 20 min or until a constant dry weight was obtained. The sample was then placed in a desiccator for a further 20 min until cool and then was weighed. All samples were analyzed in triplicate. The filtrate, which was required for further analyses, was immediately frozen at 220°C to prevent deterioration of the sample. All further analyses were performed as rapidly as possible.
Exit gas analysis The concentration of oxygen and carbon dioxide in the inlet and outlet gas streams were measured using a paramagnetic oxygen
Materials and methods Microorganism and bioreactor
Table 2 g dm23
Batch fermenter medium in
An industrial strain of P. chrysogenum was supplied by SmithKline Beecham Pharmaceuticals (Irvine, UK) in the form of liquid spore suspensions contained in sealed 10-ml glass bottles. These were stored at 220°C and brought to room temperature before use as the inoculum for seed flasks. Approximately 1 ml of spore suspension was added to each seed flask (fermenter inoculum). This was equivalent to a spore concentration of 2.1 3 105 ml21 in
Corn steep liquor Lactose KH2PO4 K2HPO4 MgSO4 z 7H2O Rape seed oil
75.0 70.0 2.5 2.5 0.4 0.5(ml)
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Papers analyzer (Model 540A, Servomex Ltd., Sussex, UK) and an infrared CO2 analyzer (Model 7000, Analytical Development Co. Ltd., Cambridge, UK), respectively. Gases were passed over a drying column of medium grade silica gel before measurements were taken.
Viscosity measurements Apparent viscosity of the whole broth was measured using a Ferranti concentric cylinder viscometer, model VL. Readings were taken using 0.1 dm3 broth samples at 25°C and a shear rate of 68 s21.
digitized on the basis of each pixel greyscale level (where 0 is black and 255 is white) and then converted to a binary two-color image. Objects within the image were then analyzed; however, objects shown in the binary image were determined by the greyscale threshold level set by the operator. Whole broth samples (2 ml) were taken from the fermenter at regular intervals and added to 1 ml of lactophenol blue and then made up to a 20 ml volume using a fixative. This was comprised of 5.6% formaldehyde and 2.5% glacial acetic acid made up to a 200 ml volume with 50% ethanol. Samples were stored at 4°C before analysis.25 For each sample, 50 free mycelial elements (organisms) were analyzed although it has been proposed that 40 would be adequate.26
Total carbohydrate assay The total amount of sugars present in the fermentation broth was determined using the method of Dubois et al.23 Standards were prepared using a concentration range between 10 –100 mg dm23. All measurements were made in triplicate.
Ammonium ion assay The ammonium ion concentration was measured using an assay kit by Sigma (No. 640, St. Louis, MO). Standards of ammonium chloride were prepared in the range 3.125–100 mg dm23. Samples were diluted 1:10 before use.
Penicillin V analysis Penicillin V concentration was assayed by HPLC analysis. The following method (modified from that of Deo and Gaucher)24 was employed. Apparatus. A Perkin-Elmer 250LC pump with a LC90J detector connected to a Perkin-Elmer Nelson 900 series interface with PC Integrator (software version 5.1.5., Perkin-Elmer Nelson Systems Inc.) was used. The column was a Nucleosil C18, 250 mm 3 4.6 mm with 5 mm particle packing (Burke Analytical, Glasgow, UK). The guard column was 20 mm 3 2 mm (internal diameter) packed with Spherisorb (3 g) Reverse Phase 18 (Anachem). The operating pressure was between 1,500 –2,500 psi. Mobile phase. This was an isocratic system maintaining constant eluent composition using a mobile phase of acetonitrile:sodium phosphate buffer (25:75 v/v) at room temperature. The mobile phase was filtered through a 0.22 mm Whatman filter paper and was degassed daily with helium before use. Sample preparation. All samples and standards were filtered through 0.2 mm Whatman nylon filter papers (diameter 13 mm). Samples were diluted 1 in 20 with mobile phase prior to filtration and injection. Absorbance was measured at 220 nm and the range setting was 0.5. The sample/standard was injected using a 20 ml injection loop and a flow rate of 1 ml min21. The internal standard used was penicillin G since this had a distinct retention time from that of penicillin V.
Image analysis Morphological characteristics of the culture were examined using a Seescan image analysis system. This produced digitized monochrome images via a Nikon Optiphot-2 microscope on which was mounted a Sony CCD video camera (Model XC-77CE). The magnifications used were 3100 and 3200 depending on the dimensions of the mycelial elements. The images of dispersed mycelial elements were then analyzed manually. The image obtained on the monochrome video camera was
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Measurements. Mean main hyphal length, number and length of branches, and autolytic lengths were measured. The mean hyphal growth unit was calculated for each hyphal tree (organism) by dividing the total hyphal length by the number of branches.
Enzymology Approximately 50 g of whole broth sample was weighed (noting volume) and centrifuged at 20,000 rpm for 10 min at 4°C. From this, 20 ml of supernatant was removed and stored for further analyses. The pellet obtained was transferred to a preweighed centrifuge tube, washed with 50 ml of 50 mm phosphate buffer pH 6.2, vortex mixed, and then re-centrifuged as above. This process was repeated a further four times. The pellet obtained was weighed and then transferred to a cell disrupter. A high pressure cell disrupter (Model 4000, Constant Systems Ltd., Warwick, UK) which gave very rapid sample disruption was used to disrupt mycelial samples before further sample treatment and analysis. After centrifugation (as above), the supernatant was then transferred to dialysis tubing (M. W. cut-off, 12,000 –14,000; pore size, 2.4 nm) and dialyzed for 3 3 8 h in 10 mm phosphate buffer pH 6.2 at 4°C. After dialysis, samples were stored at 220°C until required for analysis.10
Total proteolytic activity Determination of total proteolytic activity. The method of Santamaria and Reyes10 was employed. Casein (0.1% w/v) was dissolved in 0.25 m phosphate buffer pH 6.0, made up to 0.1 dm23, and used as the substrate. The solution was stored at 4°C. To 0.5 ml of substrate solution, 0.5 ml of suitably diluted enzyme (sample) solution was added. This was then incubated for 30 min at 30°C. The reaction was terminated by boiling for 5 min. The a-amino nitrogen released was determined using the ninhydrin method. The activity of the samples is expressed in terms of mg amino acid equivalents released min21 of the reaction (the conversion rate). Activity is also expressed in terms of specific activity where the conversion rate is divided by the total protein or biomass. This is then expressed as mg amino acid equivalents released min21 unit21 protein or unit21 biomass. Determination of b-glucanase activity. The method of Mulenga and Berry27 was used. Activity is expressed as mg glucose equivalents released min21 (conversion rate). Specific activity is in terms of mg glucose equivalents released min21 unit21 total protein or unit21 biomass in the sample.
Results and discussion Many studies have examined mycelial fragmentation in P. chrysogenum17,18,20,28 –31 and although it has been shown that changes in morphological parameters of the dispersed
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al. form are generally related to stirrer speed, Nielsen et al.17 have shown that a steady reduction in mean main hyphal length, taken as an indicator of mycelial fragmentation, occurs even at low impeller speeds. Few of these studies consider the physiology of the microorganism itself as being the factor likely to contribute to the frequency/location of fracture points. Given recent studies relating to differences in fungal cell wall structure along the hyphal length, this could be a significant oversight.32 Our initial view was that the process of autolysis might well influence that of mycelial fragmentation with areas undergoing autolysis being more likely to fragment. We thus examined, at a fixed minimum dissolved oxygen level of 40%, a range of impeller speeds using a complex medium. (Since dissolved oxygen (DO) is critically important in P. chrysogenum in terms of growth and penicillin synthesis,21,33 DO was controlled at 40% in all processes). Without medium supplementation, it was expected that this medium would be depleted of the readily available C and/or n source between 5–7 days, which based on literature reports on other organisms, should lead to autolysis in this culture.2– 6 This should permit examination of effects of impeller speed on autolysis and the effect of exhaustion of a major nutrient independently of dissolved oxygen tension.
800 rpm Process, pelleted inoculum The time course of this process is shown in Figures 1a–1c and 2a and 2b (enzymology). In this process the lag, rapid growth phase, stationary phase, and decline (autolytic) phase are clearly apparent in Figure 1a. NH1 4 was absent from the filtrate from 48 h onwards. Penicillin V synthesis and the commencement of ‘‘stationary’’ phase (a much reduced growth rate) begin around 72 h. At this stage, some hyphal segments were beginning to show signs of autolysis despite the bulk of the culture having a healthy appearance (Figure 1b). Trinci and Righelato5 noted that such autolytic segments may well occur in the middle of otherwise healthy hyphae. This was a point also emphasised by Fencl.14 Cellular breakdown was thus occurring while culture cell mass was still increasing. Around 164 h the available C source was exhausted. From this point, NH1 4 reappeared in the filtrate presumably from deamination of proteins.5,34,35 Biomass declined sharply (Figure 1a) and autolysis (as measured by image analysis) in the culture continued (Figure 1b). These events coincided with a peak in intracellular protease activity and specific intracellular protease activity at 213 h (Figure 2a) and peaks in b-1,3 glucanase activity and specific activity at 190 h (Figure 2b). Image analysis measurements (Figure 1b) showed significant signs of autolysis in the culture before other indicators such as dry weight decline and NH1 4 release; thus, these measurements of micromorphology may well be a more sensitive indicator of the onset of autolysis than biomass decline throughout the culture as a whole. The exhaustion of NH1 4 did not, in this instance, lead to rapid autolysis despite other studies showing this to be the case.14,34,36 This may be due to uptake of other n sources (e.g., amino acids) by the cells from the complex medium or, more probably, from the results obtained, autolysis may
be related primarily to the disappearance of the energy source. A number of authors have indicated that exhaustion of an exogenous energy source in fungi rapidly leads to a depletion in cellular storage compounds (e.g., glycogen) and then to a suspension of normal metabolism.2,5 These results show that when the C source is exhausted, a series of events occurs involving morphological change, proteolysis/ release of NH1 4 and increased b-glucanase activity. Despite the latter, no free C source appeared in the filtrates. This confirmed the results of Cohen36 and Pitson et al.34 who indicated that b-glucanase activity in autolysis generally did not lead to release of glucose but did lead either to partial wall restructuring (loosening) or the observation that any monosaccharides generated were utilized for cryptic growth. In this study, intrahyphal growth was noted in the autolytic phase as was the appearance of apparently healthy hyphal tips from degraded hyphal masses. This is strong evidence for the occurrence of cryptic growth in certain culture elements. Comparing C consumption (Figure 1a) with C.E.R. (Figure 1c), it can be seen that for most of the decline phase (164 h), C.E.R. remained steady. This indicated that energy generation/consumption were probably still proceeding in a fraction of the culture at this stage. The collapse in C.E.R. after 218 h is due to the fact that the great bulk of the culture is completely degraded by this stage as confirmed by microscopic and IA examination. As can be seen in Figure 2a, specific intracellular protease activity (g21 dry weight and g protein) is maximal near the end of the rapid growth phase (;80 h). It thereafter falls sharply. This may well reflect the rapid protein turnover in the growing culture at this point. Later peaks in specific intracellular proteolytic activity and specific activity (occuring between 180 –214 h) are almost certainly related to the processes of autolysis which, as a number of authors have indicated, is predominantly a proteolytic event.21 It is possible that assaying for total proteolytic activity may be obscuring our view of autolysis somewhat in that the high proteolytic activity seen in the growth phase may represent different types of protease from those in the autolytic phase. In the latter case, vacuolar proteases have been implicated9 and, in general, this fits well with the observed increase in culture vacuolation noted in this study and in yeast;2 however, another group of proteases has been shown to be especially important to the cell during transitional periods, e.g., the transition from exponential to stationary phase when nutritional status rapidly changes.37 Figures 1a and 2a show that the early specific protease peak occurs very close to the entry into ‘‘stationary’’ phase (based on the biomass curve). These proteases are generally agreed to be largely concerned with stress responses in cells while the vacuolar hydrolases seem largely to be involved during major changes in nutritional status. Based on our results, such a pattern is plausible with the release/activation of vacuolar hydrolases (especially proteolytic enzymes) being associated with C exhaustion leading to the syndrome noted in the decline/autolysis phase in this culture. In this process, the sequence of events in late stationary/ early decline phase may thus be exhaustion of energy source, depletion of endogenous reserves, failure to maintain membrane function leading to release of compartmentalized hydrolases (largely proteases), activation of hydroEnzyme Microb. Technol., 1998, vol. 22, May 1
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Figure 1 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (pelleted inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (pelleted inoculum) (B). Carbon dioxide evolution rate and dissolved oxygen concentration vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (pelleted inoculum) (C)
lytic enzymes (based on the literature, this may be about the pH shift on release from the storage compartment),2,9 proteolysis, deamination of amino acids, partial glucan hydrolysis, wall weakening, loss of structural integrity, and 450
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hyphal disintegration. The results of this fermentation process seem to point toward such a sequence. The IA results (Figure 1b) generally indicate the trend in morphology noted in P. chrysogenum processes by oth-
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al.
Figure 2 Intracellular protease activity found in disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm with a pelleted inoculum. Activity is expressed as mg amino acid equivalent released min21 and specific activity mg21 protein or g21 dry cell weight (A). Intracellular b-1,3 glucanase activity found in the disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm with a pelleted inoculum. Activity is expressed as mg of reducing sugars released min21 and specific activity mg21 protein or g21 dry cell weight (B)
ers18,28 –30 with a steady decline in the value of key parameters including mean main hyphal length with time due to mycelial fragmentation and possibly also to the increased tendency of the longer dispersed hyphal elements to form aggregates.30 The general engineering view of this process of fragmentation is that it is an event which occurs randomly and is related solely to power input. In our view, it is wrong not to consider the physiological condition of the microorganism with respect to mycelial fragmentation, and Neilsen35 indicates that it may be wise to do so. The earlier suggestion by Righelato et al.13 that there could be a link between vacuolation and fragmentation (which was confirmed by Paul et al.20) also indicates the relevance of considering the physiological aspects of fragmentation. If fragmentation increased in highly autolytic cultures, it might be expected to be reflected in a sharp decrease in,
e.g., mean main hyphal length and branch length during the autolytic phase. From Figure 1b, this did not appear to happen with the decline in both values being approximately linear from the time of entry to the stationary phase (76 – 80 h) onward; however, visual examination showed a high proportion of cellular debris (particles , 30 mm in length) especially after 28 h. The values of morphological parameters measured were based on freely dispersed organisms which became far less common in the autolytic phase. One of the major drawbacks to IA measurements in the late stages of the process was the lengthy time spent locating 50 well-dispersed ‘‘organisms’’ upon which to make measurements. Even those few ‘‘organisms’’ which remained intact by late autolytic stage showed very extensive signs of autolysis. IA techniques are useful in measurement of regions which are autolysing, but since the end process of
Table 3 Time taken to filter a given volume (10 ml) of whole broth and to wash filter cake 800 800 400 200
rpm pellet inoculum rpm rpm rpm
Up Up Up Up
to to to to
190 190 163 162
h–immediate h–immediate h–immediate h–immediate
194 214 186 186
h–56 h–20 h–60 h–90
min s s s
236 h–56 min 236 h–60 s 210 h–60 s –
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Figure 3 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (dispersed mycelial inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (dispersed mycelial inoculum) (B). Carbon dioxide evolution rate and dissolved oxygen concentration vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 800 rpm (dispersed mycelial inoculum) (C)
autolysis is cellular disintegration, IA clearly has practical limits. Consideration of micromorphology is very valuable especially as a very early and sensitive indicator of the process 452
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of autolysis in cultures which, based on conventional analysis (dry weight and NH1 4 release) and enzymology are not yet clearly in the decline or autolytic phases, but quantitative information in micromorphology must be inter-
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al.
Figure 4 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (B). Carbon dioxide evolution rate, dissolved oxygen concentration, and apparent viscosity vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (C)
preted very carefully and, if possible, integrated with some consideration of macromorphology. The onset of autolysis in culture elements coincides closely with that of penicillin synthesis (Figures 1a and 1b) and entry to the stationary phase. A striking feature of the autolytic phase (from 164 h on) is the marked decline in penicillin V concentration. This analysis was repeated on three separate occasions to confirm it. Nielsen35 indicates that the degradation of penicillin V in a complex medium is
largely to penicilloic acid (40%); the remainder is to compounds as yet unknown. The striking decrease in penicillin V titer may represent not so much an increased degradation rate as a cessation of synthesis, since the penicillin V to penicilloic acid reaction proceeds even during the penicillin synthetic period with a first-order degradation rate based on penicillin concentration;38 however, the rate of degradation reactions are heavily dependent on operational conditions. It is possible that degradation Enzyme Microb. Technol., 1998, vol. 22, May 1
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Figure 5 Intracellular protease activity found in disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm with a dispersed mycelial inoculum. Activity is expressed as mg amino acid equivalent released min21 and specific activity mg21 protein or g21 dry cell weight (A). Intracellular b-1,3 glucanase activity found in the disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm with a dispersed mycelial inoculum. Activity is expressed as mg reducing sugars released min21 and specific activity mg21 protein or g21 dry cell weight (B)
during the autolytic phase may be due to release of peptidases or amidohydrolases39 which are normally physically separated from the penicillin V, but may come into contact on membrane breakdown. Another aspect of autolysis in P. chrysogenum which may have considerable economic impact is the increase in filtration time (Table 3), i.e., the time taken to separate the cells/cellular debris from the filtrate. This increased very substantially, thus any recovery process involving initial solids separation would be lengthened and suffer a further decrease in penicillin V titer. Based on morphological examination, the decrease in filtrability may be due to the disintegration of the ordered mycelial network which would tend to maintain a relatively open fibrous structure on filtering into a largely degraded material with large quantities of cellular debris which forms a densely packed filter cake.
800 rpm Dispersed mycelial sample A younger vegetative inoculum was used in this and subsequent processes. The inoculum was of a dispersed mycelial nature as opposed to the pellet inoculum of the first process. The results of the processes are shown in Figures 3a–3c. 454
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In this process, the rate of biomass increase was lower and the growth phase more prolonged. Free NH1 4 did not disappear from the filtrate until 120 h; some of the C source was present until the end of the process. The decline in the biomass concentration (past 180 h) was again accompanied by the reappearance of free NH1 4 ions in the culture fluid. Signs of autolysis were apparent in the culture in the growth phase. The percentage of total hyphal length showing signs of autolysis rose linearly from 90 –213 h. This again emphasizes that autolysis of some hyphal regions occurs even during the growth processes. As with the previous process, widespread mycelial disintegration occurred from 180 h onward. It became time consuming to find organisms for measurement of morphological indices; however, those organisms examined were generally healthier with less apparent autolytic regions than in the previous process. This may well have been due to the presence of the available C source in this phase. Overall, this phase makes it clear that the previous history of the culture has an influence on subsequent culture behavior and the process of autolysis, and that NH1 4 limitation/exhaustion does not increase the occurrence of autolysis in hyphae in this batch process. The presence of an available energy source late in the process
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al.
Figure 6 Biomass, total carbohydrate, NH1 4 , and penicillin V concentrations vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm (dispersed mycelial inoculum) (A). Mean main hyphal length, mean branch length, mean hyphal growth unit, and percentage of total hyphal length showing autolysis vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 400 rpm (dispersed mycelial inoculum) (B). Carbon dioxide evolution rate, dissolved oxygen concentration, and apparent viscosity vs. time in a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm (dispersed mycelial inoculum) (C)
did not prevent widespread culture disintegration, but those organisms present were maintained in a healthier condition than when no C source was available (process 1). A number of reports have indicated the key role of the C/energy source in the maintenance of cell viability and the process of autolysis.2,5,13
It is possible that autolysis may be an age-related phenomenon and that cells of a particular age within a hyphal network may become metabolically inactive leading to release of vacuolar hydrolases and autolysis. Many authors refer to age and age distribution in fungal cultures usually in a rather fleeting fashion.2,30,33 This cursory Enzyme Microb. Technol., 1998, vol. 22, May 1
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Figure 7 Intracellular protease activity of a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm with a dispersed mycelial inoculum without dissolved oxygen control. Activity is expressed as mg amino acid equivalent released min21 and specific activity mg21 protein or g21 dry cell weight (A). Intracellular b-1,3 glucanase activity found in the disrupted mycelial samples of a batch fermentation of P. chrysogenum at an agitation rate of 200 rpm with a dispersed mycelial inoculum without dissolved oxygen control Activity is expressed as mg reducing sugars released min21 and specific activity mg21 protein or g21 dry cell weight (B)
treatment is understandable in dealing with the filamentous fungi since physiological (culture) age and the age of individual compartments within the hyphae may differ.14 The distinction between the age of individual hyphae and culture age must be borne in mind when considering age. Nutritional status, the process of aging, and autolysis may well be linked in the fungi. As our results show, in young growing cultures, some hyphal regions showing autolysis are present, and as might be expected, as the mean age of cells in the culture increases, autolysis becomes more extensive regardless of whether excess C substrate is available or not. Similarly, cultures may be subject to some form of nutrient exhaustion. Both these effects may contribute to the extent of autolysis during the rapid growth phase. It therefore is clearly important to establish which forms of nutrient limit autolysis in a system where culture age is more readily controlled. The final values of mean main hyphal length in processes 1 and 3 are comparable (56 – 62 mm). This may be the effective equilibrium length of active hyphae for this strain18 below which further fragmentation due to mechanical damage does not occur; however, our results indicate that autolysis does lead to increased particle breakdown leading to generation of much cellular debris. 456
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400 rpm The time course of this process is shown in Figures 4a– 4c and 5a and 5b. Comparing the results in Figures 4a and 4a, it can be seen that there is again a close correlation between the early peak in specific intracellular proteolytic activity and the onset of the stationary phase. As with process one (800 rpm), a later peak in the mid-decline phase was apparent in protease and specific protease activity with the usual increase in broth NH1 4 ion concentration. In this process, there was a very large increase in intracellular b-1,3-glucanase activity between 182–213 h (Figure 5b). This occurred in the presence of the available C source. Values of morphological parameters in this process fell sharply in the phase 24 –72 h, which may have been due to entanglement of the longer mycelial particles to form pellets or aggregates30 while the proportion of the culture showing signs of autolysis rose until 185 h when it fell sharply. Two processes may have contributed to this: firstly, new or cryptic growth5 at the expense of autolysing regions, and secondly, the disintegration of autolysed regions to generate cellular debris. The latter process was particularly marked on microscopic examination of late process samples. From Figure 4b, it can be seen that signs of autolysis were apparent in some culture elements during the stationary
Autolysis in batch cultures of Penicillium chrysogenum: L. M. Harvey et al. phase (96 –164 h); thus, IA techniques can be used to detect and measure autolysis in advance of other methods (biomass decline, NH1 4 release). Some penicillin degradation occurred very late in the process associated with widespread disintegration of culture. Figure 4c indicates that C.E.R. declined only slightly at 132 and 186 h, a period when biomass was sharply declining and autolysis was proceeding; however, this CO2 evolution was not associated with C source consumption after 164 h. Autolysis is clearly an active (energy-consuming) process. The final mean main hyphal lengths are very similar to the processes at 800 rpm at around 60 mm.
perhaps indicating a ‘‘threshold’’ effect or an intrinsic characteristic of this strain.
Acknowledgments The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council, Chemicals and Pharmaceuticals Directorate.
References 1. 2.
200 rpm Process In this process, an early rapid growth phase (up to 64 h) was associated with NH1 4 decrease and rapid C utilization. As NH1 4 concentration reached a minimum (64 h), the rate of biomass increase slowly and penicillin V titer rose (Figure 6a). Morphologically, the early rapid growth phase coincided with pronounced decreases in the value of the key morphological indices (Figure 6b) and maximum apparent viscosity occurred at 64 h. Until 92 h, autolysis was scarcely present, but increased from this point onwards (Figure 6b). The onset of autolysis as measured by IA was associated with a sharp decrease in the penicillin V titer, and preceded biomass decline (post 120 h) and NH1 4 release (164 h). After 164 h, the culture extensively disintegrated into cellular debris, leading to the decrease in IA-measured autolysis. Final mean main hyphal length in these cultures was similar to those at higher impeller speeds, namely around 60 mm. The close similarity of the final mean main hyphal lengths in processes ranging from 800 rpm down to 200 rpm may indicate that this may be the result of an interaction between an innate characteristic of this particular strain and its physical environment. The difference between the findings of this study and others demonstrating a fall in morphological indices with increased stirring may be due to the fact that in the present study, all cultures were permitted to extensively autolyse; thus, the mean main hyphal length reported may be a consequence of that process and even modest agitation. Figure 7a and 7b again show two specific protease peaks— one early in the rapid growth phase and the other in the mid-autolytic phase as before while b-glucanase activity peaks very late in the process.
3.
4. 5.
6.
7. 8.
9. 10. 11. 12. 13.
14. 15.
Conclusions IA techniques can provide a sensitive indicator of the onset and progress of autolysis in fungal cultures. Autolysis may be effected by C and n limitation and/or culture aging. Culture history and inoculum type can have marked effects on the process. Autolysis P. chrysogenum may lead to loss of penicillin V and problems in broth filtration, and is associated with pronounced increases in the activity of both proteases and b-1,3 glucanases. Regardless of impeller speed, final mean main hyphal lengths in these autolysing cultures were very similar,
16. 17.
18. 19. 20.
de Pedro, M. A., Ho¨ltje, J. V. and Lo¨ffelhardt, W. (Eds.). Bacterial Cell Growth and Lysis. Plenum Press, New York, 1– end. 1993 Hernawan, T. and Fleet, G. Chemical and cytological changes during the autolysis of yeasts. J. Ind. Microbiol. 1995, 14, 440 – 450 Nombela, C., Molero, G., Martin, H., Cenamor, R., Molina, M., and Sanchez, M. Genetic control of fungal cell wall autolysis. In: Bacterial Cell Growth and Lysis (de Pedro, M. A., Ho¨ltje, J. V. and Lo¨ffelhardt, W., Eds.). Plenum Press, New York, 285–294. 1993 Lahoz, R., Reyes, F., and Perez Leblic, M. I. Lytic enzymes in the autolysis of filamentous fungi. Mycopathologia 1976, 60, 45– 49 Trinci, A. P. J. and Righelato, R. C. Changes in constituents and ultrastructure of hyphal compartments during autolysis of glucosestarved Penicillium chrysogenum. J. Gen. Microbiol. 1970, 60, 239 –249 Gordon, L. J. and Lilly, W. W. Quantitative analysis of Schizophyllum commune metalloprotease ScPrB activity in sds-gelatin PAGE reveals differential mycelial localization of nitrogen limitationinduced autolysis. Curr. Microbiol. 1995, 30, 337–343 Lahoz, R., Ballesteros, A. M., and Jimeno, L. Influence of temperature on the autolytic phase of growth of Aspergillus niger. Ann. Bot. 1974, 38, 661 Rober, B., Riemay, K. H., and Hilpert, A. Growth and autolysis of the ascomycete Hypomyces ochraceus. Metabolism of U-14c-lleucine, 2-14c-dl-threonine and U-14c-l-aspartic acid. J. Basic Microbiol. 1986, 26(8), 475– 482 Klionsky, D. J., Herman, P. K., and Emr, S. D. The fungal vacuole: Composition, function, and biogenesis. Microbiol. Rev. 1990, Sept, 266 –292 Santamaria, F. and Reyes, F. Proteases produced during the autolysis of filamentous fungi. Trans. Br. Mycol. Soc. 1988, 91(2), 217–220 Pitson, S. M., Seviour, R. J., and McDougall, B. M. Noncellulolytic fungal b-glucanases: Their physiology and regulation. Enzyme Microb. Technol. 1993, 15, 178 –192 Perez-Leblic, M. I., Reyes, F., and Lahoz, R. Autolysis of Penicillium oxalicum with special reference to its cell walls. Can. J. Microbiol. 1982, 28, 1289 –1295 Righelato, R. C., Trinci, A. P. J., and Pirt, S. J. The influence of maintainance energy and growth rate on the metabolic activity, morphology, and conidiation of Penicillium chrysogenum. J. Gen. Microbiol. 1968, 50, 399 – 412 Fencl, Z. Cell ageing and autolysis. In: The Filamentous Fungi III, Developmental Mycology. Edward Arnold, London, 389 – 405, 1974 (Smith, J. E. and Berry, D. R., (Eds.). Bilbrey, R. E., Penheiter, A. R., Gathman, A. C., and Lilly, W. W. Characterization of a novel phenylalanine-specific aminopeptidase from Schizophyllum commune. Mycol. Res. 1996, 100(4), 462– 466 Paul, G. C. and Thomas, C. R. Applications of image analysis in cell technology. Curr. Opinion Biotechnol. 1996, 7, 35– 45; Penicillium chrysogenum. Trans. I. Chem. E. 1994, 72C, 95–105 Nielsen, J., Johansen, C. L., Jacobsen, M., Krabben, P., and Villadsen, J. Pellet formation and fragmentation in submerged cultures of Penicillium chrysogenum and its relation to penicillin production. Biotechnol. Prog. 1995, 11, 93–98 Nielsen, J. and Krabben, P. Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures. Biotechnol. Bioeng. 1995, 46, 588 –598 Paul, G. C., Kent, C. A., and Thomas, C. R. Image analysis for characterizing differentiation of Penicillium chrysogenum. Trans. I. Chem. E. 1994, 72C, 95–105 Paul, G. C., Kent, C. A., and Thomas, C. R. Hyphal vacuolation and
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fragmentation in Penicillium chrysogenum. Biotechnol. Bioeng. 1994, 44, 655– 660 Vardaar, F. and Lilly, M. D. Effect of cycling dissolved oxygen concentrations on product formation in penicillin production. Eur. J. Appl. Microbiol. Biotechnol. 1982, 14, 203–211 Jorgenesen, H., Nielsen, J., and Villadsen, J. Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations. Biotechnol. Bioeng. 1995, 46, 117–131 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 1956, 28, 350 –356 Deo, Y. M. and Gaucher, G. M. Semicontinous and continous production of penicillin G by Penicillium chrysogenum cells immobilized in K-carrageenan beads. Biotechnol. Bioeng. 1983, 26, 285–295 Tucker, K. G. and Thomas, C. R. Effect of biomass concentration and morphology on the rheological parameters of Penicillium chrysogenum fermentation broths. Trans. I. Chem. E. 1993, 71, 111–117 Vanhoutte, B., Pens, M. N., Thomas, C. R., Louvel, L., and Vivier, H. Characterization of Penicillium chrysogenum physiology in submerged cultures by color and monochrome image analysis. Biotechnol. Bioeng. 1995, 48, 1–11 Mulenga, D. K. and Berry, D. R. Characteristics and role of b-glucanase enzymes associated with blastospore formation in Saccharomycopsis fibuligera NCYC 451. Microbios 1995, 82, 75– 86 Packer, H. L. and Thomas, C. R. Morphological measurements on filamentous microorganisms by fully automatic image analysis. Biotechnol. Bioeng. 1990, 35, 870 – 881 Tucker, K. G., Kelly, T., Delgrazia, P., and Thomas, C. R. Fully
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automatic measurement of mycelial morphology by image analysis. Biotechnol. Prog. 1992, 8, 353–359 Makagiansar, H. Y., Ayazi Shamlou, P., Thomas, C. R., and Lilly, M. D. The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum. Bioproc. Eng. 1993, 9, 83–90 Cronenberg, C. C. H., Ottengraf, S. P. P., van den Heuvel, J. C., Pottel, F., Sziele, D., Schugerl, K., and Bellgardt, K. H. Influence of age and structure of Penicillium chrysogenum pellets on the internal concentration profiles. Bioproc. Eng. 1994, 10, 209 –216 Wessels, J. G. H. Gene expression during fruiting of Schizophyllum commune. Mycol. Res. 1992, 18, 609 – 620 Nielsen, J. and Jorgensen, H. S. Metabolic control analysis of the penicillin biosynthetic pathway in a high-yielding strain of Penicillium chrysogenum. Biotechnol. Prog. 1995, 11, 299 –305 Pitson, S., Sevoiur, R. J., Bott, R., and Stasinopoulos, S. J. Production and regulation of b-glucanases in Acremonium and cephalosporin isolates. Mycol. Res. 1991, 95, 352–356 Nielsen, J. Physiological engineering aspects of Penicillium chrysogenum. D.Sc. thesis, Danish Technical University, Lyngby, 1994 Cohen, B. L. Regulation of protease production in Aspergillus. Trans. Br. Mycol. Soc. 1981, 76, 447– 450 Gottesman, S. and Maurizi, M. R. Regulation of proteolysis: Energy-dependent proteases and their targets. Microbiol. Rev. 1992, 56, 592– 621 Christensen, L., Nielsen, J., and Villadsen, J. Degradation of penicillin V in fermentation media. Biotechnol. Bioeng. 1994, 44, 165–169 Bilbery, R. E., Penheiter, A. R., Gathman, A. C., and Lilly, W. W. Characterisation of a novel phenylalanine-specific aminopeptidase from Schizophyllum commune. Mycol. Res. 1996, 100, 462– 466