Im working on polyhydroxyalkanoates for my M.Sc. project, and need protocols on
1)staining PHAs using nile blue directly on the plate
2)extraction and estimation of PHA content/yield using sodium hypochlorite ....
Der-Shyan Sheu described this staining method in this 2000 articleRapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR.
Nile blue A staining
PHA-positive strains identified by PCR were cultured in 50 ml MSM containing an appropriate carbon source at 30 °C with reciprocal shaking at 150 r.p.m. for 3 d. The bacterial cells were stained with Nile blue A and observed as described by Ostle & Holt (1982)Down . The fluorescence microscope used was an Olympus AX 70; the excitation filter, barrier filter and dichroic mirror sets were BP450480, BA515 and DM500, respectively. The staining results were photographed in colour on Kodak Kodacolor print film (400 ASA).
This method references the following paper...
Ostle, A. G. & Holt, J. G. (1982). Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate. Appl Environ Microbiol 44, 238-2.
Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.
... and here is a reference for extraction of PHA
Kshama Lakshman and T. Ramachandriah Shamala. Extraction of polyhydroxyalkanoate from Sinorhizobium meliloti cells using Microbispora sp. culture and its enzymes. Enzyme and Microbial Technology. Volume 39, Issue 7, 3 November 2006, Pages 1471-1475
Sinorhizobium meliloti produced 50% polyhydroxyalkanoate (PHA) in the biomass in the presence of sucrose as carbon substrate. Isolation of the intracellular PHA was achieved through a secondary fermentation involving a cell lytic actinomycetes species namely Microbispora sp. without further supplementation of nutrients to the S. meliloti fermented broth, at 30 °C, 150 rpm up to 72 h. Microbispora sp. cells that showed pelleted growth was removed by filtration and the released polymer contained in the filtrate was extracted by chloroform or an admixture of Triton X 100 (0.6%) a surfactant and ethylene diamine tetra acetic acid (EDTA) a chelating agent. Yield of PHA obtained was 49, 41 and 7% of biomass weight after 24, 48 and 72 h of lytic culture fermentation, respectively. Corresponding recovery of the polymer was 94, 82 and 15% of 90% purity. Alternatively Microbispora sp. lytic enzyme was obtained by its cultivation in nutrient broth with S. meliloti cells as substrate and the supernatant was used for the hydrolysis of the PHA containing biomass to release PHA. A620 lytic activity value for the broth was 200 at 72 h. The enzyme showed optimized activity at 50 °C, pH 7 and this was used to hydrolyze 5 g/l of thermally inactivated biomass of S. meliloti to recover 94% of total PHA present in the cells and the polymer produced was 92% pure. Decreased cell lytic activity in the presence of soluble protein added in the form of bovine serum albumin indicated that the hydrolytic activity may be due to proteases. The polymer was characterized by GC, NMR and DSC and was found to be polyhydroxybutyrate-co-hydroxyvalerate (97:3 mol%) with a melt temperature of 169 °C.
Here is another PHA extraction reference...
M. Usman Arshad, Nazia Jamil,Nighat Naheed and Shahida Hasnain. Analysis of bacterial strains from contaminated and non-contaminated sites for the production of biopolymers. African Journal of Biotechnology, Vol. 6, No. 9, 2 May 2007, pp. 1115-1121.
A total 18 strains were collected from non-contaminated and contaminated environments, and were purified. All purified strains were characterized for Gram reaction and biochemical analysis. Screening for bioplastic production was done by Sudan black staining. Strains isolated from non-contaminated sites showed no polyhydroxyalkanoate (PHA) production. Biochemical analysis showed that PHA producing strains belong to Pseudomonas, Citrobacter, Enterobacter, Klebsiella, Escherichia and Bacillus genera. PHA extraction was done by sodium hypochlorite digestion method. Strain MS2D showed maximum percentage (65%). Production of PHA was optimized for different temperatures and pH. Selected strains were also tested for exopolysaccharides (EPS) production on EPS detection medium by solvent precipitation method. Four out of nine strains exhibited EPS production ability. EPS production was also optimized for different temperatures and pH.
Here is the paraphrase PHA extraction method...
PHA extraction was done by sodium hypochlorite digestion method after 72 h on PHA detection medium. Cells were collected by centrifugation at 4000 rpm for 15 min. Biomass of 0.2 g was suspended in 5 ml of 0.4% sodium hypochlorite. After 1 h at 37°C, PHA granules were collected by centrifugation. Palette was washed by acetone and water. The palette was dissolved in chloroform, allow-ed to evaporate, and PHA weight was noted (Arnold et al., 1999).
... and here is the Arnold 1999 reference.
Arnold L, Demain J, Davis E (1999). Polyhydroxyalkanoates. Manual of Microbiology and Biotechnology. Washington, Am Soc. Microbiol. 2:616-627.
Also, if you go to the Google Book Search preview for Chemistry and Technology of Biodegradable Polymers By G. J. L. Griffin
There is a review of PHA isolation at section 4.4 pg 55. The following methods are discussed - solvent extraction (4.4.1), Sodium hypochlorite digestion (4.4.2), and Enzymatic digestion (4.4.3).
And a patent that may be of some interest as well....
A PROCESS FOR THE EXTRACTION OF POLYHYDROXYALKANOATES FROM BACTERIA
LAKSHMAN, Kshama [IN/IN]; Central Food Technological Research Institute, Mysore-570 020, Karnataka (IN).
SHAMALA, Tumkur, Ramachandriah [IN/IN]; Central Food Technological Research Institute, Mysore-570 020, Karnataka (IN).
Polyhydroxyalkanoate (PHA) is gaining importance as an alternative source to synthetic polymers because of similar properties possessed by it compared to synthetic plastics and its biodegradability. The polymer is produced intracellularly and hence various methods are reported for its extraction. In order to minimize the changes in the polymer that occurs during chemical extraction and to avoid multiple enzyme usage, a biological method for the extraction of polyhydroxyalkanoates is proposed, which comprises of cultivation of PHA producing bacteria such as Sinorhizobium meliloti (MTCC 100) in nutrient medium, thermal inactivation of PHA containing cells along with the culture medium at 80° C, digestion of cell materials by growing any actinomycetes culture which can lyse the bacterial cells or by using its culture filtrate, and isolation of the released polymer by the use of water immiscible solvent or an admixture of a surfactant and a chelating agent. This method involves simple and easier extraction of PHA from the cells.
Field of Invention:
The present invention relates to a process for the extraction of polyhydroxyalkanoates from bacteria. The main usage of the invention is in the production and modified extraction of the biopolymer for various packaging applications.
And one more reference...
J. A. Ramsay, E. Berger, B. A. Ramsay and C. Chavarie. Recovery of poly-3-hydroxyalkanoic acid granules by a surfactant-hypochlorite treatment. Biotechnology Techniques. Volume 4, Number 4 / July, 1990. 221-226.
When Alcaligenes eutrophus biomass was treated with a surfactant and then washed with hypochlorite, the recovered poly-3-hydroxyalkanoic acid (PHA) granules were 97 to 98% pure with a molecular weight (MW) between 730,000 and 790,000, depending on the surfactant used. When treated with only surfactant, the MW was slightly higher than that obtained with the surfactant-hypochlorite treatment but the purity was 10% lower. PHA of higher purity but lower MW was obtained with just a hypochlorite treatment.
Ojumu, T.V., Yu, J. and Solomon, B.O. Production of Polyhydroxyalkanoates, a bacterial biodegradable polymer. African Journal of Biotechnology Vol. 3 (1), pp. 18-24, January 2004
There has been considerable interest in the development and production of biodegradable polymer to solve the current problem of pollution caused by the continuous use of synthetic polymer of petroleum origin. Polyhydroxyalkanoates (PHAs) are known to be accumulated as intracellular inclusion in some
bacteria. The materials properties exhibited by PHAs, ranging from stiff, brittle to rubber-like makes it a close substitute for the synthetic plastic. The high cost of PHAs production has restricted its applications. The possibility of producing this polymer commercially and at comparable cost has been
the main focus in this area.
We are planning to setup a lab of 30 lit to produce P(3HB4HB) in Singapore. Can anyone advice us where can we obtain a good micro-organism? Cooperation in any form can be discussed. Please reply to eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%67%35%5f%32%31%40%68%6f%74%6d%61%69%6c%2e%63%6f%6d%22%3e%67%35%5f%32%31%40%68%6f%74%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b')). Thanks.
I am in MSc biotech and involved in a project of making bioplastic from PHA bacterial source.. can any one help me out by giving a standard protocol for that...???