BIOTECHhTOLOGY

TECHNIQUES

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

STAINING METHOD OF POLY(3-HYDROXYALKANOIC ACIDS) 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, Japan 21 Polymer Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN),Hirosawa 2- 1, Wako-shi, Saitama 35 l-01, Japan SUMMARY Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar 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 the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells. Alcaligenes eutrophus and A./am colonies with poly(3hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand, Pseudomonas oleovoram and P.putida with medium-chain-length (mcl-) PHA copolymers of C6, CS and Cl0 units exhibited an emission maximum at 570nm. INTRODUCTION A wide variety of bacteria accumulate PHA within cells as an intracellular storage polymer. Some bacteria produced not only PHB homopolymer but also PHA copolymers of different 3-hydroxyalkanoic acids (Anderson and Dawes, 1990). The isolated PHA are a partially crystalline thermoplastic with biodegradable properties (Doi. 1990). PHA are produced by bacteria under conditions of depletion of a nutrient such as oxygen, ammonium, sulfate, magnesium, phosphate, potassium, or iron (Steinbtichel and Schlegel, 1989). The structure of PHA produced is dependent on the kind of bacteria used and the type of carbon substrates fed. Several screening methods of PHA producing bacteria have been developed. Accumulation of PHA granules in bacterial cells can be detected by electron or phase-contrast microscopic studies. PHA polymers are extracted from bacterial cells with organic solvents such as chloroform

345

and dichloromethane, and the extracted polymers are characterized by NMR and GCMS. A gas chromatographic method is used to determine the content and compositions of PHA polymers without extraction from cells. In this method, bacterial celIs are reacted with a methanol solution of sulfuric acid, and intracellular PHA granules are methanolyzed to the methyl esters of 3-hydroxyalkanoic acids (Brand1 et al., 1988). These methods are very useful for the characterization of PHA producing bacteria, but they are not efficient as a screening method to isolate PHA producing bacteria from environments. Sudan black B has been used as a dye for the isolation or detection of PHA producing colonies on the nitrogen-limiting plate (Schlegel et al., 1970), but Sudan black B showed a low selectivity to PHA granules. Nile blue sulfate is a basic oxazine dye and its aqueous solution was used to stain the intracellular PHA granules in the microscopic study (Ostle and Holt, 1982). In this communication we report the staining method of PHA producing bacterial colonies by an ethanol solution of nile blue sulfate. The stained colonies with PHA polymers fluoresce bright orange on the irradiation of UV light and its light can be detected by a fluorescence spectrophotometer. MATERIALS AND METHODS

Bacterial strains and media Alcaligenes eutrophus (ATCC17699), A.lntus (ATCC29713), Pseudomonas oleovorans (ATCC29347), and P.putidu (IF014464) were used in this study. A.eutrophus was cultivated in a mineral medium (Repaske et nf., 1976) containing 0.5g/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) containing 1.7g/l of sodium octanoate or OSg/l of fructose. These mineral media with different C/N mole ratios (carbon/nitrogen) were prepared by varying the amount of nitrogen sources to carbon sources. Solid medium was prepared by the addition of 2% agar. Cell growth Bacterial cells were grown on solid agar media at 30C. Agar plates were incubated for 48-72h until the colony diameter was increased to over 5mm. Nile blue staining of colonies on agar plate A staining solution of nile blue sulfate was prepared by dissolving 0.05g of nile blue sulfate (Chroma Gesellschaft Schmud & Co.) in lOOm1of ethanol. Colonies on the agar plate were stained with 5ml of staining solution and shaken gently at room temperature. After 20 minutes, staining solution was removed from the agar plate, and the plate was stood to dry the surface. Detection of PHA producing colonies Nile blue stained colonies were irradiated with a short wave ultraviolet light at 254 nm (distance of about 1Ocm) from a Mineralight UV lamp (Ultra-violet Products Inc., San Gabriel, CA).

346

Fluorometric analysis of nile blue stained colonies Nile 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 fluorescence spectra were detected. Slit width was lOnm, and excitation or fluorescence wavelength was scanned at 60nm/min. RESULTS AND DISCUSSION The PHA production by several bacterial strains from different carbon substrates was carried out on the agar plate with different C/N mole ratios of carbon to nitrogen sources, since bacteria are known to accumulate PHA granules in cells on the nitrogenlimiting agar plate. After staining with ethanol solution of nile blue sulfate, colonies and agar plate were stained blue. There was no difference between the colonies with and without PHA granules under conditions without UV light irradiation. When the stained colonies were irradiated, with a short wave (254nm) UV light in a dark room, the colonies with PHA fluoresced bright orange, but the other colonies without PHA remained unchanged (Fig.1). This result shows that Fig. 1. Nile blue stained colonies of A.eutropl1u.s without PHB (A) and with PHB (B) on the irradiation of UV light: the staining method by nile blue (A), C/N=lO; (B), C/N=70. UV light was irradiated from is useful to detect the PHA upper side. producing bacteria on the agar plate. Then, the fluorescence spectra were recorded on a fluorescence spectrophotometer equipped with a thin-layer chromatography accessory. In this system, excitation light from 250nm to 570nm was irradiated to a bacterial colony on the agar plate. Fig.2 z-u. shows the luminescence excitation spectrum of nile blue stained colony of A.eutrophs with PHB homopolymer grown on a nitrogen-limiting agar plate containing fructose as the sole carbon source. In this study, 490nm was used as an excitation wavelength to enhance sensitivity of detection. Fin.3 shows the fluorescence emission spectra of the nile blue stained colonies of A.eutrophs and P.oleovorans on the agar -E 3 cl
250 300 350 400 450

500

550

Wavelength (nm) Fig.2. Excitation spectrum of nile blue stained colony of A.eurrophus with PHB homopolymer, recorded with emission at 580nm.

347

(A)

(B)

C/N=70

500

550

600 650 Wavelength (nm)

700 500

550

600 650 Wavelength (nm)

700

Fig.3. Relationships betweell fluorescence intensity and C/N mole ratio of carbon to nitrogen sources in the agar plates of P.oleovam~s (A) and A.cutmplm (B).

plates with various C/N mole ratios of carbon to nitrogen sources. In this experiment, fructose was used as the sole carbon source for A.eutr-oplrus, while octanoate was used fol P.oleovnrms. A.eutropf~us accumulated PHB honiopolymer within cells, while P.oleovor-nns accumulated a mcl-PHA copolymer of 3-hydroxyoctanoate (90 mol %) and 3-hydroxyhexanoate (10 niol %). In the two strains, polyester contents within cells were confirmed to increase with the C/N mole ralio in the liquid media (data not shown). the fluorescence intensities
In 500 550 600 650 Wavelenght (nm) 700

Fig.3,

increase

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

with an increase in the C/N mole ratio in the solid media, indicating that the fluorescence intensities of the stained colonies increase with the polyester contents of the bacterial colonies on the agar plate. However, it was difficult to determine the quantitative relation between fluorescence intensity and polyester content, because the colonies were grown on the solid media. Fig.4 shows a difference in the fluorescence emission spectra between nile blue When nile blue stained colony of stained colonies of A.Eulr0j1l1l1.Y and I-).oleol~o171i1.~. A.culr@zrs with PHB homopolymer was excited at 49011111,the fluorescence
spectrum enlission

peaked at SSOnm.On the other hand, the fluorescence emission spectrum of

348

P.ofeovortrm colony with ml-PHA

copolyn~

of

C6

and CS units exhibited a peak at

570nlll. A.latus produced PHB homopolymer from fructose, while P.j~ri~la produced a mclPHA copolymer of 3-hydroxyoctanoate and 3-hydroxydecanoate from fructose (Haywood et trf.. 1990). Fig.5 shows the fluorescence emission spectra or nile blue stained colonies of four different bacterial strains. A.elrtrophus, A.latus, and Ppticln were grown on the agal piate containing fructose as the sole carbon source, while P.ofeorwcrr~s was grown on the agar plate containing octanoate. There was an apparent difference ( 101m) in emission maximun between the colonies with PHB honiopolymer (A.eutrophcrs and A.lut~s) and the colonies with ml-PHA and P.puti&~). copolymers
(f.oleovoror~s

500

k
B
550

600

650

700

500

550

600

650

700
-I

Wavelength Fio

(nm)

Wavelength

(nm)
500 550 600 650

700

5 Fluorescence emission spectra of nite blue stz;led colonies: (A), A.e~tr-oph~s with PIIB ; (13).

Wavelength

(nm)

A./afru

with

PMB with

: (C),

P.o/eo~~orn~r.r

wiih

met-PttA

(I>), P.p~irkr

mcl-PHA.

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

A.eurr-ophrrs produced a copolymer of 3HB (70mol%) and 3-hydroxyvalerate (3011101%) P(3HB-co-3HV), from pentanoic acid (Doi ETnl., 1987). Fig.6 shows the fluorescence emission spectra of slained A.elt/roplrm colonies with PHB homopolymer and 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 of enlission peak is dependent on the struclure of PHA polymers acculwlated in bacterial
cells.

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

for

the

dcteclion

349

ACKNOWLEDGMENTS This work was performed under the management of Research Institute of Innovative Technology for the Earth (RITE) as a part of the Development of Biodegradable Plastics supported by New Energy and Industrial Technology Development Organization (NEDO). REFERENCES Anderson, A. J. and Dawes, E,A. (1990). Microbid.
Rev., 54,450-472.

Doi, Y. (1990). Microbial polyesters, VHC, New York. Doi, Y., Tamaki. A., Kunioka, M., and Saga, K. (1987). J. Cizem. Sm., Clzenz. Convnm., 1635 1636. Haywood, G.W., Anderson, A.J., Ewing, D.F., and Dawes, E.A. (1990). Appl. E~rlvirw.
Micro&d., 56, 3354-3359.

Lageveen, R.G., Huisman, G.W., Preusting, H., Ketelaar, P., Eggink, G., and Witholt, 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., Schlegel, H.G., Lafferty, R., and Krauss, I. (1970). Arch. Micrubiol., Steinbiichel, A. and Schlegel, H.G. (1989). Appl. Micrubiol.
32,. 585-59 1.

71, 283-294.
3 1, 16% 175.

Biutechol.,

350