Development of a rapid method to isolate polyhydroxyalkanoates from bacteria for screening studies

Abstract

We describe a novel method of Polyhydroxyalkanoate (PHA) extraction using dimethyl sulphoxide (DMSO) for use in screening studies. Compared to conventional chloroform extraction, the DMSO method was shown to release comparable quantities of PHA from Cupriavidus necator cells, with comparable properties as determined using Fourier transform infrared spectroscopy and differential scanning calorimetry.

Flow cytometry as a rapid analytical tool to determine physiological responses to changing O2 and iron concentration by Magnetospirillum gryphiswaldense strain MSR-1

“…3 . Similarly to our findings, recent studies performed with Cupriavidus necator observed sub-populations with more and less PHA 47 . Other works with Pseudomonas putida have recently observed an asymmetric PHA distribution during cell division under carbon limiting conditions suggesting that this could be explained by different cellular growth rates, distinct ability to degrade PHA or uneven distribution of PHA granules to daughter cells 49 .…”

“…Here we have used FCM to investigate PHA accumulation within individual bacteria of starved non-magnetic and magnetic MTB cultures. Cells were stained with the lipophilic dye 1,3,5,7,8-pentamethylpyrromethene-difluoroborate complex (pyrromethene-546 or Pyr-546) which on entering bacteria stains PHA green 47 . Previous studies have shown that Pyr-546 fluorescence correlates to intracellular PHA content 48 and is superior to Nile red as a dye for PHA.…”

Bacterial Strain Isolated from High-Salt Environments Can Produce Large Amounts of New Polyhydroxyalkanoate (PHA)

“…The PHA-positive isolates opted after Nile blue A staining and then grown in an E2 broth medium containing 2% (w/v) glucose in 100-ml flasks, and were employed to extract PHA after two days of incubation on a rotary shaker. The PHA from the isolates was extracted by the chloroform method, developed by Vizcaino-Caston et al (35). The concentration of PHA in the E2 medium and PHA% of cell dry weight along with cell dry weight achieved for various positively stained isolates are depicted in Table 2.…”

Development of a simple intensified fermentation strategy for growth of Magnetospirillum gryphiswaldense MSR-1: Physiological responses to changing environmental conditions

“…2 ) revealed that cells fed with different concentrations of lactic acid contained differing amounts of PHA granules, FCM was used to determine cellular PHA accumulation by staining of the cells with the green lipophilic dye Pyrromethene-546 (Pyr546; Fig. 4 d) [ 37 , 38 ]. As with the FSC and SSC data ( Fig.…”