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pH effects on exopolysaccharide and oxalic acid production in cultures of Sclerotium glucanicum
Sclerotium glucanicum Yuchun Wang and Brian McNeil Department
of Bioscience
The effect of culture
Sclerotium glucanicum for biomass was 3.5.
and Biotechnology,
pH upon exopolysaccharide was examined
the available
carbon
source
3.5 during the growth phase, was significantly
(EPS),
increased
dehydrogenase)
could be diverted
Glasgow,
oxalic acid production,
in a stirred tank reactor.
Oxalate accumulation
of a synthetic enzyme (glyoxylate
University of Strathclyde,
activity of a degradativc
to oxalate formation
at pH 5.5.
was reduced
to
was 4.5;
by that
as a result of stimulation enzyme.
By operating
and at pH 4.5 during the synthetic phase (after NH:
improved and oxalate formation
and biomass formation
The optimum pH for EPS formation
linearly above pH 3.5, probably
and reduced
UK
exhaustion),
of the concentration
Up to 27% of
the process
at pH
EPS formation
achieved
at a fixed
pH of 4.5.
Keywords:
Sclerotium glucanicum; scleroglucan; oxalic acid; pH; stirred tank reactors
Introduction interest is being shown in the production and t.se of microbial biopolymers. 1.2 One polysaccharide that is 1 eld to have much promise is the exopolysaccharide (EPS) produced by the fungus Sclerotium glucanicum. This polyrneir is known as scleroglucan. Scleroglucan is a neutral glucan, composed of a linear chain of B-~(1,3)-linked r)-glucopyranosyl residues with single D-glucopyranosyl groups linked l3(1,6) to about every third residue of the main chain.3 The molecular weight of scleroglucan varies somewhat with values of 2 to 12 lo6 dalton having been reported.4 Structurally and functionally similar glucans have also been produced from other fungi, such as schizophyllan from Schizophyllum commune, and others from the genera Corticium and Sclerotinia ‘A By virtue of its structure and high molecular weight, scleroglucan has a number of uses. It has been widely studiled for use as a viscosifier in enhanced oil recovery, a role in which its closest rival in terms of technical qualities is xanthan gum.’ Because up to a third of the world’s known oil deposits are trapped in “watered-out” reservoirs, the potential market in this role alone is vast.8’9 Considerable
Address reprint requests to Dr. Brian McNeil, Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow Gl lXW, UK Received 12 January 1994; revised 12 May 1994; accepted 12 May 1994
Enzyme and Microbial Technology 17:124-130. 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Scleroglucan has also been examined as a biologic response modifier (BRM). lo Although a number of P-glucans have been shown to have a stimulatory effect on the immune system, scleroglucan exhibits significantly reater immune stimulatory activity than any of the others. ,B It has been shown that scleroglucan increases in vivo macrophage phagocytic function. Scleroglucan’s antineoplastic activity is believed to be mediated by this mechanism.” Despite this promise and continued industrial interest by a number of companies, relatively little has been published on the fundamental factors affecting polysaccharide synthesis by S. glucanicum and the problems associated with the fermentation process. Two areas of particular interest relate to culture pH, and the formation of the toxic by-product oxalic acid during the fermentation process. Culture pH can have profound effects on the synthesis of polysaccharides by filamentous fungi. 12.13Despite the likely importance of culture pH, most studies on EPS formation by Sclerotium sp. have adopted a fixed initial pH of 4.5 for their fermentation processes in stirred tank reactors. l4 Previous studies of the effect of pH have involved shake flask cultivation, and pH after initial adjustment was therefore uncontrolled. 15,16 A comparison of such results to the situation pertaining in STRs is difficult, especially because it has recently been shown that Sclerotium rolfsii responds in a markedly different fashion to changes in a single process variable dependent on whether cultivation was carried out in shake flasks or in an STR.”
0141-0229/95/$10.00 SSDI 0141-0229(94)00053-T
Scleroglucan, oxalate, and pH: Y. Wang and B. McNeil In many studies on EPS formation by Sclerotium sp. formation of oxalic acid during the fermentation process is not considered.15 This is in contrast to studies on plant pathogenic species where oxalate formation has been examined in relation to its role in pathogenicity. ‘*,19 However, such acid formation during a fermentation presents a two-fold difficulty: First, it represents a diversion of the C-source away from EPS formation, and second, there is a need to separate the oxalate from the product. Because oxalic acid formation in other filamentous fungi has also been shown to be influenced by environmental pH,*’ there is a clear need to examine EPS and oxalate formation by S. glucanicum under controlled pH conditions in an STR in a systematic fashion.
The overall aim of this study was to understand how culture pH influences the processes of EPS and oxalate synthesis, with a view to developing a strategy aimed at maximizing the former and controlling the latter activity. A range of culture pHs was therefore examined in this work, from 3.0 to 5.5 in steps of 0.5 pH units. Culture pH was automatically controlled at the set value throughout the process, except where indicated. In addition, because a previous study on another glucan synthesizing fungus, Aureobasidium pullulans, had demonstrated the worth of a bistaged pH process,*’ a similar process strategy was investigated for S. glucanicum.
Materials and methods Microorganism The organism used in this study was S. glucanicum NRRL 3006, (US Department of Agriculture, Peoria, IL). This was supplied as a freeze-dried culture and was resuscitated on Potato Dextrose Agar plates (Oxoid Ltd., Basingstoke, UK). These were incubated at 28°C for 5 days. We used 0.01 dm3 of a cell suspension prepared from such plates to inoculate 0.2 dm3 of sterile liquid medium in a 0.5 dm3 Erlenmeyer flask, which was subsequently incubated at 28°C at 250 rpm for 2 days on a rotary shaker. One flask was used to inoculate the fermenter vessel. The composition of the liquid medium was as follows (Kg ma3): sucrose, 30.0; (NH,), SO,, 1.0; KH,PO, . 7H,O. 1.0; KCI, 0.5; yeast extract (Oxoid Ltd.), 1.0; FeSO,, 0.01.
Fermentation process Fermentations were carried out in a 12 dm3 total volume stirred tank reactor. (SK Fermenter, Princes Street, Manchester, UK) with a working volume of 8 dm3. The fermenter was equipped with one six-bladed Rushton turbine impeller of 0.5 times the vessel diameter. A single tubular sparger located immediately below the impeller was used. The stirrer speed was 300 rpm, the temperature was 28°C * 0.2, and the airflow rate was 1 vol air per vol culture per min. Culture-dissolved oxygen was monitored using a galvanic oxygen electrode linked to a type 4022 0, monitor (both Uniprobe Ltd., Cardiff, UK). Culture pH was maintained at the chosen set point by the automatic addition of t&rants (either 1 M NaOH or 1 M H,SO,).
Analytic methods The biomass concentration was measured by means of dry weight estimation involving filtration of broth samples through pre-
weighed GF-C filter discs (Whatman Ltd., Maidstone, UK). The biomass was then washed, dried (15 min in a microwave over [600 W] at low power), cooled in a desiccator. and weighed. In all the fermentations in this study, after 96 h the apparent viscosity of the broth had risen to in excess of 180 CP (as measured using a Ferranti model VL concentric cylinder typ viscometer, at a shear rate of 68 s ~ ‘) and this necessitated a 1:3 dilution of the broth sample with distilled water before filtration. The mean error in the estimation of biomass by this method was +3%. Exopolysaccharide concentration was measured by precipitation of scleroglucan from cell-free broth samples by the addition of 2 vol of absolute ethanol. The precipitated scleroglucan was then filtered, washed, dried, and weighed. Sucrose concentration in the cell and EPS-free filtrate was measured using the method of Dubois et a1.22 The mean error in the estimation of sucrose by this method was +2%. Calibration curves prepared using known sucrose standards in the range of 0 to 70 mg I- ’ had correlation coefficients in excess of 0.99 (r 3 0.99). NH: ion concentration was measured using an assay kit by Sigma (no. 640). The concentration of oxalic acid and formic acid in the filtrates was estimated using kits from Boehringer Mannheim (nos. 755699 and 979732. respectively).
Results and discussion Figure 1 shows the time course of a fermentation carried out at a controlled pH of 5.5. As can be seen, the start of oxalic acid and scleroglucan formation occurred approximately 24 h after the start of the exponential growth phase. This sequential pattern with growth preceding synthesis of polysaccharide has been noted for other fungal glucans, such as pullulan.21~23,‘4 At this pH, the by-product (oxalate) concentration exceeded that of EPS between 50 and 120 h, although by the end of the fermentation the maximum concentrations of each were very similar (7.98 Kg me3 of EPS and 7.68 Kg m -3 of oxalic acid). Although oxalic acid excretion by fungi such as S. rolfsii may aid pathogenicity in the “natural” environment-for example, by sequestration of calcium ions and synergistic action with a number of enzymes25.26-formation of such large quantities in a fermentation process represents a considerable waste of C-substrate, and for some applications will incur increased product recovery costs, and should thus be minimized. The conditions under which oxalate accumulation by fungi occurs have been variously described by a number of authors, no doubt reflecting the wide variety of experimental systems used. Generally, oxalate formation is considered to occur in C-rich and N/P-deficient media.18.27-29 Such media and conditions are also those that would be chosen to stimulate EPS synthesis. The amount of oxalic acid accumulated under these conditions is high, with almost 27% of the sucrose consumed ending up as oxalate. It is noteworthy that significant oxalate formation occurs before complete exhaustion of the N-source. Oxalic acid has been described as a stationary phase product26 and also as a nongrowthassociated product or secondary metabolite.15 On the basis of the data in Figure I, oxalic acid formation appeared to be a largely growth-associated process in this organism under these conditions. In this respect, the kinetics of oxalate synthesis are very similar to those reported for formation of fungal polysaccharides.6.2’ Enzyme Microb. Technol.,
1995, vol. 17, February
125
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Biomass, scleroglucan,
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In many fermenter studies examining scleroglucan formation, culture pH is uncontrolled and often falls to relatively low levels (pH 3.0 or below). This is also the case in shake flask studies. l7 Since it is known that the buffering capacity of the medium may affect oxalate accumulation in Basidiomycetes, I9 it is likely that marked changes in pH during a batch fermentation may have a significant impact upon oxalate accumulation, and thus formation of the fermentation product, scleroglucan. To examine whether this is so, a fermentation with culture pH preadjusted to 5.5 was conducted, without subsequent pH control; the results are shown in Figure 2. The contrast between the controlled pH process and un-
controlled pH was marked. When culture pH was uncontrolled it rapidly fell as a result of utilization of NH: ions (from [NH&SO,) for growth, and also of acid production. Culture pH fell to 2.7 by 72 h, and was unchanged thereafter (data not shown). Growth was closely accompanied by oxalate accumulation in the culture, confirming the growth associated nature of this by-product under these conditions. Culture pH reached a minimum value at approximately the same time (70 h) that oxalate concentration was maximal, after which oxalate declined. Maximal oxalate concentration in the pH-uncontrolled process was very low compared with that when pH was controlled. There are a number of possible reasons for the reduced
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residual (NH,+),
Technol.,
ii5
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residual sucrose, and oxalate versus time, at an initial pH of 5.5 (pH uncontrolled
1995, vol. 17, February
Scleroglucan,
oxalate, and pH:
Y. Wang and B. McNeil
pH 6.3 was only 10% of the maximum.” If the downward trend in activity of this oxalate synthesizing enzyme continued below pH 6.0, then one might reasonably expect markedly decreased oxalate formation as culture pH declined . Thus, oxalate accumulation in pH uncontrolled fermentations is reduced relative to fermentations controlled at pH 5.5. for two probable reasons: first, diminished activity of a synthetic enzyme (probably glyoxylate dehydrogenase), and second, activation of an oxalate-degrading enzyme (possibly oxalate decarboxylase). Not controlling the culture pH seems an excellent means of minimizing diversion of the C-source into oxalate formation, but as can be seen in Figure 2, polysacchatide formation is also much reduced relative to the controlled fermentation, though biomass formation is relatively unaffected. This cannot simply be due to inhibition of polysaccharide-synthesizing enzymes by low culture pHs, because in the absence of any additional means of using the C-source there would be unused C-source remaining at the end of the process. As this is not so, one likely explanation is a significant increase in the amount of the energy (C) source required for maintenance purposes at very low external pHs. An increase in the pH difference (ApH) between the external medium and the cytoplasm would produce such an effect.3’ It is clear that culture pH significantly influences many processes within the fermentation. The influence of this upon the process was examined in a systematic fashion by carrying out a number of fermentations under pH-controlled conditions at pHs from 3.0 up to 5.5. The results of this series of fermentations are summarized in Figures 3 and 4. Figure 3 shows that the pH optima for biomass and scleroglucan formation are quite distinct; the former occurs around 3.5, whereas the latter is around 4.5. This difference in the optimal conditions for growth and polysaccharide
overall oxalate concentration and the decline in oxalate concentration after 70 h. The first possibility is reuse of excreted oxalate. This is very unlikely because oxalate is usable as a C-source by very few microbes,30 and there is still a considerable quantity of readily usable C-source (sucrose) available at that point. Another possibility is the activation of a degradative enzyme such as oxalate decarboxylase (ODC, E.C. 4.1.1.2) by the low pH achieved in this process. (The presence of an active oxalate decarboxylase is usually advanced as one reason why cultures of white-rot fungi do not accumulate oxalic acid.25 In other fungi, such as Collybia velutipes and Coriolus versicofor, the pH range of this enzyme is said to lie between 2.5 and 4.5, with an optimum around 3.0.26 Under controlled pH conditions at 5.5 this enzyme, if present, would display much reduced activity allowing accumulation of oxalate. Conversely, when pH falls rapidly to 2.7, as in the uncontrolled situation, activation might occur, leading to reduction in broth concentration of oxalate. This is consistent with the results shown in Figure 2. No free formate was detected in any samples from these fermentations, indicating that if ODC were active, formate dehydrogenase would perhaps also be active (E.C. 1.2.3.4). An alternative route to CO, via an oxidation reactionz6 cannot, however, be excluded. The nature of the degradative reaction in 5. glucanicum clearly merits further study. A third possibility for the observed difference is diminished activity of one or more enzymes involved in oxalate synthesis. This possibility cannot be excluded based on the results in this study, but reduced synthetic activity is insufficient in itself to explain the decrease in oxalate concentration after 72 h in the uncontrolled pH situation (Figure 2). However, in shake flask studies of S. rolfsii it was shown that the enzyme glyoxylate dehydrogenase, which appeared to be the major route of oxalate formation in this species, had a pH optimum of around 9.0, and activity at
PH Figure 3
The effect of pH upon the maximum
concentration
of biomass, scleroglucan,
Enzyme
Microb.
and oxalate
Technol.,
1995, vol. 17, February
127
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Figure 3 clearly illustrates a rise in maximum oxalate concentration in the broth as culture pH rises. At pH 3.5 and below no oxalate is present in the broth. The observed trend is consistent with the explanation advanced earlier, involving activation of a degradative enzyme and/or progressive inhibition of a synthetic enzyme. Figure 4 shows the effect of pH upon the optimization parameters, productivity, specific productivity, and yield of product on sucrose (Yp/c), which have important implications for process design. It can be seen that all of these have maximal values at pH 4.5; thus, from all considerations a controlled pH of 4.5 appears to be the optimum. The only drawback to operation at this pH is the pres-
formation has been noted for a range of organisms.32 These results are similar to these reported for other glucansynthesizing fungi such as Aureobasidium pullulans, for which the pH optimum for growth has been shown to be lower than that for EPS synthesis.6T21 This suggests the possibility of a fermentation process in which pH is initially suited to growth to hasten the period of biomass formation, followed by a phase at higher pH, where polysaccharide synthesis at a high rate is permitted. Such a strategy has been successfully adopted for pullulan formation on a small scale.*’ In the scleroglucan fermentation the effect of such a strategy upon oxalate would also have to be considered.
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Enzyme Microb. Technol.,
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residual sucrose, and pH versus time in bistaged pH process (3.6-96 h, 4.5 thereafter)
1995, vol. 17, February
Scleroglucan, ence of a significant amount of oxalate in the broth (3.6 Kg me3>. In addition to attempting to improve the growth phase and boost polysaccharide formation by operating a b&aged pH fermentation, such a process, operated initially at pH 3.5, might also reduce oxalate formation. The results of a process operated at pH 3.5 until the N-source was exhausted (around 96 h) and thereafter at pH 4.5 is shown in Figure 5. This operational strategy leads to both higher maximal biomass and maximal scleroglucan concentrations compared with operation at a fixed, single pH. In addition to these improvements, oxalate concentration before the pH shift (in the “growth” phase, at pH 3.5) is, as might be expected very low, at only 0.0016 Kg rnp3 immediately before the switch occurs, and rises to only 0.275 Kg me3 at 196 h (data not shown). This is approximately 10% of the maximal value for oxalate when the fermentation is carried out at pH 4.5. This indicates that conditions during the “growth phase” may be an important influence upon subsequent oxalate formation, perhaps by determining the amount or activity of GDH or degradative enzyme(s) present.
1
2
4
6
7 8
10
11 12
13
18
polysaccharide Y,,., =
19
produced (kg m - ‘)
sucrose consumed
Nardin. P. and Vincendon. M. Isotopic exchange study of the scleroglucan chain in solution. Macromolecules 1989, 22, 3551-3554 Lecacheux. D.. Mustiere. Y. and Panaras. R. Molecular weight of scleroglucan and other extracellular microbial polysaccharides by size exclusion chromatography and low angle laser light scattering.
(kg mV3)
(Pr):
Polym.
1984. 76, 947-950
Takao, S. Organic acid production by Basidiomycetes. Appl. Microbiol. 1965. 13, 732-737 Lacroix, C.. LeDuy, A.. Noel, G. and Choplin, L. Effect of pH on the batch fermentation of pullulan from sucrose medium. Biotechno/. Bioeng.
max, polysaccharide Pr =
concentration
fermentation
time
(kg mp3)
22
(h) 23
Specific productivity
(Pr/x): 24
Pr Prlx =
max. biomass
concentration
(kg m-3)
1986. 6, 411-493
Rau. U.. Miller. R.-J., Cordes, K. and Kiein, J. Process and molecular data of branched 1.3 p D glucans in comparison with xanthan. Bioproress. Eng. 1990. 5, 89-93 Harvey, L. M. Production of microbial polysaccharides by continuous culture of fungi. PhD thesis, University of Strathclyde. UK, 1984 Holzwarth, G. Xanthan and scleroglucan: structure and use in enhanced oil recovery. Dev. Intf. Microbial. 1985, 26, 271-280 Kulicke, W. M.. Bose. N. and Bouldin. M. The role of polymers in enhanced oil recovery In: Water Soluble Polymers for Petroleum Recovery (Stahl, G. A. and Schulz, D. A.. eds.1. Plenum Press. London, 1988 Sutherland. I. W. and Kierulf. C. Downhole use of biopolymers. In: Microbial Problems in the Offshore Oil Industry (Hill, E. C., Schennan. J. L. and Watkinson, R. J.. eds 1. John Wiley, and Sons. Chichester. 1982, 93 Pretus. H. A., Ensley, H. E.. McNamee. R. B., Jones, E. L., Browder. I. W. and Williams. D. L. Isolation, physicochemical characterisation and preclinical efficacy evaluation of soluble scleroglucan. J. Pharmacol E.rp. Ther. 1991. 257, 5OG510 Jong, S. C. and Donovick, R. Antitumour and antiviral substances from fungi. Adv. Appl. Microbial. 1987, 34, 188-262. McNeil, B.. Kristiansen. B. and Seviour. R. J. E’olysaccharide production and morphology of Aureobasidium pulluluns in continuous culture. Biorechnol. Bioeng. 1989. 33, 1210-1212 Kato. K. and Shiosaka, M. Pullulan. In: Chemicals by Enzymatic and Microbial Processes: Recent Advances (Duffy. 1. I.. ed.1. Noyes. New Jersey, 1980. 220-230 Halleck. F. E. Polysaccharides and methods for production thereof. 1967. US Patent no. 3,301.848 Wernau. W. C. Fermentation methods for the production of polysaccharides. Dev. Ind. Microbial. 1985, 26. 263-269 Maxwell, D. P. and Bateman, D. F. Oxalic acid biosynthesis by 5. rolfsii. Phytopathology 1968. 58, 163&1634 Pilz, F.. Auling. Cl., Stephan. D.. Rau. V. and Wagner, F. A high affinity Zn’ + uptake system controls growth and biosynthesis of an extracellular. ranched B-I .3-p 1.6-glucan in Sclerotium rolfsii ATCC 15205. Exp. Mycol. 1991. 15, 181-192 Pierson. P. E. and Rhodes. L. H. Effect of culture medium on the production of oxalic acid by Sclerotinia rr[fohorum. Mycologia 1992. 84. 467469 Punja. Z. K. and Jenkins. S. F. Influence of medium composition on mycelial growth and oxalic acid production in S. roffsii. Mycologia
20 21
Productivity
Sinskey A., Jamas, S., Easson, D. Jr. and Rha, C. K. Biopolymers and modified polysaccharides. In: Biotechnology in Food Processing CHarlander, S. K. and Labuza, T. P., eds.). Noyes, New Jersey. 1986.73-114 Paul, F., Morin. A. and Monsan, P. Microbial polysaccharides with actual potential industrial applications. Eiofechnol. Adv. 1986, 4.
Carhohydr.
5
Nomenclature on sucrose (Yr,J:
Y. Wang and B. McNeil
245-259
3
Conclusions
Yield factor for scleroglucan
and pH:
References
9
Culture pH critically influences many aspects of the scleroglucan fermentation process-in particular, formation of the product scleroglucan and the unwanted by-product, oxalate-but at a low pH increased diversion of the energy source to maintenance may occur. The optimum controlled pH for biomass formation is around 3.5, whereas that for EPS is around 4.5. Lowering the culture pH leads to decreased oxalate formation but also to decreased product levels. Reduced oxalate concentrations at low pH may be the consequence of reduced GDH activity, an&or reduced activity of degradative enzyme such as ODC. By attempting to understand the mechanisms by which the observed effects may be mediated, an operational strategy that maximizes product formation and minimizes byproduct accumulation can be designed. This “bistaged pH” process offers considerable advantages, combining high EPS formation with very low oxalate synthesis.
oxalate,
1985. 27, 202-207
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. Calorimetric methods for the determination of sugars and related substances. Analyt. Chem. 1956, 28, 350-356 Rau, U., Gura. E., Olszewski, E. and Wagner. F. Enhanced glucan formation of filamentous fungi by effective mixing, oxygen limitation and fed-batch processing. J. Ind. Microbial 1992, 9, I%25 Chul Shin, Y.. Ho Kim, Y., Soo Lee. H., Nam Kim, Y. and Myung Byun. S. Production of pullulan by a fed-batch fermentation. Biotechnol.
Lett 1987, 9. 621-624
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Dutton, M. V., Evans, C. S., Atkey, P. T. and Wood, D. A. Oxalate production by Basidiomycetes, including the white-rot species Coriolus versicolor and Phonerochaete chrysoporium. Appl. Microbiol. Biotechnol. 1993, 39, 5-10 Kuan, I.-C. and Tien, M. Stimulation of Mn peroxidase activity: a possible role for oxalate in lignin biodegradation. Proc. Nutl. Acad. Sci. USA 1993, 90, 1242-1246 Punja, Z. K., Huang, J. S., Jenkins, S. F. Relationship of mycelial growth and production of oxalic acid and cell wall degrading enzymes to virulence in Sclerotium rolfsii. Can. J. Plant Pathol. 1985, 7, 109-117
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Maxwell, D. P. and Bateman, D. F. Oxalic acid biosynthesis by Sclerotium rolfsii. Phytoputhology 1968, 58, 1635-1639 Espejo, E. and Agosin, E. Production and degradation of oxalic acid by brown rot fungi. Appl. Environ. Microbial. 1991,57, 1980-1986 Quayle, J. R. Carbon assimilation by Pseudomonas oxaloticus (0X1) reactions of oxalyl coenzyme A. Biochem. J. 1%3, 87,368-373 Sinclair, C. G. and Kristiansen, B. Fermentation Kinetics and Modelling. (Bu’Lock, J. D., ed.). Open University Press, Milton Keynes, 1987, 28-30 Sutherland, I. W. Biosynthesis of microbial polysaccharides. Adv. Microb. Physiol. 1982, 23, 79-145
of Bioscience
The effect of culture
Sclerotium glucanicum for biomass was 3.5.
and Biotechnology,
pH upon exopolysaccharide was examined
the available
carbon
source
3.5 during the growth phase, was significantly
(EPS),
increased
dehydrogenase)
could be diverted
Glasgow,
oxalic acid production,
in a stirred tank reactor.
Oxalate accumulation
of a synthetic enzyme (glyoxylate
University of Strathclyde,
activity of a degradativc
to oxalate formation
at pH 5.5.
was reduced
to
was 4.5;
by that
as a result of stimulation enzyme.
By operating
and at pH 4.5 during the synthetic phase (after NH:
improved and oxalate formation
and biomass formation
The optimum pH for EPS formation
linearly above pH 3.5, probably
and reduced
UK
exhaustion),
of the concentration
Up to 27% of
the process
at pH
EPS formation
achieved
at a fixed
pH of 4.5.
Keywords:
Sclerotium glucanicum; scleroglucan; oxalic acid; pH; stirred tank reactors
Introduction interest is being shown in the production and t.se of microbial biopolymers. 1.2 One polysaccharide that is 1 eld to have much promise is the exopolysaccharide (EPS) produced by the fungus Sclerotium glucanicum. This polyrneir is known as scleroglucan. Scleroglucan is a neutral glucan, composed of a linear chain of B-~(1,3)-linked r)-glucopyranosyl residues with single D-glucopyranosyl groups linked l3(1,6) to about every third residue of the main chain.3 The molecular weight of scleroglucan varies somewhat with values of 2 to 12 lo6 dalton having been reported.4 Structurally and functionally similar glucans have also been produced from other fungi, such as schizophyllan from Schizophyllum commune, and others from the genera Corticium and Sclerotinia ‘A By virtue of its structure and high molecular weight, scleroglucan has a number of uses. It has been widely studiled for use as a viscosifier in enhanced oil recovery, a role in which its closest rival in terms of technical qualities is xanthan gum.’ Because up to a third of the world’s known oil deposits are trapped in “watered-out” reservoirs, the potential market in this role alone is vast.8’9 Considerable
Address reprint requests to Dr. Brian McNeil, Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow Gl lXW, UK Received 12 January 1994; revised 12 May 1994; accepted 12 May 1994
Enzyme and Microbial Technology 17:124-130. 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Scleroglucan has also been examined as a biologic response modifier (BRM). lo Although a number of P-glucans have been shown to have a stimulatory effect on the immune system, scleroglucan exhibits significantly reater immune stimulatory activity than any of the others. ,B It has been shown that scleroglucan increases in vivo macrophage phagocytic function. Scleroglucan’s antineoplastic activity is believed to be mediated by this mechanism.” Despite this promise and continued industrial interest by a number of companies, relatively little has been published on the fundamental factors affecting polysaccharide synthesis by S. glucanicum and the problems associated with the fermentation process. Two areas of particular interest relate to culture pH, and the formation of the toxic by-product oxalic acid during the fermentation process. Culture pH can have profound effects on the synthesis of polysaccharides by filamentous fungi. 12.13Despite the likely importance of culture pH, most studies on EPS formation by Sclerotium sp. have adopted a fixed initial pH of 4.5 for their fermentation processes in stirred tank reactors. l4 Previous studies of the effect of pH have involved shake flask cultivation, and pH after initial adjustment was therefore uncontrolled. 15,16 A comparison of such results to the situation pertaining in STRs is difficult, especially because it has recently been shown that Sclerotium rolfsii responds in a markedly different fashion to changes in a single process variable dependent on whether cultivation was carried out in shake flasks or in an STR.”
0141-0229/95/$10.00 SSDI 0141-0229(94)00053-T
Scleroglucan, oxalate, and pH: Y. Wang and B. McNeil In many studies on EPS formation by Sclerotium sp. formation of oxalic acid during the fermentation process is not considered.15 This is in contrast to studies on plant pathogenic species where oxalate formation has been examined in relation to its role in pathogenicity. ‘*,19 However, such acid formation during a fermentation presents a two-fold difficulty: First, it represents a diversion of the C-source away from EPS formation, and second, there is a need to separate the oxalate from the product. Because oxalic acid formation in other filamentous fungi has also been shown to be influenced by environmental pH,*’ there is a clear need to examine EPS and oxalate formation by S. glucanicum under controlled pH conditions in an STR in a systematic fashion.
The overall aim of this study was to understand how culture pH influences the processes of EPS and oxalate synthesis, with a view to developing a strategy aimed at maximizing the former and controlling the latter activity. A range of culture pHs was therefore examined in this work, from 3.0 to 5.5 in steps of 0.5 pH units. Culture pH was automatically controlled at the set value throughout the process, except where indicated. In addition, because a previous study on another glucan synthesizing fungus, Aureobasidium pullulans, had demonstrated the worth of a bistaged pH process,*’ a similar process strategy was investigated for S. glucanicum.
Materials and methods Microorganism The organism used in this study was S. glucanicum NRRL 3006, (US Department of Agriculture, Peoria, IL). This was supplied as a freeze-dried culture and was resuscitated on Potato Dextrose Agar plates (Oxoid Ltd., Basingstoke, UK). These were incubated at 28°C for 5 days. We used 0.01 dm3 of a cell suspension prepared from such plates to inoculate 0.2 dm3 of sterile liquid medium in a 0.5 dm3 Erlenmeyer flask, which was subsequently incubated at 28°C at 250 rpm for 2 days on a rotary shaker. One flask was used to inoculate the fermenter vessel. The composition of the liquid medium was as follows (Kg ma3): sucrose, 30.0; (NH,), SO,, 1.0; KH,PO, . 7H,O. 1.0; KCI, 0.5; yeast extract (Oxoid Ltd.), 1.0; FeSO,, 0.01.
Fermentation process Fermentations were carried out in a 12 dm3 total volume stirred tank reactor. (SK Fermenter, Princes Street, Manchester, UK) with a working volume of 8 dm3. The fermenter was equipped with one six-bladed Rushton turbine impeller of 0.5 times the vessel diameter. A single tubular sparger located immediately below the impeller was used. The stirrer speed was 300 rpm, the temperature was 28°C * 0.2, and the airflow rate was 1 vol air per vol culture per min. Culture-dissolved oxygen was monitored using a galvanic oxygen electrode linked to a type 4022 0, monitor (both Uniprobe Ltd., Cardiff, UK). Culture pH was maintained at the chosen set point by the automatic addition of t&rants (either 1 M NaOH or 1 M H,SO,).
Analytic methods The biomass concentration was measured by means of dry weight estimation involving filtration of broth samples through pre-
weighed GF-C filter discs (Whatman Ltd., Maidstone, UK). The biomass was then washed, dried (15 min in a microwave over [600 W] at low power), cooled in a desiccator. and weighed. In all the fermentations in this study, after 96 h the apparent viscosity of the broth had risen to in excess of 180 CP (as measured using a Ferranti model VL concentric cylinder typ viscometer, at a shear rate of 68 s ~ ‘) and this necessitated a 1:3 dilution of the broth sample with distilled water before filtration. The mean error in the estimation of biomass by this method was +3%. Exopolysaccharide concentration was measured by precipitation of scleroglucan from cell-free broth samples by the addition of 2 vol of absolute ethanol. The precipitated scleroglucan was then filtered, washed, dried, and weighed. Sucrose concentration in the cell and EPS-free filtrate was measured using the method of Dubois et a1.22 The mean error in the estimation of sucrose by this method was +2%. Calibration curves prepared using known sucrose standards in the range of 0 to 70 mg I- ’ had correlation coefficients in excess of 0.99 (r 3 0.99). NH: ion concentration was measured using an assay kit by Sigma (no. 640). The concentration of oxalic acid and formic acid in the filtrates was estimated using kits from Boehringer Mannheim (nos. 755699 and 979732. respectively).
Results and discussion Figure 1 shows the time course of a fermentation carried out at a controlled pH of 5.5. As can be seen, the start of oxalic acid and scleroglucan formation occurred approximately 24 h after the start of the exponential growth phase. This sequential pattern with growth preceding synthesis of polysaccharide has been noted for other fungal glucans, such as pullulan.21~23,‘4 At this pH, the by-product (oxalate) concentration exceeded that of EPS between 50 and 120 h, although by the end of the fermentation the maximum concentrations of each were very similar (7.98 Kg me3 of EPS and 7.68 Kg m -3 of oxalic acid). Although oxalic acid excretion by fungi such as S. rolfsii may aid pathogenicity in the “natural” environment-for example, by sequestration of calcium ions and synergistic action with a number of enzymes25.26-formation of such large quantities in a fermentation process represents a considerable waste of C-substrate, and for some applications will incur increased product recovery costs, and should thus be minimized. The conditions under which oxalate accumulation by fungi occurs have been variously described by a number of authors, no doubt reflecting the wide variety of experimental systems used. Generally, oxalate formation is considered to occur in C-rich and N/P-deficient media.18.27-29 Such media and conditions are also those that would be chosen to stimulate EPS synthesis. The amount of oxalic acid accumulated under these conditions is high, with almost 27% of the sucrose consumed ending up as oxalate. It is noteworthy that significant oxalate formation occurs before complete exhaustion of the N-source. Oxalic acid has been described as a stationary phase product26 and also as a nongrowthassociated product or secondary metabolite.15 On the basis of the data in Figure I, oxalic acid formation appeared to be a largely growth-associated process in this organism under these conditions. In this respect, the kinetics of oxalate synthesis are very similar to those reported for formation of fungal polysaccharides.6.2’ Enzyme Microb. Technol.,
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In many fermenter studies examining scleroglucan formation, culture pH is uncontrolled and often falls to relatively low levels (pH 3.0 or below). This is also the case in shake flask studies. l7 Since it is known that the buffering capacity of the medium may affect oxalate accumulation in Basidiomycetes, I9 it is likely that marked changes in pH during a batch fermentation may have a significant impact upon oxalate accumulation, and thus formation of the fermentation product, scleroglucan. To examine whether this is so, a fermentation with culture pH preadjusted to 5.5 was conducted, without subsequent pH control; the results are shown in Figure 2. The contrast between the controlled pH process and un-
controlled pH was marked. When culture pH was uncontrolled it rapidly fell as a result of utilization of NH: ions (from [NH&SO,) for growth, and also of acid production. Culture pH fell to 2.7 by 72 h, and was unchanged thereafter (data not shown). Growth was closely accompanied by oxalate accumulation in the culture, confirming the growth associated nature of this by-product under these conditions. Culture pH reached a minimum value at approximately the same time (70 h) that oxalate concentration was maximal, after which oxalate declined. Maximal oxalate concentration in the pH-uncontrolled process was very low compared with that when pH was controlled. There are a number of possible reasons for the reduced
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Scleroglucan,
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Y. Wang and B. McNeil
pH 6.3 was only 10% of the maximum.” If the downward trend in activity of this oxalate synthesizing enzyme continued below pH 6.0, then one might reasonably expect markedly decreased oxalate formation as culture pH declined . Thus, oxalate accumulation in pH uncontrolled fermentations is reduced relative to fermentations controlled at pH 5.5. for two probable reasons: first, diminished activity of a synthetic enzyme (probably glyoxylate dehydrogenase), and second, activation of an oxalate-degrading enzyme (possibly oxalate decarboxylase). Not controlling the culture pH seems an excellent means of minimizing diversion of the C-source into oxalate formation, but as can be seen in Figure 2, polysacchatide formation is also much reduced relative to the controlled fermentation, though biomass formation is relatively unaffected. This cannot simply be due to inhibition of polysaccharide-synthesizing enzymes by low culture pHs, because in the absence of any additional means of using the C-source there would be unused C-source remaining at the end of the process. As this is not so, one likely explanation is a significant increase in the amount of the energy (C) source required for maintenance purposes at very low external pHs. An increase in the pH difference (ApH) between the external medium and the cytoplasm would produce such an effect.3’ It is clear that culture pH significantly influences many processes within the fermentation. The influence of this upon the process was examined in a systematic fashion by carrying out a number of fermentations under pH-controlled conditions at pHs from 3.0 up to 5.5. The results of this series of fermentations are summarized in Figures 3 and 4. Figure 3 shows that the pH optima for biomass and scleroglucan formation are quite distinct; the former occurs around 3.5, whereas the latter is around 4.5. This difference in the optimal conditions for growth and polysaccharide
overall oxalate concentration and the decline in oxalate concentration after 70 h. The first possibility is reuse of excreted oxalate. This is very unlikely because oxalate is usable as a C-source by very few microbes,30 and there is still a considerable quantity of readily usable C-source (sucrose) available at that point. Another possibility is the activation of a degradative enzyme such as oxalate decarboxylase (ODC, E.C. 4.1.1.2) by the low pH achieved in this process. (The presence of an active oxalate decarboxylase is usually advanced as one reason why cultures of white-rot fungi do not accumulate oxalic acid.25 In other fungi, such as Collybia velutipes and Coriolus versicofor, the pH range of this enzyme is said to lie between 2.5 and 4.5, with an optimum around 3.0.26 Under controlled pH conditions at 5.5 this enzyme, if present, would display much reduced activity allowing accumulation of oxalate. Conversely, when pH falls rapidly to 2.7, as in the uncontrolled situation, activation might occur, leading to reduction in broth concentration of oxalate. This is consistent with the results shown in Figure 2. No free formate was detected in any samples from these fermentations, indicating that if ODC were active, formate dehydrogenase would perhaps also be active (E.C. 1.2.3.4). An alternative route to CO, via an oxidation reactionz6 cannot, however, be excluded. The nature of the degradative reaction in 5. glucanicum clearly merits further study. A third possibility for the observed difference is diminished activity of one or more enzymes involved in oxalate synthesis. This possibility cannot be excluded based on the results in this study, but reduced synthetic activity is insufficient in itself to explain the decrease in oxalate concentration after 72 h in the uncontrolled pH situation (Figure 2). However, in shake flask studies of S. rolfsii it was shown that the enzyme glyoxylate dehydrogenase, which appeared to be the major route of oxalate formation in this species, had a pH optimum of around 9.0, and activity at
PH Figure 3
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1995, vol. 17, February
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Figure 3 clearly illustrates a rise in maximum oxalate concentration in the broth as culture pH rises. At pH 3.5 and below no oxalate is present in the broth. The observed trend is consistent with the explanation advanced earlier, involving activation of a degradative enzyme and/or progressive inhibition of a synthetic enzyme. Figure 4 shows the effect of pH upon the optimization parameters, productivity, specific productivity, and yield of product on sucrose (Yp/c), which have important implications for process design. It can be seen that all of these have maximal values at pH 4.5; thus, from all considerations a controlled pH of 4.5 appears to be the optimum. The only drawback to operation at this pH is the pres-
formation has been noted for a range of organisms.32 These results are similar to these reported for other glucansynthesizing fungi such as Aureobasidium pullulans, for which the pH optimum for growth has been shown to be lower than that for EPS synthesis.6T21 This suggests the possibility of a fermentation process in which pH is initially suited to growth to hasten the period of biomass formation, followed by a phase at higher pH, where polysaccharide synthesis at a high rate is permitted. Such a strategy has been successfully adopted for pullulan formation on a small scale.*’ In the scleroglucan fermentation the effect of such a strategy upon oxalate would also have to be considered.
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Enzyme Microb. Technol.,
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residual sucrose, and pH versus time in bistaged pH process (3.6-96 h, 4.5 thereafter)
1995, vol. 17, February
Scleroglucan, ence of a significant amount of oxalate in the broth (3.6 Kg me3>. In addition to attempting to improve the growth phase and boost polysaccharide formation by operating a b&aged pH fermentation, such a process, operated initially at pH 3.5, might also reduce oxalate formation. The results of a process operated at pH 3.5 until the N-source was exhausted (around 96 h) and thereafter at pH 4.5 is shown in Figure 5. This operational strategy leads to both higher maximal biomass and maximal scleroglucan concentrations compared with operation at a fixed, single pH. In addition to these improvements, oxalate concentration before the pH shift (in the “growth” phase, at pH 3.5) is, as might be expected very low, at only 0.0016 Kg rnp3 immediately before the switch occurs, and rises to only 0.275 Kg me3 at 196 h (data not shown). This is approximately 10% of the maximal value for oxalate when the fermentation is carried out at pH 4.5. This indicates that conditions during the “growth phase” may be an important influence upon subsequent oxalate formation, perhaps by determining the amount or activity of GDH or degradative enzyme(s) present.
1
2
4
6
7 8
10
11 12
13
18
polysaccharide Y,,., =
19
produced (kg m - ‘)
sucrose consumed
Nardin. P. and Vincendon. M. Isotopic exchange study of the scleroglucan chain in solution. Macromolecules 1989, 22, 3551-3554 Lecacheux. D.. Mustiere. Y. and Panaras. R. Molecular weight of scleroglucan and other extracellular microbial polysaccharides by size exclusion chromatography and low angle laser light scattering.
(kg mV3)
(Pr):
Polym.
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Takao, S. Organic acid production by Basidiomycetes. Appl. Microbiol. 1965. 13, 732-737 Lacroix, C.. LeDuy, A.. Noel, G. and Choplin, L. Effect of pH on the batch fermentation of pullulan from sucrose medium. Biotechno/. Bioeng.
max, polysaccharide Pr =
concentration
fermentation
time
(kg mp3)
22
(h) 23
Specific productivity
(Pr/x): 24
Pr Prlx =
max. biomass
concentration
(kg m-3)
1986. 6, 411-493
Rau. U.. Miller. R.-J., Cordes, K. and Kiein, J. Process and molecular data of branched 1.3 p D glucans in comparison with xanthan. Bioproress. Eng. 1990. 5, 89-93 Harvey, L. M. Production of microbial polysaccharides by continuous culture of fungi. PhD thesis, University of Strathclyde. UK, 1984 Holzwarth, G. Xanthan and scleroglucan: structure and use in enhanced oil recovery. Dev. Intf. Microbial. 1985, 26, 271-280 Kulicke, W. M.. Bose. N. and Bouldin. M. The role of polymers in enhanced oil recovery In: Water Soluble Polymers for Petroleum Recovery (Stahl, G. A. and Schulz, D. A.. eds.1. Plenum Press. London, 1988 Sutherland. I. W. and Kierulf. C. Downhole use of biopolymers. In: Microbial Problems in the Offshore Oil Industry (Hill, E. C., Schennan. J. L. and Watkinson, R. J.. eds 1. John Wiley, and Sons. Chichester. 1982, 93 Pretus. H. A., Ensley, H. E.. McNamee. R. B., Jones, E. L., Browder. I. W. and Williams. D. L. Isolation, physicochemical characterisation and preclinical efficacy evaluation of soluble scleroglucan. J. Pharmacol E.rp. Ther. 1991. 257, 5OG510 Jong, S. C. and Donovick, R. Antitumour and antiviral substances from fungi. Adv. Appl. Microbial. 1987, 34, 188-262. McNeil, B.. Kristiansen. B. and Seviour. R. J. E’olysaccharide production and morphology of Aureobasidium pulluluns in continuous culture. Biorechnol. Bioeng. 1989. 33, 1210-1212 Kato. K. and Shiosaka, M. Pullulan. In: Chemicals by Enzymatic and Microbial Processes: Recent Advances (Duffy. 1. I.. ed.1. Noyes. New Jersey, 1980. 220-230 Halleck. F. E. Polysaccharides and methods for production thereof. 1967. US Patent no. 3,301.848 Wernau. W. C. Fermentation methods for the production of polysaccharides. Dev. Ind. Microbial. 1985, 26. 263-269 Maxwell, D. P. and Bateman, D. F. Oxalic acid biosynthesis by 5. rolfsii. Phytopathology 1968. 58, 163&1634 Pilz, F.. Auling. Cl., Stephan. D.. Rau. V. and Wagner, F. A high affinity Zn’ + uptake system controls growth and biosynthesis of an extracellular. ranched B-I .3-p 1.6-glucan in Sclerotium rolfsii ATCC 15205. Exp. Mycol. 1991. 15, 181-192 Pierson. P. E. and Rhodes. L. H. Effect of culture medium on the production of oxalic acid by Sclerotinia rr[fohorum. Mycologia 1992. 84. 467469 Punja. Z. K. and Jenkins. S. F. Influence of medium composition on mycelial growth and oxalic acid production in S. roffsii. Mycologia
20 21
Productivity
Sinskey A., Jamas, S., Easson, D. Jr. and Rha, C. K. Biopolymers and modified polysaccharides. In: Biotechnology in Food Processing CHarlander, S. K. and Labuza, T. P., eds.). Noyes, New Jersey. 1986.73-114 Paul, F., Morin. A. and Monsan, P. Microbial polysaccharides with actual potential industrial applications. Eiofechnol. Adv. 1986, 4.
Carhohydr.
5
Nomenclature on sucrose (Yr,J:
Y. Wang and B. McNeil
245-259
3
Conclusions
Yield factor for scleroglucan
and pH:
References
9
Culture pH critically influences many aspects of the scleroglucan fermentation process-in particular, formation of the product scleroglucan and the unwanted by-product, oxalate-but at a low pH increased diversion of the energy source to maintenance may occur. The optimum controlled pH for biomass formation is around 3.5, whereas that for EPS is around 4.5. Lowering the culture pH leads to decreased oxalate formation but also to decreased product levels. Reduced oxalate concentrations at low pH may be the consequence of reduced GDH activity, an&or reduced activity of degradative enzyme such as ODC. By attempting to understand the mechanisms by which the observed effects may be mediated, an operational strategy that maximizes product formation and minimizes byproduct accumulation can be designed. This “bistaged pH” process offers considerable advantages, combining high EPS formation with very low oxalate synthesis.
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Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. Calorimetric methods for the determination of sugars and related substances. Analyt. Chem. 1956, 28, 350-356 Rau, U., Gura. E., Olszewski, E. and Wagner. F. Enhanced glucan formation of filamentous fungi by effective mixing, oxygen limitation and fed-batch processing. J. Ind. Microbial 1992, 9, I%25 Chul Shin, Y.. Ho Kim, Y., Soo Lee. H., Nam Kim, Y. and Myung Byun. S. Production of pullulan by a fed-batch fermentation. Biotechnol.
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Maxwell, D. P. and Bateman, D. F. Oxalic acid biosynthesis by Sclerotium rolfsii. Phytoputhology 1968, 58, 1635-1639 Espejo, E. and Agosin, E. Production and degradation of oxalic acid by brown rot fungi. Appl. Environ. Microbial. 1991,57, 1980-1986 Quayle, J. R. Carbon assimilation by Pseudomonas oxaloticus (0X1) reactions of oxalyl coenzyme A. Biochem. J. 1%3, 87,368-373 Sinclair, C. G. and Kristiansen, B. Fermentation Kinetics and Modelling. (Bu’Lock, J. D., ed.). Open University Press, Milton Keynes, 1987, 28-30 Sutherland, I. W. Biosynthesis of microbial polysaccharides. Adv. Microb. Physiol. 1982, 23, 79-145