A High Throughput Nile Red Fluorescence Method for Rapid Quantification of Intracellular Bacterial Polyhydroxyalkanoates

DOI 10.1007/s12257-012-0607-z

A High Throughput Nile Red Fluorescence Method for Rapid Quantification of Intracellular Bacterial Polyhydroxyalkanoates

Zuriani R., Vigneswari S., Azizan M. N. M., Majid M. I. A., and Amirul A. A.

Received: 12 September 2012 / Revised: 20 December 2012 / Accepted: 25 December 2012

© The Korean Society for Biotechnology and Bioengineering and Springer 2013

Abstract A rapid quantitative measurement of accumu- lated polyhydroxyalkanoate (PHA) is essential for rapid monitoring of PHA production by microorganisms. In the present study, a 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the Nile red stained cells containing PHA. The linear correlation obtained between intracellular PHA concentration and the fluorescence intensity represents the potential of the Nile red method employment to determine PHA concentration. The optimal ranges of excitation and emission wavelengths were determined using bacterial cells containing different types of PHAs, of different co-monomers and compositions. Interestingly, in spite of different co-monomers compositions in each PHA, all tested PHAs fluoresced maximally at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. The developed staining method also had successfully demonstrated a good correlation between the amount of accumulated PHA based on the fluorescence intensity measurements and that from chromatographic analysis to evaluate poly(3- hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4- hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate- co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3- hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate)

[P(3HB-co-3HV-co-4HB)], using the same calibration curve, despite of different co-monomers that the PHA consist.

Strongly supported by these experimental results, it can therefore be concluded that the developed staining method can be efficiently applied for rapid monitoring of PHA production.

Keywords: polyhydroxyalkanoate, nile red staining, excitation and emission wavelength, fluorescence intensity

1. Introduction

Polyhydroxyalkanoates (PHAs) are well known as a general class of polymers which are produced by wide variety of microorganisms under stress conditions. These PHAs are accumulated as carbon reserve materials when the microbial growth is limited due to depletion of essential nutrients such as nitrogen and oxygen, but in the excess of carbon [1]. They are biodegradable and biocompatible, thus providing them as an environmental friendly alternative to conventional plastics. Numerous studies have been carried out to improve the process of PHA synthesis which includes fermentation strategies, control in monomer compositions and polymer recovery process [2,3]. However, these polymers remain uncompetitive compared to traditional plastics due to the difficulties in obtaining the desired material properties and expensive production cost [4].

Chromatographic analysis is a commonly used conven- tional method to quantify the intracellular PHA. This method though able to produce important details on PHA such as PHA content, co-monomers types and polymer compositions, it involves tedious steps of sample preparation besides producing hazardous waste products of solvents and acids [5]. This analytical approach may not be practical

Zuriani R., Vigneswari S., Majid M. I. A., Amirul A. A.

Malaysian Institute of Pharmaceuticals & Nutraceuticals, MOSTI, Penang 11700, Malaysia

Amirul A. A.

School of Biological Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia

Tel: +604-652-1200/1222; Fax: +604-656-3016 E-mail: [email protected]

Azizan M. N. M.

Universiti Kuala Lumpur, Kuala Lumpur 50205, Malaysia

RESEARCH PAPER

in real time monitoring of PHA accumulation especially when PHA content becomes the sole interest. Therefore, the application of Nile red fluorescence as a rapid PHA quantification method has been a great substitute that offers advantages of real time monitoring while fermentation is in progress.

Due to the fact that Nile red fluorescence is strongly influenced by environmental conditions, Nile red is an appropriate dye that can be used as a probe to determine the polarity of organic solvents, solvent mixtures, supercri- tical fluids and ionic liquids [6]. To date, Nile red fluorescence method has been used extensively to determine the presence of PHAs and their quantification in Gram negative bacteria [7-9]. Nile red fluorescence method has also been applied in isolating strains which are capable of producing neutral lipids, the quantification of hydrophobic proteins and emulsan [6,10-12].

In this study, we aimed to develop a staining method which uses Nile red dye to quantitatively measure PHA content as a function of fluorescence intensity in Cupriavidus sp. USMAA1020, a locally isolated Gram negative bacte- rium [9]. The fluorescence was measured by means of 96- well plate using a multilabel plate reader which could provide a rapid quantitative measurement of PHAs. The fluorescence intensity measurement enhances the study of fermentation kinetics as the accumulated PHA can be estimated during the accumulation phase without the needs of tedious sample preparation steps which is time consuming.

2. Materials and Methods 2.1. Synthesis of various PHAs

The productions of PHAs containing different co-monomers were carried out by culturing Cupriavidus sp. USMAA1020 in the presence of different precursors through one stage cultivation [9]. The microorganism was initially grown in 30 mL nutrient rich (NR) medium, containing 10 g of peptone, 10 g of beef extract, and 2 g of yeast extract in 1 L of distilled water, agitated at 200 rpm for 12 h at 30

C.

Then, 1 mL of the precultured cells was transferred into 50 mL NR medium and again agitated at 200 rpm for 12 h at 30

C. Approximately, 0.1 g/L of the seed culture were subsequently inoculated into 1.0 L mineral salts medium (MSM) in a 1.5 L bioreactor (B. Braun Biotech.). The MSM, containing 3.70 g/L KH

PO

, 5.80 g/L K

HPO

, 1.1 g/L (NH

)

SO

, 0.2 g/L Mg SO

· 7H

O and 1.0 mL/L microelements solution (2.78 g/L FeSO

· 7H

O, 1.98g/L MnCl

· 4H

O and 2.81 g/L CoSO

· 7H

O, 1.67 g/L CaCl

· 2H

O, 0.17 g/L CuCl

· 2H

O and 0.29 g/L ZnSO

· 7H

O per liter of 0.1 M HCl, was supplemented with various carbon precursors as listed in Table 1, all to a final

concentration of 0.56 wt% C. The cultivation was carried out at 30

C, agitated at 250 rpm and aerated at 1 vvm.

2.2. Nile red staining of cells

The staining method was carried out essentially as described by Spiekerman et al. [8]. The cell suspension (1 mL) was centrifuged at 12,000 g for 5 min and the pellet was resuspended in 1 mL of distilled water. Subsequently, 40 µL of Nile red (80 µg/mL dissolved in dimethyl sulfoxide [DMSO]) was added to the suspension to give a final concentration of 3.1 µg Nile red per mL suspension, and was incubated at room temperature for 30 min. The stained suspension was then centrifuged at 12,000 g for 5 min and the supernatant was discarded. Distilled water (1 mL) was added and the resulting pellet was vigorously vortexed. An aliquot of suspension was pipetted into 96-well microplate.

The fluorescence was then read at excitation and emission wavelength 535 and 605 nm respectively using Wallac EnVision

Manager 1.12 software program in EnVision Multilabel Plate Reader (Perkin Elmer, Waltham, United States) using monochromator.

2.3. Correlation between PHA concentration and Nile red fluorescence intensity

To ascertain the correlation between PHA concentration and Nile red fluorescence intensity, a preliminary study was carried out using lyophilized cells containing 30 wt%

of P(3HB-co-4HB). The cell suspensions were prepared at concentrations ranging from 0.5 to 5.0 mg/mL and were stained with Nile red as described previously. Cells not containing PHAs were also stained as described above and were used as control. The optimal concentration of Nile red used to stain the cells containing PHA was investigated using various amount of Nile red ranging from 5 to 80 µL of 80 µg/mL concentration.

The optimal excitation and emission wavelengths for fluorescence measurements were determined by staining seven different types of PHAs of different polymer content and types of co-monomers. The stained cells were subse- quently scanned at excitation wavelength ranging from 450 to 600 nm and emission wavelength ranging from 550 to 650 nm which were comparable to that reported by Gorenflo Table 1. Various carbon precursors used in the synthesis of various PHAs

Types of PHAs Types of carbon sources / precursors

P(3HB) Oleic acid

P(3HB-co-4HB) Oleic acid, γ-butyrolactone

P(3HB-co-3HV) Oleic acid, 1-pentanol

P(3HB-co-3HHx) Palm kernel acid oil (PKAO)

P(3HB-co-3HV-co-4HB) Oleic acid, γ-butyrolactone, 1-pentanol

et al. [7]. Table 2 lists all types of PHAs used to determine the excitation and emission wavelength.

2.4. Rapid monitoring of PHA accumulation

In order to demonstrate the application of Nile red staining for PHA estimation, four different batch of fermentations were carried out to produce P(3HB), P(3HB-co-4HB), P(3HB-co-3HV) and P(3HB-co-3HV-co-4HB) through one stage cultivation [9]. All fermentations were carried out in the 1.5 L bioreactor as described in 2.1. Samples were taken for measurements of cell growth, fluorescence intensity and PHA content by gas chromatographic analysis [13].

3. Results and Discussion

3.1. Correlation of PHA content and fluorescence intensity

A preliminary study was carried out using different concentrations of cells containing P(3HB-co-4HB) of 30 wt% PHA content, ranging from 0.5 to 5.0 mg/mL. The excitation and emission wavelength used were 540 and 613 nm respectively, which were determined through wavelength optimization using Assay Start Wizard in Wallac EnVision

Manager 1.12 software program. Based on Fig. 1, it was shown that the fluorescence intensity is linearly correlated with increasing cell concentration from 0.5 to 1.5 mg/mL. Further increase in cell concentrations results in a plateau graph which could possibly be due to the limitation of the microplate reader or limited concentration of Nile red used for staining. Since the linearity was obtained at concentrations ranging from 0.5 to 1.5 mg/mL, cell concentration of 0.5 mg/mL was chosen to study other parameters, considering that cells of higher PHA content will relatively exhibit higher fluorescence intensity which probably reaches the linear limitation.

As the underlying principle of Nile red interaction to the PHA granules is a crucial concern, the optimal concentration

of Nile red used to stain the cells was investigated here.

Fig. 2 shows a significant increase of fluorescence intensity when amount of Nile red increase from 5.0 to 40 µL of 80 µg/mL. Further increase to 80 µL resulted in similar fluorescence intensities. For this reason, Nile red amount of 40 µL was used to stain the cells in further experiment. This optimal Nile red amount which produces final concentration of 3.1 µg/mL was considerably higher than the use of 0.5 µg/mL Nile red reported by Spiekerman et al. [8].

The efficacy of Nile red permeability into cells was studied by introducing few solvent pretreatments using ethanol, methanol, sodium hydroxide and DMSO to improve the partition of dye and effectively stain the PHA. Prior to staining with Nile red, the cell pellets were initially vortexed and suspended in the solvents for 30 min, followed by centrifugation and were subsequently stained as mentioned Table 2. Range of excitation wavelength tested with ANOVA to

determine the significance of difference between the tested wavelengths

Types of PHAs

Range of tested excitation wavelength (nm)

Lack of fit F value p-value

P(3HB) 520 ~ 550 0.54 0.7698

P(3HB-co-4HB) 520 ~ 550 3.11 0.0819

P(3HB-co-3HV) 520 ~ 550 0.49 0.8042

P(3HB-co-3HHx) 520 ~ 550 0.32 0.9162

P(3HB-co-3HHx) 520 ~ 550 2.38 0.0849

P(3HB-co-3HV-co-4HB) 520 ~ 550 0.41 0.8606 P(3HB-co-3HV-co-4HB) 520 ~ 550 1.46 0.2604

Fig. 1. The correlation of cell concentration and fluorescence intensity of 30 wt% P(3HB-co-4HB). The excitation and emission wavelengths used were 540 and 613 nm respectively. Data expressed were the mean values and standard deviations of six replicates.

Fig. 2. The effect of amount of Nile red on fluorescence intensity of the stained cells containing 30 wt% P(3HB-co-4HB). The excitation and emission used were 540 and 613 nm respectively.

Data expressed were the mean values and standard deviations of

six measurements.

previously. The effect of staining temperature was also studied by incubating the stained cells at different temperatures ranging from 4 to 80

C. However, none of the treatments and the manipulated staining condition had significantly increased the fluorescence intensity of the stained cells. The introduction of these solvents is believed to affect the permeability of cell membrane and could further facilitate the penetration of dye into cell. However, the insignificant effect towards the fluorescence intensity elucidate that the current staining method is adequate for an effective fluorescence measurement without prior solvent pretreatment.

In contrast, these solvent treatments as reported by Chen et al. [10] had significantly increased the permeability of Nile red to stain the neutral lipids in algal cells. The efficacy of Nile red to stain the lipids was further improved when the staining conditions regarding the staining temperature, incubation time and dye concentration were optimized to obtain the optimum condition for the lipid staining in microalgae [10]. This could be explained by the difference in the cell wall structure as Gram negative bacteria cell wall contains only a thin peptidoglycan adjacent to the cytoplasmic membrane. Therefore, the dye is readily permeable into the cell without the aid of cell pretreatments with the solvents. On the other hand, the presence of thick and fairly rigid cell walls in algae prevents Nile red from entering the cell hence, requires cell pretreatments to facilitate the partition of the dye into cell.

3.2. Determination of optimal excitation and emission wavelength

The optimum excitation and emission wavelength were studied to determine the suitable excitation and emission wavelength which can be used to measure the fluorescence of all types of PHAs. The stained cell was scanned at excitation wavelength ranging from 450 to 600 nm and emission wavelength, ranging from 550 to 650 nm using the Assay Start Wizard in Wallac EnVision

Manager 1.12 software. Based on Fig. 3, all investigated PHAs, including two medium chain length (mcl) PHAs consist of hydroxyhe- xanoate (HHx) monomer, fluoresce maximally at excitation wavelength between 520 and 550 nm and emission wave- length between 590 and 630 nm. This is slightly different to that reported by Wu et al. [14] that revealed a maximum fluorescence for short chain length (scl) PHAs when measured at emission wavelength of 590 nm, whereas mcl PHAs fluoresce maximally at emission wavelength of 575 nm.

Gorenflo et al. [7] in their finding had also demonstrated that all tested PHAs revealed a clear fluorescence at excitation wavelength between 540 and 560 nm and emission wavelength between 570 and 605 nm.

A statistical analysis of Design-Expert 8.0.0 one-way

analysis of variance (ANOVA) was performed to statistically prove that the range of excitation (520 ~ 550 nm) and emission wavelength (590 ~ 630 nm) is suitable for all types of PHAs for the fluorescence measurement. The analysis was carried out to determine the significance of intensity difference produced between the tested wavelength ranges. In this analysis, p-value is used to represent the probability of obtaining a mean difference between the wavelengths as high as what is observed by chance. The Fig. 3. The scanning of investigated PHAs to obtain optimal (A) excitation wavelength and (B) emission wavelength. Data expressed were the mean values and standard deviations of three measurements.

Table 3. Range of emission wavelength tested with ANOVA to determine the significance of difference between the tested wavelengths

Types of PHAs

Range of tested emission wavelength (nm)

Lack of fit F value p-value

P(3HB) 600 ~ 620 0.99 0.4568

P(3HB-co-4HB) 605 ~ 630 1.14 0.4332

P(3HB-co-3HV) 590 ~ 610 3.11 0.0661

P(3HB-co-3HHx) 590 ~ 620 0.81 0.5761

P(3HB-co-3HHx) 595 ~ 615 1.50 0.2755

P(3HB-co-3HV-co-4HB) 590 ~ 620 1.42 0.2739

P(3HB-co-3HV-co-4HB) 595 ~ 615 2.84 0.0825

difference between the tested wavelengths is significant when the p-value is less than the significance level, often 0.05 or 0.01. Based on Table 2, it was found that the p-value for all PHAs is more than 0.05, revealing no statistical significant difference in intensity exhibited between the tested wavelength ranges. The excitation wavelength of 535 nm is therefore selected to be used in further experiments. On the other hand, the range of emission wavelength slightly varied between all tested PHAs in the range of 590 ~ 630 nm (Table 3). However, the wavelength 605 nm is within the range of all PHAs and therefore emission wavelength of 605 nm is selected. Based on this ANOVA analysis, excitation and emission wavelength of 535 and 605 nm are used to measure the fluorescence intensity of all PHAs.

3.3. Rapid monitoring of PHA accumulation

In order to demonstrate the application of Nile red staining, four fermentation profiles producing P(3HB), P(3HB-co- 45%4HB) and P(3HB-co-7%3HV) and P(3HB-co-8%3HV- co-10%4HB) had been carried out where the accumulated polymer was estimated using the developed staining method.

A general calibration curve generated from known amount of PHA concentration (Fig. 4), was used for the estimation of accumulated PHA, both in concentration (g/L) and content (wt%). The following equations were used in the evaluation of PHA concentration [1] and PHA content [2]:

C

= (K

I

) / C

× CDW [1]

C

= (K

I

) / C

× 100 [2]

where C

being the concentration of PHA in g/L for equation [1] and wt% for equation [2], K

is the calibration constant, I

is the fluorescence intensity of the stained suspension, C

is the cell concentration and CDW is the cell dry weight.

The presented equations were efficiently applied in all fermentations as shown in Fig. 5. In P(3HB) production (Fig. 5A), a good correlation was obtained between P(3HB) content determined from GC analysis and that from calibration curve in the accumulation phase. A final P(3HB) content of 47.6 wt% (11.9 g/L) and 48.0 wt% (12.0 g/L) were obtained based on the calibration curve and GC analysis respectively, demonstrating a close correlation between Nile red fluorescence and the chromatographic method. The similar correlation was also observed in the production of P(3HB-co-45%4HB) copolymer shown in Fig. 5B, where the final copolymer content calculated from the standard curve and GC analysis were 71.1 wt% (3.3 g/L) and 63.4 wt% (2.90 g/L) respectively. In P(3HB-co-7%3HV)

production shown in Fig. 5C, the final copolymer content were 23.9 wt% (1.2 g/L) and 34.8 wt% (1.6 g/L), whereas for P(3HB-co-8%3HV-co-10%4HB) (Fig. 5D), the terpolymer content were 40.7 wt% (2.4 g/L) and 46.1 wt% (2.7 g/L), which were determined based on the calibration curve and GC analysis respectively.

Fascinatingly, the same calibration curve had been used to quantitatively determine the accumulated PHA in the production of P(3HB), P(3HB-co-45%4HB), P(3HB-co- 7%3HV) and P(3HB-co-8%3HV-co-10%4HB) regardless of the co-monomers compositions. This signifies the simplicity of this method as no new standard curve was required for each respective PHA. As for previous studies, many had reported on the application of Nile red staining to estimate P(3HB) content [7,15-17] but none had reported on the evaluation of other PHAs incorporated with different co-monomers by employing the Nile red staining method.

Therefore, these experimental results have constituted strong evidence that this fluorescence method depends exclusively on the PHA content and can be successfully employed for the evaluation of polymer content during the accumulation phase.

Previously, Gorenflo et al. [7] had reported similar trend in the production of PHA in Pseudomonas putida SK 6138 using Nile red method. It was reported that the fluorescence signal increased with the increase of PHA content. The Nile red fluorescence method had also been reported by Vidal-Mas et al. [18] to detect the PHA accumulation in Pseudomonas aeroginosa 47T2 in conjunction with flow cytometry to quantify the PHA produced based on the fluorescence intensity.

Fig. 4. The correlation of PHA concentration and fluorescence of

strain Cupriavidus sp. USMAA1020. The PHA concentration was

calculated by multiplying the average PHA content (wt%) determined

from chromatographic analysis with the cell concentration. The

excitation and emission wavelength used were 535 and 605 nm

respectively. Data expressed were the mean values and standard

deviations of six replicates.

Based on assumption that the dye binds preferentially to the surface layer of granules as reported by Gorenflo et al.

[7], the resulting fluorescence probably depends on the surface area and on the shape of the granules, and not particularly on their mass. Chee et al. [19] reported that the prolonged cultivation time of wild-type Burkholderia sp.

up to 48 and 72 h may results in the coalescence of neighboring granules to form one large granule. This was supported by previous reports by Tian et al. [20] and Gerngross et al. [21] that the increasing granular size would gradually force the granules to coalesce together, thus decreasing the surface area available for Nile red staining. The dependence of fluorescence emission on the surface area available for dye staining could perhaps provide an explanation towards the discrepancy that exists between Nile red fluorescence and the chromatographic method.

The presented method requires less than an hour and

involves only five simple steps for sample preparation and fluorescence measurement which results in a drastic reduction of analysis time compared to conventional chromatographic analysis. This particularly offers time advantage especially when the polymer content becomes the sole interest. On the other hand, the introduction of 96-well plate has been very beneficial to replace the conventional one sample at a time measurement to produce a rapid and reliable fluorescence measurement.

4. Conclusion

In conclusion, Nile red staining is a great tool to rapidly estimate PHA during the accumulation phase. The developed method is also readily performed on wet cells, allowing the rapid monitoring of PHA production that could facilitate the optimization of production period and the polymer

*Determined from gas chromatography

**Determined from calibration curve

Fig. 5. Rapid monitoring of PHA accumulation by Cupriavidus sp. USMAA1020 incubated at 30

C in 1.5 L bioreactor. (A) P(3HB), (B)

P(3HB-co-45%4HB), (C) P(3HB-co-7%3HV), (D) P(3HB-co-8%3HV-co-10%4HB). The excitation and emission wavelength used for

fluorescence measurement were 535 and 605 nm respectively. Data expressed represent the mean values and standard deviations of three

replicates.

concentration. This study has also highlighted the successful identification of suitable excitation and emission wavelength to be used for fluorescence measurement of all PHAs which is 535 and 605 nm respectively. All four fermentation profiles producing different types of PHA demonstrate a successful application of Nile red staining method to estimate the PHA concentration during the accumulation phase, highlighting the potential of this method to be applied in rapid monitoring of PHA accumulation. Through the introduction of 96-well plate technology, this high throughput method can be developed into a rapid kit to determine the presence of PHA and its quantification hence, enhancing the screening development for PHA accumulating strains and the real time monitoring of polymer production.

Acknowledgement

This work was supported by grant given by Ministry of Science, Technology and Innovation (MOSTI) Malaysia (09-05-IFN-BPH-002).

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