APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1981, p. 261-267 0099-2240/81/010261-07$02.

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Vol. 41, No. 1

Symbiotic Utilization of Polyvinyl Alcohol by Mixed Cultures

CHIKAHIRO SAKAZAWA, MASAYUKI SHIMAO,* YOSHIFUMI TANIGUCHI, AND NOBUO KATO Department of Environmental Chemistry and Technology, Tottori University, Tottori 680, Japan

Polyvinyl alcohol (PVA)-utilizing cultures were obtained from various sources. They were mixed cultures even after cyclical transfer to liquid and plate media with PVA as a sole source of carbon. Component bacteria were isolated from the several mixed cultures, and it was shown that PVA was utilized symbiotically by two bacterial members which could not utilize PVA in each respective pure culture. From a mixed culture, strains VM15, VM15A (Pseudomonasputida) and VM15C (Pseudomonas sp.) were isolated as members essential for PVA utilization. VM15C was the predominant strain in the mixed-culture population and produced PVA-degrading enzyme. The culture supernatant of VM15A enabled VM15C to grow on PVA. VM15A was presumed to supply VM15C with a unique growth stimulant which was distinct from usual growth factors.
In recent years, the biodegradation of polyvinyl alcohol (PVA), a water-soluble synthetic polymer used widely in industry, has been studied by several workers. PVA-utilizing bacteria so far reported by these workers belong to the genera Pseudomonas and Xanthomonas, and these organisms have been assumed to utilize PVA in pure cultures (8, 10, 12). In the course of studies on microbial degradation of water-soluble synthetic polymers, we found that all of the PVA-utilizing isolates were mixed cultures. The present report is concerned with the finding of symbiotic utilization of PVA and some properties of the symbionts.
MATERIALS AND METHODS Media and growth conditions. The basal medium used throughout this work was essentially the same as that used by Suzuki et al. (10) for PVA utilization of Pseudomonas 0-3 in a pure culture, except for the addition of 10 mg of FeSO4. 7H20, 0.5 mg of MnSO4-4-6H20, 0.4 mg of pyridoxine hydrochloride, and 0.4 mg of thiamine hydrochloride as substitutes for FeSO4, MnSO4, pyridoxine, and thiamine in 1 liter of the medium. Unless otherwise noted, 5 g of PVA (Wako Pure Chemical Industry, Osaka, Japan; degree of polymerization, 500; degree of saponification, 88.7%) was added as a sole source of carbon to 1 liter of the medium. For the solid medium, 15 g of agar was added to 1 liter of the medium. The nutrient broth contained the following (in 1 liter of deionized water at pH 7.0): peptone (Difco), 10 g; beef extract (Difco), 5 g; NaCl, 5 g; and vitamin mixture, 1 ml. The vitamin mixture contained the following (in milligrams per liter of deionized water): calcium pantothenate, 400; inositol, 200; niacin, 400; p-aminobenzoate, 200; pyridoxine hydrochloride, 400; thiamine hydrochloride, 400; biotin, 2; and vitamin B12, 0.5. The nutrient agar was commercial dehydrate medium (Nissui Seiyaku, Tokyo, Japan). The cytophaga agar was essentially the same as that described by Anacker and Ordal (1), except for the addition of 1 ml of the vitamin
261

mixture to 1 liter of the medium. The liquid culture was grown in a test tube with 5 ml of medium or in a 500-ml flask with 200 ml of medium at 30C under continuous reciprocal shaking. A loopful of cells grown on a slope medium was suspended in 1 ml of physiological saline, and a 0.1-ml portion of the suspension was inoculated into each test tube. Each 500-ml flask was inoculated with a loopful of cells taken directly from a slant. Growth was estimated by reading the optical density at 660 rm. Cultivation on solid medium was carried out at 30C. Analyses. The culture supematant was prepared by centrifugation at 18,000 x g for 20 min at 4C. PVA concentration in the supernatant was estimated by the method of Finley (4). Relative viscosity of the supernatant to water was measured at 30C with an Ostwald viscometer. PVA-degrading enzyme activity in the culture supernatant was assayed by measuring the decrease of viscosity of a reaction mixture containing 50 mM phosphate buffer (pH 7.5), 0.5% PVA (Nippon Synthetic Chemical Industry, Osaka; degree of polymerization, 1,350; degree of saponification, 86.5%), and culture supematant in a total volume of 20 ml. After reciprocal shaking at 30C for 5 h, the viscosity of the reaction mixture was measured and the enzyme activity was determined by the method of Watanabe et al.

Enumeration of bacteria. The number of viable bacteria was determined from the plate colony count with nutrient agar. The total number of cells was measured with a bacterial counting chamber under a microscope. The most probable number (MPN) of a culture was estimated by the following procedure. Five 1-ml portions of a 10-fold dilution series of culture broth were each inoculated into test tubes containing 5 ml of medium and cultivated at 30C for 14 days under reciprocal shaking. The number of test tubes showing growth was recorded, and MPN was estimated by the method of Taylor (11). Taxonomic study. The taxonomic study was carried out by standard methods (3, 5) and Bergey's Manual of Determinative Bacteriology, eighth edition

(13).

(2).

Materials. PVA was used after extraction of so-

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APPL. ENVIRON. MICROBIOL.

dium acetate, which is contained as a impurity in commercial PVA, by methanol in a Soxhlet extractor. Other chemicals used were commercial products.

RESULTS

Isolation of PVA-utilizing mixed cultures. About 1,000 samples were collected from soil, sludge, factory waste, etc., and subjected to repeated enrichment cultures in PVA medium. A portion of each enrichment culture was spread on a plate of PVA medium and cultivated for 14 days. The colonies appearing on each plate were picked and inoculated into PVA medium and cultivated for 14 days. For the succeeding isolation, liquid and plate cultures were repeated alternately about 10 times. As the result of the above screening procedure, about 30 PVA-utilizing cultures were isolated. But, in this process, the colonies which showed growth in liquid cultures on PVA could not be obtained as single colonies apart from other types of colonies. PVA-utilizing colonies always overlapped either one or two types of colonies. All of the single colonies on the plates showed poor or no growth on PVA in liquid cultures. Therefore, in spite of the repetition of the isolation procedure, pure cultures which utilize PVA could not be obtained, and all of the isolates were mixed cultures.

nies on nutrient agar plates prepared for PVA culture broths, but strains obtained from these colonies did not utilize PVA in pure and remixed cultures of them. Isolation of component strains. Among the PVA-utilizing cultures, a mixed culture designated as VM15 was the most potent PVA utilizer (Fig. 1). Growth of the mixed culture was accompanied by decreases in the PVA concentration and in the relative viscosity of the medium. The decrease in the relative viscosity showed that the mixed culture could split molecular chains of PVA. After 7 days of cultivation, the maxiTABLE 1. Effect of surface area of plate of PVA medium on the frequency of the appearance of PVAutilizing coloniesa No. of PVA-utilizing coloSurface area of plate (cm2
58C
165d

nies' 2,105
206

Colony formation on PVA medium. Each mixed culture isolated formed a few types of non-PVA-utilizing colonies on plates of PVA medium, in addition to one type of PVA-utilizing colony. Non-PVA-utilizing colonies were small, and it was thought that these colonies develop on the plate by utilization of impurities of the solid medium. PVA-utilizing colonies appeared late and developed slowly at a position on a plate where many small non-PVA-utilizing colonies had been already formed. When cultivation of a PVA-utilizing mixed culture was carried out on a plate of PVA medium in such a way that all colonies were apart from each other, a PVA-utilizing colony did not appear. Replicates of a suspension of a mixed culture were spread and cultivated on plates of PVA medium with different surface areas, and the numbers of PVAutilizing colonies were counted (Table 1). Fewer colonies developed on large plates than on small plates. These facts suggested that formation of a PVA-utilizing colony resulted from overlapping of different bacteria. It was, therefore, presumed that all obtained PVA-utilizing cultures were symbiotic mixed cultures. But, remixing of non-PVA-utilizing colonies picked from PVA plates of the mixed cultures did not produce the ability to utilize PVA in liquid cultures. Also, each mixed culture forned a few types of colo-

a A mixed culture was plated on PVA medium. After 14 days of cultivation, the PVA-utilizing colonies were counted by taking advantage of morphological differences from non-PVA-utilizing ones. The PVA-utilizing colonies of this mixed culture were yellow, and the non-PVA-utilizing colonies were white. 'Average number of colonies on five plates. 'Plate diameter, 86 mm. d Plate diarneter, 145 mm.

4 6 8 Time (days) FIG. 1. PVA-utilization by VM15. Cultivation was carried out in 200 ml of PVA medium in a 500-ml shaking flask. Symbols: 0, growth; 0, concentration of PVA; A, relative viscosity of supernatant; A, pH of the medium.

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mum extent of growth and almost complete degradation of PVA were attained. Two kinds of bacteria, VM15A and VM15B, were isolated from a direct plate culture of the mixed culture on nutrient agar. As described above, neither strain could utilize PVA in pure or mixed cul-

TABLE 2. MPN of VM15 culture grown on PVA'

meration

erua-

No. of tubes showing growth' at dilution ratio

MPN
(cells/mi)

media

culture *__ 10 109 1010 lol

of VM15

mPN

1012

ture.

The number of cells was examined during growth of VM15 on PVA (Fig. 2). After 7 days of cultivation, the total number of cells counted under a microscope was about 10 times higher than the sum of the viable counts of strains VM15A and VM15B. This discrepancy between the total cell number and the viable cell number suggested the presence of another strain, although colonies other than those of VM15A and VM15B did not appear on the plates used for the viable counts even after 30 days of cultivation. To isolate the unknown strain, plate cultures of the mixed culture with various carbon sources were carried out, but the unknown strain was not detected. To detect and isolate the unknown strain, the MPN method was applied for the VM15 culture grown on PVA for 7 days (Table 2). From Fig.

5 5 1 0 0 3.3 X 109 0 5 5 4 0 1.3 x 1010 5 5 3 Ce 0 0 7.9 x 109 a MPN was measured for VM15 cultivated as described in Fig. 1 for 7 days. b Cultivation time: 14 days. c PVA medium. d PVA medium supplemented with viable VM15A

Ae

Bd

and VM15B. e Nutrient broth.

10

E 0

108

4 6 Time (days)

FIG. 2. Time course of cell number during growth of VM15 on PVA. Cultivation was carried out as described in Fig. 1. Symbols: numbers of total cells (A), of viable VM15A (0), and of viable VMJ5B (-).

2, it can be seen that the unknown strain in the mixed culture numbers about 1010 cells per ml. MPN was measured with three kinds of media: A, PVA medium; B, PVA medium supplemented with viable VM15A and VM15B grown on nutrient agar; and C, nutrient broth. MPN measured with medium B and that with medium C were in fair agreement with the total number of cells in the mixed culture. This indicated that the unknown strain should be present in the mixed culture. The distinct difference between MPN measured with medium A and that with medium B suggested that the unknown strain could not grow on PVA in the absence of VM15A or VM15B or both. On the other hand, MPN measured with medium C suggested that the unknown strain could grow in nutrient broth. From Fig. 2, it could be estimated that VM15A and VM15B were rarely present in 1 ml of a 1010 dilution of the mixed culture, and therefore the unknown strain grew in a pure culture in nutrient broth inoculated with replicates of 1010 dilution. Actually, when the cultures grown in nutrient broth inoculated with replicates of 1010 dilution were plated on nutrient agar, only one kind of colony, differing from those of VM15A and VM15B, appeared very slowly and developed up to only 1 mm in diameter after 10 days of cultivation. The pure culture of the strain thus obtained, designated VM15C, was maintained on nutrient agar after some successive plate cultures on nutrient agar. Effect of other component strains on colony formation by VM15C on nutrient agar. As described above, colonies of VM15C could not be detected on nutrient agar by direct plating of the mixed culture, although VM15C grew in a pure culture on the medium. To examine this phenomenon, mixed suspensions containing a given population of VM15C and various populations of VM15A or VM15B were spread and

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cultivated on plates of nutrient agar, and the VM15C colonies were counted (Table 3). The suspensions were prepared by mixing pure cultures of each strain grown on nutrient agar. No colonies of VM15C were formed on a plate such as the one on which only 1 colony of VM15A or about 10 colonies of VM15B appeared. This result agreed with the fact that VM15C could not be detected on direct plating of the mixed culture on various media. The disappearance of VM15C colonies in the presence of other component strains may result from competition for nutrients or inhibition of the growth of VM15C by toxins which VM15A and VM15B produce on nutrient agar plates, because the growth rate of VM15C was very slow compared with those of VM15A and VM15B. Reconstruction of PVA-utilizing communities. The reconstruction of VM15 strains was carried out with the isolated bacterial members. The component strains VM15A, VM15B, and VM15C were remixed and cultivated in PVA medium (Table 4). Although PVA was not utilized for growth in each pure culture, the remixed culture containing VM15A and VM15C could utilize PVA at almost the same extent as the original mixed culture. These facts show that the mixed culture, VM15, utilizes PVA symbiotically, and VM15A and VM15C are the members essential for PVA utilization. From four mixed cultures other than VM15, the component bacteria were also isolated by application of the MPN method described above. These PVA-utilizing communities could be reconstructed by their isolated component strains, and it was confirmed that PVA utilization by these mixed cultures is caused symbiotically by two component strains. Roles of component strains. As described above, VM15C was the most numerous strain in the mixed culture grown on PVA. Therefore, it is thought that VM15C utilizes PVA predominantly as a carbon source. The pH range for growth of each component strain in nutrient broth was examined (Fig. 3). The pH range of VM15C, pH 6.5 to 7.5, agreed with that for PVA utilization by the mixed culture. This result supported the finding described above.
Strain

To determine the producer of the PVA-degrading enzyme, each essential bacterial strain, VM15A or VM15C, was cultivated in nutrient broth, and the enzyme activity of the culture supernatant was assayed. The PVA-degrading enzyme was detected only in the culture supernatant of VM15C in the activity of 55 units per liter after 14 days of cultivation. This fact showed that VM15C is the producer of the PVAdegrading enzyme for the mixed culture. A possible role of VM15A during PVA utilization by the mixed culture was presumed to be as a producer of a growth stimulant for VM15C. PVA utilization by VM15C was examined with PVA medium supplemented with the culture supernatant of VM15A. The culture supernatant was prepared from 4-day culture of VM15A incubated in the basal medium containing one of following carbon sources: 0.5% PVA, 0.1% glucose, or 0.1% succinate. After removal of the cells by centrifugation, the supernatant was autoclaved at 120C for 20 min. VM15C was cultivated in 5 ml of medium prepared by mixing 1 ml of the basal medium containing 2.5% PVA (or 0.5% PVA in the case of the culture supernatant of VM15A on PVA) with 4 ml of each culture supernatant. Addition of each culture
supernatant enabled VM15C to grow on PVA in a pure culture. VM15C did not show growth in the medium which was prepared by mixing the basal medium containing no carbon sources with the culture supernatant of VM15A on glucose or succinate. These facts showed that VM15A plays as the producer of a growth stimulant for VM15C in the mixed culture on PVA. The nutritional requirement of VM15C was studied in connection with the growth stimulant supplied by VM15A. VM15C required thiamine hydrochloride for growth on glucose. The concentration of thiamine hydrochloride in the basal medium (0.4 mg/liter) was enough for VM15C to grow on glucose, but VM15C could not grow on PVA in the basal medium which contained the higher concentration of thiamine hydrochloride (4,40, or 400 mg/liter). Also, PVA utilization by VM15C was examined with media containing 0.05% of one of the following growth factors: peptone, yeast extract, beef extract, or
No. of colonies per plate

TABLE 3. Effect of the component bacteria on growth of VM15C on nutrient agara

-b VM15A 212 183 23 15 1 1 _ _ _ _ _ VM15B 244 198 36 26 9 7 0 0 0 0 0 VM15C 0 0 0 0 0 0 442 480 0 a Portions (0.1 ml) of mixed cell suspensions containing a given number of VM15C cells and various numbers of VM15A cells or VM15B cells were spread on the agar. After 20 days of cultivation, the colonies of each strain were counted. b_, The corresponding strain was not added.

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Casamino Acids. VM15C could not utilize PVA in these media. From these results, the growth stimulant of VM15C supplied by VM15A is anticipated to be unlike usual growth factors. Taxonomic study. The taxonomic characteristics of VM15A and VM15C were studied (Table 5). Electron micrographs of both strains are shown in Fig. 4. From these characteristics, VM15A was identified as Pseudomonas putida, and VM15C tentatively could be assigned as Pseudomonas sp., but the very slow growth rate, thiamine requirement, and lack of ability to utilize acetate are different from usual Pseudomonas species. Several taxonomic characteristics of VM15B were examined, and this strain was assigned as achromogenic Pseudomonas sp. DISCUSSION No example of PVA utilization by a symbiotic mixed culture has been reported so far. All cultures isolated in this work were able to utilize PVA only in mixed cultures even in the medium used by other workers for PVA utilization in a pure culture (10). This fact indicates that symbiotic mixed cultures are not exceptional in PVA utilization by microorganisms. The predominant strains, VM15C and the other bacteria, of the mixed cultures were difficult to detect and isolate by usual plate culture techniques such as direct streaking or spreading a mixed culture on a plate. In this work, the MPN method was applied to detect and isolate these strains from the mixed cultures. This method may be applicable not only for a PVAutilizing mixed culture but also for other mixed cultures to isolate a predominant strain in cases when the colony formation is inhibited by coexisting strains. Also, cultivation of replicates of the mixed culture on plates with various surface
TABLE 4. Reconstruction of VM15 with component bacteria
Bacteria Bacteria

~0

B
0 ~
4

o~ ~ ~ ~ ~ 0
~ ~ ~
0

265

50 e

7 10 9 8 Initial pH FIG. 3. Effect of pH on PVA utilization by VM15 and growth of the component bacteria in nutrient broth. Cultivation was carried out in 5 ml of medium in a test tube. Cultivation time: 24 h (VM15A, VM15B), 7 days (VM15C, VM15). Symb,pls: PVA medium (A); nutrient broth (B); growth of VM15A (0), of VM15B (0), of VM15C (A), and of VM15 (A); and PVA concentration of supernatant (5).

Growth'

~(OD66o)
0.12 0.16 0.04 0.14 0.38 1.73 1.91 1.85

Degraded PVA
(%
1.4 1.6

Ab
Bc

Cd

A+B B+C A+C A+B+C VM15e

26.7 2.0 81.0 100 100 100 was carried out in 5 ml of PVA medium in a 'Cultivation test tube for 7 days. ODr6o, Optical density at 660 rnm. bA, VM15A. 'B, VM15B. d C, VM15C. 'Original mixed culture.

areas may be useful for determining whether a mixed culture is symbiotic or a simple mixture of bacteria. Suzuki et al. (10) examined PVA degradation by Pseudomonas 0-3 grown on various carbon sources. On the basis of the observation that only the cells grown on PVA could degrade PVA, they presumed that PVA-degrading enzyme was inducibly produced. However, VM15C, isolated in this work, produced PVA-degrading enzyme in nutrient broth even in the absence of PVA. This fact indicates that the PVA-degrading enzyme of VM15C is constitutive or at least not an enzyme induced by only PVA. Watanabe et al. (6, 7) and Suzuki (9) purified PVA-degrading enzymes of Pseudomonas strains, and they found that the enzyme is active, not only for PVA but also for various low-molecular-weight secondary alcohols. These facts suggest that microbial PVA-degrading ability was not gained specially for PVA utilization, but resulted from the broad substrate specificity of a kind of enzyme which catalyzes a reaction other than PVA

degradation inherently. VM15A plays a minor role in the mixed culture for PVA biodegradation. It was found that

the culture supematant of VM15A enables VM15C to grow on PVA in a pure culture be-

TABLE 5. Taxonomic characteristics of VM15A and VM15C

Characteristic
VM15A
VM15C

Shape Motility Gram stain Oxidase Catalase 0-F test Pigmentation Gas production' Indole production Nitrate reduction Denitrification Voges-Proskauer reaction Methyl red reaction Gelatin hydrolysis Starch hydrolysis Growth at 41C Nutritional requirement Carbon source utilization test Utilized

Rods, 0.3-0.6 by 0.7-1.2 um Motile by polar flagella Negative Positive Positive Oxidative Fluorescent pigment Negative Negative Positive Negative Negative Negative

Rods, 0.2-0.4 by 1.1-2.4 pLm Motile by a polar flagellum Negative Positive Positive Oxidative Diffusible brown pigment

Negative Negative
Positive Not tested

Negative Negative Negative None

Negative Negative Negative Negative Negative Thiamine

D-Fructose, D-galactose, D-glucose, Dlactose, D-mannose, D-trehalose, Dxylose, L-alanine, L-glutamate L-Arabinose, D-ribose, meso-inositol, glycerol, L-arginine, acetate, citrate

L-Arabinose, D-fructose, D-glucose, Dribose, glycerol, ,8-alanine, DLarginine, L-valine, acetate, citrate Not utilized D-Galactose, D-lactose, D-mannose, D-trehalose, D-xylose, mesoinositol, geraniol a Gas production from D-glucose or glycerol.

FIG. 4. Electron micrographs of VM15A and VM15C. VM15A was cultivated on nutrient agar for 2 days, and VM15C was cultivated on cytophaga agar for 5 days. Cells were negatively stained with 0.5% sodium phosphotungstate at pH 7.0.
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cause it supplies a growth stimulant, which may not be a usual growth factor. The isolation and the identification of the growth stimulant produced by VM15A, now in progress, will greatly help elucidation of the mechanism of symbiotic PVA utilization.
ACKNOWLEDGMENTS
We are grateful to H. Yamada and Y. Tani, Kyoto University, for their interest and valuable discussion in this work. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture and Science of Japan.

LITERATURE CITED 1. Anacker, R. L., and E. J. Ordal. 1959. Studies on the myxobacterium Chondrococcus columnaris. J. Bacteriol. 78:25-32. 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 3. Cowan, S. T., and K. J. Steel. 1974. Cowan & Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, Cambridge. 4. Finley, J. H. 1961. Spectrophotometric determination of polyvinyl alcohol in paper coatings. Anal. Chem. 33: 1925-1927.

5. Komagata, K. 1975. Saikin no bunrui to dotei, p. 203245. In T. Hasegawa (ed.), Biseibutsu no bunrui to dotei. Tokyo Daigaku Shuppankai, Tokyo. 6. Morita, M., and Y. Watanabe. 1977. A secondary alcohol oxidase: a component of a polyvinyl alcohol degrading enzyme preparation. Agric. Biol. Chem. 41:1535-1537. 7. Morita, M., N. Hamada, K. Sakai, and Y. Watanabe. 1979. Purification and properties of secondary alcohol oxidase from a strain of Pseudomonas. Agric. Biol. Chem. 43:1225-1235. 8. Nishikawa, H., and Y. Fujita. 1975. Polyvinyl alcohol degradation techniques using microorganisms. Chem. Econ. Eng. Rev. 7(no. 4): 33-41. 9. Suzuki, T. 1978. Oxidation of secondary alcohols by polyvinyl alcohol-degrading enzyme produced by Pseudomonas 0-3. Agric. Biol. Chem. 42:1187-1194. 10. Suzuki, T., Y. Ichihara, M. Yamada, and K. Tonomura. 1973. Some characteristics of Pseudomonas 0-3 which utilizes polyvinyl alcohol. Agric. Biol. Chem. 37: 747-756. 11. Taylor, J. 1962. The estimation of numbers of bacteria by tenfold dilution series. J. Appl. Bacteriol. 25:54-61. 12. Watanabe, Y., N. Hamada, M. Morita, and Y. Tsujisaka. 197.6. Purification and properties of a polyvinyl alcohol-degrading enzyme produced by a strain of Pseudomonas. Arch. Biochem. Biophys. 174:575-581. 13. Watanabe, Y., M. Morita, N. Hamada, and Y. Tsujisaka. 1975. Formation of hydrogen peroxide by a polyvinyl alcohol degrading enzyme. Agric. Biol. Chem. 39:2447-2448.