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BIOTECHhTOLOGY TECHNIQUESVolume 8 No.5 (May 1994) pp.345-350Received 15th Makh

STAINING METHOD OF POLY(3-HYDROXYALKANOICACIDS) PRODUCING BACTERIA BY NILE BLUE

ShiroKitamura 1 and Yoshiharu Doi l)*1)Research Institute of Innovative Technology for the Earth (RITE), Hirosawa 2-I.

Wako-shi,Saitama 351-01, Japan21Polymer Chemistry Laboratory, The Institute o f Physical and Chemical Research(RIKEN),Hirosawa 2- 1, Wako-shi, Saitama 35 l-01, Japan

SUMMARYUsing an ethanol solution of nile blue, we have developed an efficient method todetect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on theagar plate. When the bacterial colonies with PHA granules were stained with nile blue,

the stained colonies fluoresced bright orange on the irradiation of UV light. In thefluoresce emission spectra, fluorescence intensity increased with an increase in thePHA content of bacterial cells. Alcaligenes eutrophus and A./am colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nmon the excitation at 490nm. On the other hand, Pseudomonas oleovoram and P.putidawith medium-chain-length (mcl-) PHA copolymers of C6, CS and Cl0 units exhibitedan emission maximum at 570nm.INTRODUCTION

A wide variety of bacteria accumulate PHA within cells as an intracellular storagepolymer. Some bacteria produced not only PHB homopolymer but also PHAcopolymers of different 3-hydroxyalkanoic acids (Anderson and Dawes, 1990). Theisolated PHA are a partially crystalline thermoplastic with biodegradable properties(Doi. 1990). PHA are produced by bacteria under conditions of depletion of a nutrientsuch as oxygen, ammonium, sulfate, magnesium, phosphate, potassium, or iron(Steinbtichel and Schlegel, 1989). The structure of PHA produced is dependent on thekind of bacteria used and the type of carbon substrates fed. Several screening methodsof PHA producing bacteria have been developed. Accumulation of PHA granules inbacterial cells can be detected by electron or phase-contrast microscopic studies. PHApolymers are extracted from bacterial cells with organic solvents such as chloroform

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and dichloromethane, and the extracted polymers are characterized by NMR and GC-MS. A gas chromatographic method is used to determine the content and compositionsof PHA polymers without extraction from cells. In this method, bacterial celIs arereacted with a methanol solution of sulfuric acid, and intracellular PHA granules aremethanolyzed to the methyl esters of 3-hydroxyalkanoic acids (Brand1 et al., 1988).These methods are very useful for the characterization of PHA producing bacteria, butthey are not efficient as a screening method to isolate PHA producing bacteria fromenvironments. Sudan black B has been used as a dye for the isolation or detection ofPHA producing colonies on the nitrogen-limiting plate (Schlegel et al., 1970), butSudan black B showed a low selectivity to PHA granules. Nile blue sulfate is a basicoxazine dye and its aqueous solution was used to stain the intracellular PHA granulesin the microscopic study (Ostle and Holt, 1982).

In this communication we report the staining method of PHA producing bacterialcolonies by an ethanol solution of nile blue sulfate. The stained colonies with PHApolymers fluoresce bright orange on the irradiation of UV light and its light can bedetected by a fluorescence spectrophotometer.

MATERIALS AND METHODS

Bacterial strains and mediaAlcaligenes eutrophus (ATCC17699), A.lntus (ATCC29713), Pseudomonasoleovorans (ATCC29347), and P.putidu (IF014464) were used in this study.A.eutrophus was cultivated in a mineral medium (Repaske et nf., 1976) containing0.5g/l of fructose or pentanoic acid. A. latus was cultivated in a mineral medium(Ramsay et al., 1990) containing lg/l of fructose as the sole carbon source.Pseudomonas strains were grown in a mineral medium (Lageveen, 1988) containing1.7g/l of sodium octanoate or OSg/l of fructose. These mineral media with differentC/N mole ratios (carbon/nitrogen) were prepared by varying the amount of nitrogensources to carbon sources. Solid medium was prepared by the addition of 2% agar.Cell growthBacterial cells were grown on solid agar media at 30C. Agar plates were incubatedfor 48-72h until the colony diameter was increased to over 5mm.Nile blue staining of colonies on agar plateA staining solution of nile blue sulfate was prepared by dissolving 0.05g of nile bluesulfate (Chroma Gesellschaft Schmud & Co.) in lOOm1of ethanol. Colonies on the agarplate were stained with 5ml o f staining solution and shaken gently at room temperature.After 20 minutes, staining solution was removed from the agar plate, and the plate wasstood to dry the surface.Detection of PHA producing coloniesNile blue stained colonies were irradiated with a short wave ultraviolet light at 254nm (distance of about 1Ocm) from a Mineralight UV lamp (Ultra-violet Products Inc.,San Gabriel, CA).

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Fluorometric analysis of nile blue stained coloniesNile blue stained colonies were analyzed on a fluorescence spectrophotometer(HITACHI F-3010) equipped with a thin-layer chromatography accessory 250-0037.The stained colonies were excited by excitation light directory and their fluorescencespectra were detected. Slit width was lOnm, and excitation or fluorescence wavelengthwas scanned at 60nm/min.RESULTS AND DISCUSSION

The PHA production by several bacterial strains from different carbon substrateswas carried out on the agar plate with different C/N mole ratios of carbon to nitrogensources, since bacteria are known to accumulate PHA granules in cells on the nitrogen-limiting agar plate. After staining with ethanol solution of nile blue sulfate, coloniesand agar plate were stained blue. There was no difference between the colonies withand without PHA granules under conditions without UV light irradiation.

When the stained colonieswere irradiated, with a shortwave (254nm) UV light in adark room, the colonies withPHA fluoresced bright orange,but the other colonies withoutPHA remained unchanged(Fig.1). This result shows thatthe staining method by nile blueis useful to detect the PHA

Fig. 1. Nile blue stained colonies of A.eutropl1u.s withoutPHB (A) and with PHB (B) on the irradiation of UV light:(A), C/N=lO; (B), C/N=70. UV light was irradiated fromupper side.producing bacteria on the agar plate.

Then, the fluorescence spectra were recorded on a fluorescence spectrophotometerequipped with a thin-layer chromatography accessory. In this system, excitation lightfrom 250nm to 570nm was irradiated to abacterial colony on the agar plate. Fig.2 z-u.shows the luminescence excitation spectrum -of nile blue stained colony of A.eutrophswith PHB homopolymer grown on anitrogen-limiting agar plate containingfructose as the sole carbon source. In this -Estudy, 490nm was used as an excitation 3wavelength to enhance sensitivity of cl250 300 350 400 450 500 550detection. Wavelength (nm)

Fin.3 shows the fluorescence emission Fig.2. Excitation spectrum of nile blue stainedspectra of the nile blue stained colonies of colony of A.eurrophus with PHB homopolymer,recorded with emission at 580nm.A.eutrophs and P.oleovorans on the agar

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. (A) (B)

C/N=70

500 550 600 650 700 500 550 600 650 700Wavelength (nm) Wavelength (nm)Fig.3. Relationships betweell fluorescence intensity and C/N mole ratio of carbon tonitrogen sources in the agar plates of P.oleovam~s (A) and A.cutmplm (B).

plates with various C/N mole ratios ofcarbon to nitrogen sources. In thisexperiment, fructose was used as thesole carbon source for A.eutr-oplrus,while octanoate was used folP.oleovnrms. A.eutropf~us accumulatedPHB honiopolymer within cells, whileP.oleovor-nns accumulated a mcl-PHAcopolymer of 3-hydroxyoctanoate (90mol %) and 3-hydroxyhexanoate (10niol %). In the two strains, polyestercontents within cells were confirmed toincrease with the C/N mole ralio in theliquid media (data not shown). In Fig.3,the fluorescence intensities increasewith an increase in the C/N mole ratio in

500 550 600 650 700Wavelenght (nm)

Fig.4. A comparison of fluorescence emissionspectra of P.o/eovom~.~ colony with ml-PIIA(A) ad A.e~~~p/ru.s colony with PI IB (B).

the solid media, indicating that the fluorescence intensities of the stained coloniesincrease with the polyester contents of the bacterial colonies on the agar plate.However, it was difficult to determine the quantitative relation between fluorescenceintensity and polyester content, because the colonies were grown on the solid media.

Fig.4 shows a difference in the fluorescence emission spectra between nile bluestained colonies of A.Eulr0j1l1l1.Y nd I-).oleol~o171i1.~.hen nile blue stained colony ofA.culr@zrs with PHB homopolymer was excited at 49011111, he fluorescence enlissionspectrum peaked at SSOnm. On the other hand, the fluorescence emission spectrum of

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P.ofeovortrm colony with ml-PHA copolyn~ of C6 and CS units exhibited a peak at570nlll.

A.latus produced PHB homopolymer from fructose, while P.j~ri~la produced a mcl-PHA copolymer of 3-hydroxyoctanoate and 3-hydroxydecanoate from fructose(Haywood et trf.. 1990). Fig.5 shows the fluorescence emission spectra or nile bluestained colonies of four different bacterial strains. A.elrtrophus, A.latus, and Ppticlnwere grown on the agal piate containing fructose as the sole carbon source, whileP.ofeorwcrr~s was grown on the agar plate containing octanoate. There was an apparentdifference ( 101m) in emission maximun between the colonies with PHB honiopolymer(A.eutrophcrs and A.lut~s) and the colonies with ml-PHA copolymers (f.oleovoror~sand P.puti&~).

A

Bk00 550 600 650 700 500 550 600 650 700

Wavelength (nm) Wavelength (nm)Fio 5 Fluorescence emission spectra of nite bluestz;led colonies: (A), A.e~tr-oph~s with PIIB ; (13).A./afru with PMB : (C), P.o/eo~~orn~r. r wiih met-PttA :(I>), P.p~irkr with mcl-PHA.

-I500 550 600 650 700Wavelength (nm)

Fig. 6. Fluorescence emission spectm ofnite blue stained colonies of A.e~rtroph~rswith PttB (A) and P(3HB-co-3HV) (B).

A.eurr-ophrrs produced a copolymer of 3HB (70mol%) and 3-hydroxyvalerate(3011101%) (3HB-co-3HV), from pentanoic acid (Doi ETnl., 1987). Fig.6 shows thefluorescence emission spectra of slained A.elt/roplrm colonies with PHB homopolymerand P(3HB-co-3HV) copolymer. The A.er/ti-oph~rs colo~ly with P(3HB-co-3HV)copolymer showed a red shift to 59011~11. Thus, it has been found that Ihe maxin~um ofenlission peak is dependent on the struclure of PHA polymers acculwlated in bacterialcells.

This study has denlonstrated that nile blue staining nlethod is uselul for the dcteclionof PHA producing bacterial colonies on the ngar place.

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ACKNOWLEDGMENTSThis work was performed under the management of Research Institute of InnovativeTechnology for the Earth (RITE) as a part of the Development of BiodegradablePlastics supported by New Energy and Industrial Technology DevelopmentOrganization (NEDO).

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Micro&d., 56, 3354-3359.Lageveen, R.G., Huisman, G.W., Preusting, H., Ketelaar, P., Eggink, G., andWitholt, B. (1988). Appl. Environ. Microbid., 54, 2924-2932.Ostle, A. G. and Holt, J. G. (1982). Appl. Environ. Microbid., 44, 238-24 1.Ramsay, B.A., Lornaliza, K., Chavarie, C., Dube, B., Bataille, P., and Ramsay, J.A.(1990). Appl. Environ. Microobiol., 56, 2093-2098.Repaske, R. and Repaske, A. C. (1976). Appl. Envirutl. Microbid., 32,. 585-59 1.Schlegel, H.G., Lafferty, R., and Krauss, I. (1970). Arch. Micrubiol., 71, 283-294.Steinbiichel, A. and Schlegel, H.G. (1989). Appl. Micrubiol. Biutechol., 3 1, 16% 175.

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