Appl Microbiol Biotechnol (1999) 51: 523±526

Ó Springer-Verlag 1999

SHORT CONTRIBUTION

K. Hong á S. Sun á W. Tian á G. Q. Chen á W. Huang

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy

Received: 14 October 1998 / Received revision: 29 December 1998 / Accepted: 30 December 1998

Abstract Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm)1, 1740 cm)1 or 1732 cm)1 respectively. Other accompanying bands near 1280 cm)1 and 1165 cm)1 helped identify the types of PHA. The intensity of the methylene band near 2925 cm)1 provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm)1, 1744 cm)1 or 1739 cm)1, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further con®rmed by gas chromatography analysis.

K. Hong1 á W. Tian á G. Q. Chen (&) Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China e-mail: [email protected] Tel.: +8610-62783844 Fax: +8610-62788784 S. Sun Analysis Centre, Tsinghua University, Beijing 100084, China W. Huang Microbiology Department, Nanjing Agriculture University, Nanjing 210095, China Present address: Engineering School, South China Tropical Agriculture University, Hainan 571737, China

1

Introduction Polyhydroxyalkanoate (PHA) is a family of intracellular biopolymers synthesized by many bacteria as intracellular carbon and energy storage compounds under conditions of restricted growth (Anderson and Dawes 1990; Steinbuchel 1991). Many attempts have been made to produce PHA as environmentally degradable thermoplastics (Anderson and Dawes 1990; Byrom 1992; Chen and Page 1997; Chen et al. 1991). Polyhydroxybutyrate (PHB) is the commonest member of the PHA family; it belongs to the shortchain-length PHA (sclPHA) with its monomers containing 4 or 5 carbon atoms, while PHA containing monomers consisting of 6±16 carbon atoms have been termed medium-chain-length PHA (mclPHA) (Steinbuchel 1991). It is known that PHA containing scl monomers such as hydroxybutyrate (HB) and mcl monomers such as hydroxyhexanoate (HH) show dramatically better mechanical properties than do PHB (Doi et al. 1995). Fourier-transform infrared spectroscopy (FT-IR) has been demonstrated to be a powerful tool for studying microorganisms and their cell components in intact form (Helm et al. 1991; Naumann et al. 1991; Naumann et al. 1995). It has been reported that PHB is observable in FTIR spectra in intact bacteria (Helm and Naumann 1995; Nicols et al. 1984). In our study we extended the observation to ®nd that not only PHB but also mclPHA can be detected rapidly by the FT-IR technique in intact cells.

Materials and methods Bacterial strains and growth conditions Strain Azotobacter vinelandii UWD, kindly provided by Dr. W. J. Page of the University of Alberta, Canada, was grown in molasses medium (Chen and Page 1994). Strains Pseudomonas mendocina AS 1.2329 and Pseudomonas pseudoalkaligenes AS 1.2328, respectively isolated from the Dagang oil®eld in Tianjin, China, and from a waste-water treatment plant of the Yanshan Petroleum Company

524

2968

Sample preparation and FT-IR analysis

2928 2859

1057

a

1165

1740

723 b 1281 1185

2863

2957

2983 2936

1057

1732

c

1500 2000 Wavenumber (cm-1)

3000

4000

Absorbance

1186

2932

2931

Cells were precipitated by centrifugation from shake-¯ask cultures, washed with distilled water and spread on KRS-5 windows. Cells grown on petri dishes were directly applied to the window. After air-drying or irradiation with infrared light, the dried cells were subjected to FT-IR. Puri®ed PHA were dissolved in chloroform and layered on the KRS-5 window. After evaporation of the chloroform, the PHA polymer ®lm was subjected to FT-IR analysis. A FT-IR Spectrum 2000 spectrometer (Perkin Elmer, USA) with a beam condenser was employed in this study: the scanning conditions were as follows: a spectra range of 4000±400 cm)1, KRS-5 as the window material, 64 scans, a resolution of 4 cm)1 and an MIR DTGS detector. Gas chromatography analysis was performed in a model SQ204 gas chromatograph equipped with a column of chromosorb 101 treated with polyethyleneglycol 20 000 (Braunegg et al. 1978).

1282

1728

Absorbance

in Beijing, China, were cultivated in mineral medium supplemented with 15 mg ml)1 glucose and 8 mg ml)1 sodium octanoate as carbon source respectively (He et al. 1998). [The two strains are stored in the China General Microbiological Culture Collection Centre (CGMCC) with the above collection numbers; they are freely available to the public]. All strains used in this study were grown in 500-ml conical ¯asks containing 100 ml medium and cultured for 48 h at 30 °C and 200 rpm on a rotary shaker (NBS 25D shaker, New Brunswick, USA). PHA in the biomass were extracted by a mixture of sodium hypochlorite and chloroform (Hahn et al. 1994).

1000

400 A

1185

1732

1262 a

b 1744 2957

2927

1163

1744

c

2857 1739

Results

2932 2959

2863

1185 1262 d

PHA characteristic bands in FT-IR spectra Puri®ed PHB, mclPHA and P(HB + mclHA) showed their strongest band at 1728 cm)1, 1740 cm)1 and 1732 cm)1 respectively in FT-IR spectra (Fig. 1A). The methylene CAH vibration near 2928 cm)1 had the strongest band in spectra of mclPHA, the weakest one in PHB. Other characteristic bands for PHB and mclPHA were visible near 1282 cm)1 and 1165 cm)1 respectively. In comparison, intact cells of PHB-producing A. vinelandii UWD, mclPHA-producing P. mendocina AS 1.2329 and P(HB + mclHA)-producing P. pseudoalkaligenes AS 1.2328 demonstrated their ester carbonyl bands at 1732 cm)1, 1744 cm)1 and 1739 cm)1 respectively (Fig. 1B). Other characteristic bands visible in their pure forms were all observable in the spectra of the intact cells, albeit at a di€erent position and with weaker bands. No band was observable near 1744 cm)1 when P. mendocina AS 1.2329 accumulated no PHA (Fig. 1B), further demonstrating that the band between 1728 cm)1 and 1744 cm)1 is characteristic of PHA. FT-IR and GC analysis In order to test the general suitability of the FT-IR technique for the rapid identi®cation of PHA in intact cells, strains collected from oil-contaminated soil and water were used to compare the results of FT-IR and GC. Ten of these strains showed strong carbonyl marker

4000

3000

1500 2000 Wavenumber (cm-1)

1000

400 B

Fig. 1A, B Fourier-transform infrared (FT-IR) spectra of pure polyhydroxyalkanoates (PHA) extracted from various cells. A a Polyhydroxybutyrate (PHB) from Azotobacter vinelandii UWD; b medium-chain-length (mcl) PHA from Pseudomonas mendocina AS 1.2329; c PHA containing hydroxybutyrate (HB) and mcl hydroxyacetate (HA) monomers from Pseudomonas pseudoalkaligenes AS 1.2328. B FT-IR spectra of (a) PHB-producing cells of strain A. vinelandii UWD; (b) cells of P. mendocina AS 1.2329 not containing PHA; (c) mclPHA-producing cells of P. mendocina AS 1.2329; (d) cells of P. pseudoalkaligenes AS 1.2328 producing PHA containing HB and HA monomers

bands near 1740 cm)1 in the FT-IR spectra, with characteristic accompanying bands for various PHA, indicating the possible accumulation of various PHA in the cells. GC analysis clearly demonstrated results that agreed with those obtained by FT-IR (Table 1).

Discussion Puri®ed PHB, mclPHA and P(HB + mclHA) showed their strongest ester carbonyl band at 1728 cm)1, 1740 cm)1 and 1732 cm)1 in FT-IR spectra; by contrast, intact cells of PHB-producing A. vinelandii UWD, mclPHA-producing P. mendocina AS 1.2329 and P(HB + mclHA)-producing P. pseudoalkaligenes AS

525 Table 1 Comparison of results from Fourier-transform infrared (FT-IR) spectra and gas chromatography (GC) for component analysis of polyhydroxyalkanoates (PHA) synthesized by bacteria isolated from various sources. Bands near 2925 cm)1 represent methylene CAH vibrations, and bands in between 1150 cm)1 and 1270 cm)1 stand for ester C@O vibrations. s strong band, mcl medium chain length, HB hydroxybutyrate, HA hydroxyalkanoate

Strain ID no.

Source of sample collection

UWD DG0801

Kindly provided by Dr. W.J. Page (University of Alberta) Oil-contaminated river

DG0802

Oil-contaminated river

DG0805

Oil-contaminated river

DG0806

Oil-contaminated soil

DG0815

Oil-contaminated river

DG1317

Oil-contaminated sea (the Yellow Sea)

YS2

Petroleum waste-water treatment plant

YS10

Petroleum waste-water treatment plant

YS12

Petroleum waste-water treatment plant

YS13

Petroleum waste-water treatment plant

1.2328 had their ester carbonyl bands at 1732 cm)1, 1744 cm)1 and 1739 cm)1 respectively. The band shift in intact cells could be attributed to the more complex chemical environment surrounding the PHA, compared with that of the puri®ed PHA (Fig. 1). The bands represent the ester carbonyl group C@O stretch in various PHA (Naumann et al. 1991; Helm and Naumann 1995; Nicols et al. 1984). Although other esters, such as wax esters or triacylglycerols, may also generate similar phenomena, no such disturbance was observed when cells of P. mendocina AS 1.2329 did not accumulate PHA (Fig. 1B). This further demonstrates that the band between 1728 cm)1 and 1744 cm)1 is characteristic of PHA. It is also possible that a low content of PHA in cells will not be detectable by FT-IR spectroscopy as other esters will disturb the characteristic band Several methods have been developed for qualitative analysis of PHA, including GC, nucleic magnetic resonance and pyrolysis (Braunegg et al. 1978; Cross et al. 1989; Morikawa and Marchessault 1981). These methods often require extensive and complicated sample preparation, like hydrolysis, extraction, puri®cation or methylation etc. The FT-IR method does not require extensive sample preparation and it is thus very useful

FT-IR analysis

GC analysis: PHA monomer composition (%)

Wave-number (cm)1)

Possible PHA components

1728 1262

PHB

100HB

1739 1261 2925 1739 1260 2924 1744 1165 2926(s) 1744 1162 2926(s) 1744 1165 2928(s) 1744 1165 2928(s) 1739 1257 2926 1739 1258 2926 1739 1257 2926 1744 1168 2929(s)

HB and mclHA

92HB, 8HD

HB and mclHA

98HB, 2HO

mclHA

0.3HB, 58HO, 41HD

mclHA

22HO, 78HD

mclHA

0.6HB, 19HO, 80HD

mclHA

0.4HB, 20HO, 80HD

HB and mclHA

23HB, 39HO, 38HD

HB and mclHA

14HB, 50HO, 36HD

HB and mclHA

22HB, 40HO, 38HD

mclPHA

0.3HB, 59HO, 41HD

for broad screening of PHA-producing microorganisms (Table 1). The rapid, non-destructive analysis involving minimal sample preparation a€orded by FT-IR spectroscopy o€ers a useful method that can complement existing procedures used for the analysis of PHA. The FT-IR method is particularly suitable for screening large amounts of bacteria for their abilities to synthesize various types of PHA. It must be emphasized that the above-mentioned method is still unable to distinguish whether PHA are a blend or copolymers, and a low PHA content is dicult to detect by this method. Further e€orts are being made to analyse the PHA content in cells quantitatively by the non-invasive FT-IR technique. Acknowledgements This project was partially funded by the State Cross Century Foundation of China and the State 95 Key Project Fund.

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