title: Quantification of intracellular polyhydroxyalkanoates by virtue of personalized flow cytometry protocol.
abstract: Polyhydroxyalkanoates (PHAs) are natural polyesters produced by microbes, a potential alternative to synthetic plastics. Various methods ranging from gravimetry to spectrophotometry are routinely used for qualitative analysis of extracted PHA. There is a great need for accurate quantification of intracellular PHA during bioprocess. Hence, the present study aims to improvise the existing Nile red-based flow cytometry protocol. It was achieved using respective cells in a non-PHA accumulating state as gating control to minimize non-specific staining. The optimal Nile red concentration required for PHA staining is 5 × 10(3) pg mL(-1), which is ~10(3)-fold less than that of earlier reports. Further, it was inferred that flow-based quantification was more accurate than the gravimetric method. The intracellular PHA content was highest in Pseudomonas sp. MNNG-S (52.06 %) among the Pseudomonas strains tested by the flow-based method. Both gravimetric and flow-based cell cycle analyses revealed that DNA synthesis (S phase) and PHA production (log phase) are synchronous at 24-48 h of culture. This study supports flow-based PHA quantification for real time online measurement of intracellular PHA for bioreactor monitoring, control and optimization enduing industrial applications.
authors.affiliation: PG and Research Department of Zoology and Biotechnology, Lady Doak College, Madurai, India.
authors.forename: V
authors.initials: V
authors.lastname: Saranya
authors.lastname: Poornimakkani
authors.forename: M S
authors.initials: MS
authors.lastname: Krishnakumari
authors.forename: P
authors.initials: P
authors.lastname: Suguna
authors.forename: C
authors.initials: C
authors.lastname: Binuramesh
authors.forename: P
authors.initials: P
authors.lastname: Abirami
authors.forename: V
authors.initials: V
authors.lastname: Rajeswari
authors.forename: K B
authors.initials: KB
authors.lastname: Ramachandran
authors.forename: R
authors.initials: R
authors.lastname: Shenbagarathai
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D054813:Polyhydroxyalkanoates / Q000032:analysis* / Q000378:metabolism; D011549:Pseudomonas / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D054813:Polyhydroxyalkanoates