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Utilization of Carbon Sources by Pullularia pullulans for the Elaboration of Extracellular Polysaccharides

The yeastlike fungus Pullularia pullulans utilizes simple mono- and disaccharides both in the production of cell mass and the elaboration of extracellular polysaccharide. The utilization pattern of these sugars and the effect obtained by varying the pH of the medium are studied, and the ability of the organism to utilize and elaborate extracellular polysaccharides from noncarbohydrate sources is explored.

The yeastlike fungus Pullularia pullulans utilizes simple mono-and disaccharides both in the production of cell mass and the elaboration of extracellular polysaccharide. The utilization pattern of these sugars and the effect obtained by varying the pH of the medium are studied, and the ability of the organism to utilize and elaborate extracellular polysaccharides from noncarbohydrate sources is explored.
The production of extracellular polysaccharides by the yeastlike fungus Pullularia pullulans from a variety of glycosidic carbon sources has been widely reported (6-9, 32, 34), and in some instances (6,9,34) the elaboration of pullulan has been specifically described. Pullulan is an extracellular neutral glucan, the structure of which has been determined by Bender and Wallenfels (6) and by Wallenfels et al. (34) as consisting of a-maltotriose units polymerized in linear fashion through 1 -) 6 linkages on the terminal glucose residues of the trisaccharide. Minor tetrasaccharide components occupying both internal and external positions have been reported (13,14,34), and in one case evidence for a small proportion of 1 -) 3 linkages has been established (28).
This report describes the utilization by P. pullulans of sucrose, maltose, glucose, fructose, and the nonglycosidic substrates, glycerol and acetate, for cell growth and production of pullulan. The effect of extracellular pH on polysaccharide synthesis is discussed, and the circumstances leading to the production of pullulan are considered.
MATERIAIS AND METHODS Quartermaster strain no. 3092 of P. pullulans was kindly supplied by Elwyn T. Reese of the U.S. Army Natick Laboratories, Natick, Mass. The medium used is that of Ueda et al. (32), i.e., 0.5% K2HP04, 0.1 % NaCl, 0.02% MgSO4-7H20, 0.06% (NH4)2SO4, and 0.04% yeast extract (Difco), together with the appropriate carbon source. Where the starting pH of the medium was required to be other than normal, i.e., pH 7.5, an equivalent amount of phosphoric acid replaced the potassium phosphate and was titrated to the desired pH before sterilization. Cultures were maintained at 4 C on an agar slope containing the above nutrients together with 2.5% sucrose. Suspensions of cells from these slopes were transferred to a medium containing 2.5% glucose as carbon source and grown for 3 to 4 days at room temperature (25 to 27 C). Transfers of 0.5 ml of this culture into 50 ml of starter culture containing the appropriate carbon source were then made, and these were grown for 3 to 4 days. These cultures (0.5 ml) were now used as innocula for 50-ml cultures to be subjected to analysis. In the appropriate experiments (for details see figures), these cultures were supplemented with approximately 2 ACi of 14C of the carbon source under consideration, i.e., uniformly labeled sucrose, glucose, fructose, glycerol, or acetate obtained from New England Nuclear Corp. or maltose obtained from Calatomic. Cultures were grown on a gyratory shaker at 200 rev/min in flasks fitted with septa-covered side arms, and samples were withdrawn by needle and syringe.
Cell weights were obtained by centrifugation of culture samples at 17,000 X g for 10 min, followed by three washes with distilled water and collection of the cells on 0.8-,um membrane filters (Millipore Corp., Bedford, Mass,) which were then dried for 18 hr at 50 C over phosphorus pentoxide in vacuo. Counting this filter paper by standard procedures of liquid scintillation spectrometry provided a measure of 14C incorporation into the cells. The scintillation medium for all 14C assays was 0.5% 2,5-diphenyloxazole and 0.01% 1,4-bis-2-(5,phenyloxazolyl)-benzene in toluene.
Elaboration of polysaccharide during fungal growth was monitored by two methods. A relatively unspecific technique utilized the method of Thomas et al. (30). A 100-,uliter sample of culture supernatant fluid was spotted on a 1-cmi piece of filter paper (Whatman E. T.), extracted with 66% ethanol, and dried. The paper was then counted by liquid scintillation spectrometry and provided a measure of labeled macromolecule in the supematant fluid. For a more thorough analysis of extracellular polysaccharide and to demonstrate specifically the presence of pullulan, the culture supematant fluid was made 66% with respect to ethanol, and the precipitated material was washed three times with 66% ethanol. The washed precipitate was dissolved in water at 100 C, and samples of the solution were assayed as follows. The total carbohydrate was determined either by the phenol-641 sulfuric acid procedure (18) with glucose as standard or by acid hydrolysis (25) and estimation of the liberated sugars by Nelson's method (23) or estimation of glucose by the glucose oxidase reagent (21). Pullulanolysis was carried out at pH 5.0 and 37 C as by Abdullah et al. (1) until no further increase in reducing power (23) was observed. The products of pullulanolysis were fractionated by descending paper chromatography (Whatman no. 1), irrigating with ethyl acetate-pyridine-water (10:4:3, v/v), and the sugar zones were located by the silver nitrate-sodium hydroxide dip (31). For the assay of 14C-labeled products, chromatography in the same system was performed by using Whatman 3MM paper. Material was applied to a 10-cm base line, and the developed chromatogram was cut into strips (1 by 10 cm) which were immersed in scintillation fluid and counted by standard procedures of scintillation spectrometry. Fructose was measured by the method of Dische (17). Sucrose was determined by the incubation of samples with yeast invertase at pH 5.0 and 37 C with measurement of released glucose by the glucose oxidase reagent (21). Maltose was determined by the incubation of samples with amyloglucosidase from Aspergillus niger (2) in 60 mm citrate phosphate buffer (pH 5.0) at 37 C with measurement of released glucose by the glucose oxidase method (21). The amyloglucosidase was adjusted to a final concenitration of 0.02 international units per ml based upon the hydrolysis of maltose. These conditions were sufficient to cleave maltose but were shown to have a negligible effect on an equal amount of pullulan. Uronic acids were measured by the carbazole method of Dische (16) and phosphate, by the method of Sumner (29).
Alkali-soluble material was extracted from washed lyophilized cells with 30%0 potassium hydroxide at 100 C for 30 min. The digest was diluted with 2 volumes water and centrifuged; 6 volumes of ethanol were added to the supernatant fluid to precipitate extracted material. For alcohol extraction, three extractions of washed cells were made with 95% ethanol for 20 min at 60 C. The combined extracts were evaporated to dryness at 40 C and dissolved in water.

RESULTS AND DISCUSSION
An examination of the literature describing pullulan (6,9,28,32,34) suggests that different strains of P. pullulans do not elaborate polysaccharides of a common structure. The investigations reported here define pullulan as a glucan insoluble in 33% aqueous ethanol, which is depolymerized by the a-1---6-glucosidic bond endohydrolase, pullulanase (1), to yield a single major product of maltotriose.
Carbon source utilization The growth of P. pullulans, production of extracellular polymeric material, culture pH, and utilization of carbon substrate of a shake culture grown on a medium containing 2.5 % glucose as sole carbon source are described in Fig. la. After about 100 hr, the culture gradually darkens and eventually becomes black through the elaboration of melanin pig-ments (19). This pigmentation also appeared in cultures grown on sucrose, maltose, and fructose, but was absent in those grown on glycerol or acetate.
Polymeric material isolated at 100 hr of growth from the supernatant fluid of glucosegrown cultures was dried to constant weight and subjected to acid hydrolysis and depolymerization by pullulanase. The results, together with similar analyses of polysaccharides isolated at 100 hr from cultures grown on maltose, sucrose, and fructose, are listed in Table 1. Correspondence between glucose assayed by glucose oxidase and glucose equivalents estimated by Nelson's method indicated the polymer to be essentially polyglucose in constitution, a fact confirmed by paper chromatography of these hydrolysates which indicates glucose as the major monosaccharide together with a trace of galactose. Depolymerization with pullulanase followed by a-2.5% GLUCOSE  bPolysaccharide content estimated by acid hydrolysis and measurement of released sugars by Nelson's method, I, glucose oxidase, II, and phenol-sulfuric acid procedure, III. c Per cent pullulan is based on the glucose content of the polymer and is described in text. d 14C-maltotriose isolated from chromatograms separating the pullulanolysis products of labeled polymers as described in text. bPolysaccharide estimated as glucose content of polymer; see Table 1, footnote b. c See footnote c, Table 1. paper chromatography of the products revealed maltotriose as the major oligosaccharide product together with traces of tetrasaccharide (13).
Similar chromatographic analyses of '4C-labeled polymer that had been subjected to pullulanolysis indicated that as well as triand tetrasaccharide products a proportion of the label remained at the origin (Table 1). Uronic acids or phosphate, if present in these polymers, constitute less than 5 % of their dry weight. A quantitative analysis, by pullulanolysis of polysaccharides isolated at 63, 88, 111, 135, and 160 hr from cultures utilizing sucrose, indicated that 88 to 107% of the polyglucose content could be accounted for as polymaltotriose ( Table 2).
The growth of P. pullulans utilizing 2.5%O concentration of fructose containing 0.04 ,uCi of 4C-fructose per ml is illustrated in Fig. lb. The parameters of cell weight, extracellular pH, utilization of carbon substrate, and extracellular polymer production appear to be comparable to the glucose-grown culture of Fig. la. In particular, the rate of fructose uptake is essentially the same as glucose. However, the analyses provided in Table 1 show fructose to be less efficient as a source of pullulan.
The characteristics of growth on a 5% sucrose medium are shown in Fig. 2a, and demonstrate the preference of the cell for the utilization of glucose over fructose, the rate of uptake of each sugar in the absence of the other being approximately the same (Fig. 1). The almost complete hydrolysis of sucrose before commencement of the log phase of growth is catalyzed by the elaboration of an induced invertase. This enzyme cannot be detected in cultures grown on 2.5% glucose or mixtures of 2.5% glucose and 2. fructose, a fact that probably reflects repression that has been demonstrat vertase in other systems (20). It raffinose to fructose and galactosylgl has a negligible effect on maltose. A supernatant fluid, isolated from a suci culture at 100 hr and adjusted to a su centration of 1 %O containing 14C-su incubated for 24 hr at 37 C. Portic digest sampled at 0 and 24 hr were fi on paper (Whatman 3MM). The 14C p 3a) revealed a major pattern of hy glucose and fructose with a minor por sucrose (5%o) involved in transfer r yield oligosaccharides. The complete any label at the origin indicated that no polysacc.
Fr. charide synthesis had taken place. Primer, should it have been needed, was present as unlabeled pullulan. Samples of whole supernatant fluid isolated at 40, 63, 88, 111, 135, and 160 hr from sucrosegrown cultures were fractionated on Sephadex G-200 in 10 mm phosphate buffer (pH 7.0) containing 100 mm sodium chloride. Column effluent, monitored for carbohydrate by the phenolsulfuric acid assay, revealed that polysaccharide, when present, was completely excluded by the 410 50 gel. This evidence, together with that cited above, indicates that pullulan cannot be synthesized in a cell-free system but is produced by the organism as a completed macromolecule which may later undergo modification (13).
Cultures provided with equivalent amounts of glucose and fructose exhibit growth characteristics similar to sucrose-grown cultures. With inclusion of either 14C-glucose or fructose, a preferential use of glucose is seen both in the uptake of label by the cell and the production of extracellular polymer (Fig. 4). The appearance of oligosaccharide in the sucrose-grown cultures (Fig. 3a), presumably arising through disproportionation of the substrate, was not observed in cultures grown on a combination of glucose and fructose. Examination of "IC-polymer produced 40--50 from "4C-fructose in this combination of glucose and fructose indicated that 80%, of the label was nsferase ac-found in maltotriose after depolymerization with !pon sucrose puUulanase. This is to be contrasted with the !on maltose 29% found from fructose alone ( Table 1). is described in Fig. 2b. The characteristics of cell growth are comparable with sucrose, but it is apparent that the rate of glucose utilization by the cells is at least equal to its production from maltose. Glucose is only detected in the culture medium towards the end of the log phase. It is not known from these studies whether maltose itself is transported into the cell prior to hydrolysis. Fuente and Sols (15) have provided evidence that this is so with the metabolism of lactose and maltose by Saccharomyces cerevisiae. Measurements of maltase activity in the supernatant fluid of maltose-grown Pullularia cells indicates the release of 0.11 mg of glucose per hour per ml of culture at 40 hr. The utilization of substrate by cells growing on glucose is 0.14 mg of glucose per hour per ml of culture at a comparable period of cell growth, and although this is a comparison of data from two separate cultures it would appear that the maltose is capable of providing enough glucose by way of extracellular hydrolysis for the cells' requirements. Though not looked for, there may exist a bound maltase located at the cell surface, a possible precursor of the extracellular enzyme (20), which could augment the hydrolysis of extracellular disaccharide. However, the transport of maltose into the cell prior to metabolism cannot be ruled out.
Examination of the supernatant carbohydrase activity indicates an ability to hydrolyze sucrose and raffinose as well as maltose. No maltase activity is present in cultures grown solely on glucose. The hydrolysis of maltose is accompanied by considerable transferase activity as judged by comparison of the release of glucose assayed by glucose oxidase with the increase in reducing power measured by the Nelson reagent. Figure 3b illustrates the disproportionation of a 1% maltose digest containing "4C-maltose and catalyzed by supernatant fluid enzymes isolated after 100 hr of culture on maltose. In 24 hr, 40% of the original maltose has been transformed to products of higher molecular weight with a chromatographic mobility suggesting the formation of glucosidic bonds other than a-i-. Correspondence between extracellular enzymes and those associated with the cell is well documented (20). Ueda et al. (33) have reported the property of acetone-dried cells of P. pullulans to produce panose and higher molecular weight oligosaccharides from maltose, and it is probable that this cell-bound transferase is related to that reported in this communication. Reference to Fig. 3b shows that no polysaccharide is produced by this cell-free system as evidenced by the absence of 14C label at the chromatogram origin. However, this disproportionation was observed in a cell-free system. The released glucose in maltose-grown cultures is immediately metabolized, but the extracellular transfer products, detectable at all stages of culture growth by paper chromatography, apparently are not.
Thus, in the complete system some extracellular polysaccharide may be synthesized through a shift in equilibrium of the transfer system brought about by the continuous removal of glucose. The analysis of polysaccharide material from maltosegrown cultures (Table 1) indicated that the polymaltotriose content of the polymer is but slightly lower than that obtained from glucose. dry cell weight (milligrams/milliliter); 0, X cellular glucose (milligrams/milliliter); *, cel content (counts per minute of cells/milliliter o) ture); A, extracellular polymer content (count minute per milliliter); 0, extracellular pH. elaboration of polysaccharide, and, by the u 4C-glycerol in this two-substrate culture, it established that 4.3% of the polysacch originated from the triol (Fig. 5b). This is compared with 25%7, of glucose that is divert extracellular polymer in a culture utilizing. of that substrate as carbon source. Depolym tion followed by paper chromatography o products established that 58% of this lal material was pullulan. The pH profile o. glucose-glycerol culture, unlike growth on erol alone which did not fall below 6.9, indii increasing acidity during the course of growth and was comparable to glucose-g cultures (Fig. la). It could be argued ti drop in pH of the growth medium might b initiator of pullulan elaboration and that a sequence of including glucose in a growth dium containing glycerol is the provision favorable acidic environment for the conversion 40 of glycerol into pullulan. The relationship between extracellular pH and metabolic activity I has been reported for polysaccharide synthesis in S. cerevisiae (5) and the elaboration of extrax cellular polysaccharide from Cryptococcus lau-20 rentii (3). The growth and polysaccharide production of glucose-or glycerol-grown cultures 2 adjusted to an acid pH of 5.7 or 5.4, respec-'°A . tively, are shown in Fig. 6. It is apparent that pH does indeed have an effect on glucose utiliza-0 tion with an increase in rate of 2.5 times in the log phase of growth. This is reflected in an increase in the rate of cell growth and polysaccharide production. However, comparison with analysis of which shows 90% to be pullulan. In contrast, the increased acidity of the glycerol medium does not induce pullulan elaboration. 30 Maintenance of glucose-grown cultures above°p H 8 produces adequate cell growth with a final 20 £ density of 3 mg/ml, but no extracellular material.
Reinoculation of this growth into a glucose medium that is allowed to progress to acidity 102 during growth will again produce pullulan.
The utilization of acetate, provided as 5%o 0 sodium acetate trihydrate (i.e. 2.2%-acetate), as carbon source appears to be effective for cell growth but not in production of extracellular ces of polysaccharide (Fig. 7). Cultures grown on 2.2% nedia. acetate and supplemented with a 2.5% glucose nbols: concentration do not elaborate polysaccharide 'xtrafrom either source although, as seen in the !J 14C labeling profiles of Fig. 8  used for cell growth. The pH profile of the acetate-grown culture (Fig. 7) indicates a shift to alkalinity upon commencement of the log phase of culture, a process which is but slightly delayed by the presence of 2.5% glucose (Fig. 8) and which probably accounts for the absence of extracellular polysaccharide elaboration.
Of the carbon sources investigated, whether disaccharide, monosaccharide, glycerol, or acetate, P. pullulans appears capable of their utilization for cell growth. In this investigation, it was decided to maintain the supply of carbon at approximately the same level, e.g., 2.5% glucose (40% C); 2.5% glycerol (39% C); 5% sodium acetate trihydrate (18% C), rather than maintain these substrates at equimolarity. Ethanolic extraction of cells that have been grown on glucose, glycerol, or acetate, followed by paper chromatographic separation of the extracted sugars revealed trehalose to be present in all three cells. It is apparent, then, that the reserve disaccharide of P. pullulans (22) is readily synthesized from 6-3and 2-carbon substrates. However, the production of extracellular pullulan is not so facile, and it is of interest to attempt a definition of the conditions under which it is elaborated.
Conditions for polysaccharide elaboration. Reference to growth characteristics of P. pullulans on glucose (Fig. la) and fructose (Fig. lb) indicates that pullulan elaboration appears to commence only when the cells are in the late log phase and does not stop when cell growth has reached the stationary phase. "IC-glucose added to cells in the late log phase is immediately observed in elaborated polysaccharide. It is supposed, then, that glucose transported into the cell is not diverted to an extracelular destination until the basic growth requirements of the cell are met. Pullulan is not produced by cells growing in media containing a 0.5% glucose carbon source, and, since the maximum cell density under these conditions is 47% that of growth on 2.5% glucose, it is presumed that the lower glucose concentration presents conditions of carbon limitation to the growth of the organism. The conditions for the production of pullulan then are reminiscent of the accumulation of polysaccharide in bacteria which occurs as a result of limiting growth conditions in the presence of excess carbon source (27). However, by using procedures of alkaline extraction in 30% potassium hydroxide at 100 C, pullulan has not been detected within the cell and does not appear to be metabolized upon elaboration (13). By contrast, growth upon excess glycerol does not lead to pullulan production. It is apparent from Fig. 5a that glycerol uptake is mediated by a different form of control than that of glucose utilization since, upon growth limitation by an as yet unspecified nutritional component, glycerol, unlike glucose or fructose, ceases to be metabolized. The production of extracellular polysaccharide by cells growing on maltose occurs only when the requirements of the cell for glucose are exceeded by the production of that substrate from maltose.
Pullulan, then, appears to be elaboratred under conditions favorable for the accumulation of glucose or fructose metabolites within the cells. Regulation mechanisms have been proposed for Saccharomyces cerevisiae (4) which control the initial interaction of the cell with monosaccharide, i.e., membrane transport of glucose is under control from an immediate metabolite, glucose-6phosphate. Romano and Kornberg (26), studying A. nidulans, have postulated the mediation of a glucose-transport mechanism by acetyl coenzyme A, an observation which provides another possible explanation for the lack of glucose utilization for extracellular polysaccharide production in the presence of acetate, since the uptake of hexoses by P. pullulans may now be under a more strict control. It is perhaps this type of transport control function which is missing in P. pullulans. The conversion of glycerol into pullulan in the presence of glucose undergoing a similar transformation reflects that proportion of glycerol metabolites that are channeled into polymer production under these conditions. The effect of pH on the ability of the cell to utilize glucose for extracellular polysaccharide production may reflect transport dependence on pH and provide another reason for the apparent inability of the cell to produce pullulan from glucose in the presence of acetate. Cellular pullulan. The occurrence of pullulan was sought within cells that were actively elaborating pullulan. Washed and lypholized preparations were extracted with hot 30% potassium hydroxide. Acid hydrolysis of the isolated polysaccharides indicated that 5.7% of the dry weight of glucose-grown cells could be accounted for as polysaccharide of which 50%,0 was comprised of glucose. Depolymerization of this material with pullulanase indicated that pullulan, if present, comprised less than 2% of the isolated polysaccharide. Pullulan itself was shown to tolerate these extraction conditions. Similarly, examination of polysaccharides extracted from glycerol or acetate-grown cells revealed no detectable pullulan. Correspondence between extracellular polysaccharides and the structure of the cell walls of the elaborating organism has been suggested. Such a relationship has been reported for five strains of Candida (12), and in some yeasts there appears to be a correspondence between elaborated mannans and the cell wall architecture of these organisms (24). However, Brown et al. (10,11), in the examination of cell wall constituents of P. pullulans, have not reported the presence of pullulan, and the similar alkaline extraction procedures described above have failed to demonstrate the presence of the polysaccharide.