Journal of Environmental Biotechnology
Vol. 9, No. 2, 131–134, 2009

Original paper (short paper)

Symbiotic Degradation of Polyvinyl Alcohol by Novosphingobium sp.
and Xanthobacter flavus.
DA RONG1, 3, KAORU USUI1, TOMOHIRO MOROHOSHI1*, NORIHIRO KATO2, MEI-HUA ZHOU3,
and TSUKASA IKEDA1
Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University,
7–1–2 Yoto, Utsunomiya, Tochigi 321–8585, Japan2
Department of Advanced Interdisciplinary Sciences, Graduate School of Engineering, Utsunomiya University,
7–1–2 Yoto, Utsunomiya, Tochigi 321–8585, Japan
3
College of Environmental Science and Engineering, Dong Hua University, Shanghai 201620, China

1* TEL: +81–28–689–6176 FAX: +81–28–689–6176* E-mail: morohosi@cc.utsunomiya-u.ac.jp(Received; 18 August, 2009/Accepted; 26 August, 2009)
Polyvinyl alcohol (PVA) must be removed from the waste water before discharge. In this study, we isolated some bacteria
from the activated sludge. One of them, Novosphingobium sp. strain P7, could degrade PVA in the minimal medium suppliedwith methionine. We also isolated Xanthobacter flavus strain B2, which did not have PVA-degrading activity. Co-culture ofstrain P7 and B2 could degrade PVA without supplying the methionine. It seems likely that strain B2 supplied methionine
for the growth of strain P7, which led to the degradation of PVA.
Key words: Polyvinyl alcohol, Biodegradation, Symbiosis, Novosphingobium sp., Xanthobacter flavus

Polyvinyl alcohol (PVA), a water soluble polymer prepared by the hydrolysis of polyvinyl acetate, is widely used
in industrial applications, such as adhesives and sizing agent
in textiles10). PVA is produced in greater quantities than any
other water-soluble synthetic polymer. The world productionof PVA is about 650,000 tons per year. PVA is well-known
biodegradable polymer but not easily degraded in the environment. Therefore, the large amount of used and discarded
PVA has become a significant waste problem. In previous
study, several microorganisms, which can degrade aliphatic
polyesters like as poly (hydroxyalkanoate) and poly(ε-caprolactone), are reported2,10,13). In case of PVA, somemicroorganisms, such as Pseudomonas O-312), Pseudomonas vesicularis PD7), Alcaligenes faecalis5), Bacillus megaterium6), and Sphingopyxis sp. PVA314), have been reported
as PVA-degrading bacteria. Furthermore, several reports on
PVA degradation have dealt with symbiont and the diﬃcultyof isolating PVA-degrading microorganisms has been
pointed out in earlier reports1,3,8,11). One reason may be thediﬃculty of isolating a single strain by conventional plateculture methods. This vagueness led us to seek a techniquefor isolation of individual PVA-degrading microorganisms.
The little information available regarding microbial degradation of PVA, despite the large amounts consumed, hasmade study of its degradation necessary. In the paper, we
present the study on the degradation behavior of PVA by an
acclimated PVA-degrading microorganism, and responsible

heterogeneous microbial culture involved. Degradation
patterns of PVA by the isolated strains are also discussed.
Activated sludge samples were obtained from the wastewater treatment plant. For isolation of PVA-degrading bacteria form the activated sludge, PVA minimal medium,
which contained KH2PO4 (0.4 g/l), K2HPO4 (3.2 g/l),(NH4)2SO4 (1 g/l), MgSO4 (1 g/l), NaCl (0.1 g/l), FeSO4(10 mg/l), yeast extract (0.05 g/l), and the various concentration of PVA (Wako Pure Chemicals, Osaka; degree ofpolymerization degree: 500), was used. Activated sludge was
inoculated into the 0.6% PVA medium and incubated at
30°C with shaking. The concentration of residual PVA wasmeasured by the previously described method with slight
modification1). Briefly, the culture supernatant was diluted
to 0.3 ml with distilled water, and then 0.45 ml of 4% boric
acid and 90 μl of I2-KI solution were added. Then, themixture was diluted to a volume of 1.5 ml with distilledwater and analyzed at 660 nm to estimate PVA concentration. Cell growth was measured the optical density at
600 nm (OD600). After 6 days of incubation, increasing ofcell growth and degradation of PVA were appeared, andalmost of all PVA was degraded after 10 days (Fig. 1). To
isolate PVA-degrading bacteria, bacterial cells in the culture
were collected by centrifugation, resuspended in the distilledwater, and spread on the LB agar plates9). After a week ofincubation, several colonies were formed. Formed colonies
were picked up and grown in the LB liquid medium. Grown

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RONG et al.

cells were collected, washed twice with distilled water, andtransferred to the 1.2% PVA medium. After a week of incubation, the concentration of the residual PVA was estimated.
One of these isolates, designed strain P7, slightly degraded
PVA (Fig. 2A). To identify the bacterial species, almost 16SrRNA gene was amplified by PCR using previously describedprimers, 63f (5’-CAGGCCTAACACATGCAAGTC-3’) and
1387r (5’-GGGCGGTGTGTACAAGGC-3’)4). Sequencing
was performed using BigDye Terminator ver. 3.1 and ABI
Prism 3100 Genetic Analyzer (Applied Biosystems, Foster
City, CA). 16S rRNA sequence of strain P7 was comparedwith those from the other type strains using the Ribosomal
Database Project (http://rdp.cme.msu.edu/index.jsp1). As
the results, the 16S rRNA gene sequence of strain P7
showed high similarity to that of Novosphingobium capsulatum GIFU11526 (Fig. 3A). Cells of strain P7 were yellowpigmented and rod-shaped bacteria (data not shown). Thesefeatures supported that strain P7 was a member of genus

Fig. 1. PVA concentration and bacterial growth in the PVA medium.
The microorganisms from activated sludge were inoculated
into 0.6% PVA medium and incubated with shaking at 30°C.
Residual PVA concentration (closed squares) and cell growth(open squares) were measured by the method described incontext.

Novosphingobium (Sphingomonas). The nucleotide sequenceof the 16S rRNA gene from the strain P7 has been depositedin DDBJ/EMBL/GenBank databases under accession no.
AB512109.
Although strain P7 showed obvious PVA-degrading activity,
growth and PVA degradation of strain P7 in PVA medium
were limited compared with that of activated sludge (Fig.
2A). It was considerable that strain P7 received some
growth factors or coenzymes for degradation of PVA fromthe other bacteria. To identify the co-factor for PVA degradation by strain P7, the modified PVA medium (M-PVA
medium), which did not contain yeast extract but trace
metals, was used. First, strain P7 was inoculated into the
M-PVA medium containing 0.1% glucose as a sole carbon
source. However, cell growth was not observed after a weekof incubation (data not shown). This feature showed thatstrain P7 requires the growth factor, which was not present
in the M-PVA medium. Therefore, we added various nutrientsto the M-PVA medium. When the 0.8% M-PVA medium
was supplied with 0.1% peptone, PVA was degraded completely by strain P7 after a week of incubation (Fig. 2B).
Thus, each of twenty kinds of amino acids, which were main
components of peptone, was added to the M-PVA medium
at the final concentration of 0.01%. One of twenty aminoacids, only addition of methionine induced the growth and
PVA degradation of strain P7 (Fig. 2B). Strain P7 could
also grow in the M-PVA medium containing 0.1% glucose
and 0.01% methionine (data not shown). These results
showed that methionine worked as the growth factor forstrain P7. Large number of PVA-degrading bacteria required
exogenous pyrroloquinoline quinone (PQQ) as a coenzyme
for PVA-degrading enzyme. However, strain P7 did not
require supply of PQQ externally. It was assumed that strain
P7 produced PQQ by itself or had PQQ-independent enzymes
for PVA degradation.
Strain P7 did not grow and degrade PVA in the M-PVA
medium, but did in the activated sludge. It was assumed

Fig. 2. Degradation of PVA in the PVA minimal medium by strain P7 and activated sludge.
(A) Strain P7 or activated sludge was inoculated into the 1.2% PVA medium. (B) Strain P7 was inoculated into 0.8% M-PVA medium
with or without 0.1% peptone (Pep) and 0.01% methionine (Met). Bacterial cultures were incubated with shaking at 30°C and residual
PVA concentration was measured after a week of incubation by the method described in context.

Symbiotic degradation of PVA

133

Fig. 3. Neighbor-joining trees of the 16S rRNA gene sequences.
PVA-degrading strain P7 (A) and its symbiotic strain B2 (B) were isolated from activated sludge. Bacterial isolates in this study wereshown by bold style. The scale bar represented 0.01 substitutions per nucleotide position. Bootstrap values (from 100 replicates) were indicated at the nodes.

that the putative symbiotic bacteria might support growthof strain P7 and degradation of PVA by strain P7. To identify the symbiotic bacteria from the activated sludge,
PVA-degrading activated sludge were diluted and spreadon LB agar plates. After forming colonies, eight isolates
and strain P7 were grown in the LB medium. Bacterial
cells were collected from 1 ml cultures by centrifugation,
and washed and resuspended with distilled water. The cellsuspensions of strain P7 and each isolates were co-inoculated
into 1.2% M-PVA medium. After a week of incubation, only
co-culture of strain P7 with one isolate named strain B2
could degrade PVA was almost completely (Fig. 4). Interestingly, strain B2 could not degrade PVA by itself with orwithout the amino acids (data not shown). To identify thebacterial strain of B2, 16S rRNA sequence of strain B2
was compared with those from the other type strains. As
the results, the 16S rRNA gene sequence of strain B2
showed high similarity to that of Xanthobacter flavus 301(Fig. 3B). The nucleotide sequence of the 16S rRNA genefrom the strain B2 has been deposited in DDBJ/EMBL/
GenBank databases under accession no. AB512110. Strain
P7 requires the supply of methionine, but did not either
PQQ or the other exogenous co-factors. Thus, it was assumed
that PVA was degraded by these two strains, in which strain
B2 produced a growth factor and strain P7 utilized thisFig. 4. Degradation of PVA in pure and mixed cultures of strain P7
and B2.
Bacterial strains were inoculated into 1.2% M-PVA medium
and incubated with shaking at 30°C. After a week of incubation, residual PVA concentration was measured by the method
described in context.

growth factor for PVA degradation. Previously reported
PVA-degrading Sphingomonas sp., which related to strain P7,
was also required PQQ and supplied PQQ with surrounding
symbiotic bacteria1). Our study demonstrated the new typesymbiotic degradation of PVA by strain P7 and B2.

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134

Acknowledgements
We thank Dr. Masayoshi Kojima (Fuji Film Co., Ltd.) for
providing us with activated sludge sample and technical assistance.

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