Glutamine-induced filamentous cells of Pseudomonas mediterranea CFBP-5447T as producers of PHAs
Polyhydroxyalkanoates (PHAs) are considerable biopolymers that have gained an increasing biotechnological interest in different applications, although their industrial production presents several limitations. Filamentous bacterial cells could represent a possible strategy to increase PHA yield, since more abundant PHA inclusions can be stored in elongated than in rod-shaped cells. At first, we determined the optimal batch culture conditions to induce filamentation in Pseudomonas mediterranea CFBP-5447T, using glutamine, glycerol, glucose, and sodium octanoate, as the sole carbon source, at low- (100 rpm) or high- (250 rpm) shaking speeds. Successively, a fermentative process was set up using glutamine in a co-metabolic strategy with glycerol, and the PHAs production was compared in rod-shaped and filamentous cells. High glutamine concentrations (from 28 to 56 mM) were able to induce alone filamentation, whereas at lower glutamine concentrations (5–10 mM), the shaking speeds became critical to allow or not filamentous phenotype. PHA granule production was higher in filamentous than in rod-shaped cells, when glycerol (46.6 mM) was added to glutamine (5 mM) in co-metabolism, and fermentation was performed at a low-shaking speed. After extraction and precipitation, PHA yield was about two times higher in filamentous than that rod-shaped cells. Our results provide new insights into filament-inducing conditions and indicate a potential use of filamentous P. mediterranea CFBP-5447T cells to increase PHA yield. These findings could have great advantages in PHAs recovering during downstream processes, since the harvesting of elongated cells is much less time-consuming and energy expensive than required with rod-shaped cells.
KeywordsPseudomonas mediterranea Glutamine Filamentous cells Medium-chain-length polyhydroxyalkanoates (mcl-PHAs)
The strain Pseudomonas mediterranea CFBP-5447T was a kind gift by Science and Technology Park of Sicily (PSTS). We express our gratitude to Professor Miguel Martinez of Universidad de Concepción (Chile) for providing valuable suggestions and discussions.
This work has been partially funded by the Italian Ministry of University and Research (MIUR) by means of the National Program PON R&C 2007–2013, Project “PolyBioPlast – Technologies and processes for the production of diversely functionalized sheets based on microbial biopolymers and biosurfactants (PON01_1377)”.
Compliance with ethical standards
The presented research did not involve studies with human participants or animal subjects or recombinant DNA.
Conflict of interest
The authors declare that they have no conflict of interest.
- Clark G (1981) Staining procedures. In: Clark G (ed) Biological Stain Commission (U.S.), 4th edn. Williams & Wilkins, BaltimoreGoogle Scholar
- Dinamarca MA, Aranda-Olmedo I, Puyet A, Rojo F (2003) Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals evidence from continuous cultures. J Bacteriol 185:4772–4778. https://doi.org/10.1128/jb.185.16.4772-4778.2003 CrossRefPubMedPubMedCentralGoogle Scholar
- Galán B, Dinjaski N, Maestro B, de Eugenio LI, Escapa IF, Sanz JM, García JL, Prieto MA (2011) Nucleoid-associated PhaFphasin drives intracellular location and segregation of polyhydroxyalkanoate granules in Pseudomonas putida KT2442. Mol Microbiol 79(2):402–418. https://doi.org/10.1111/j.1365-2958.2010.07450.x CrossRefPubMedGoogle Scholar
- Licciardello G, Bella P, Devescovi G, Strano CP, Sarris PF, Catara AF, Venturi V, Catara V (2014) Draft genome sequence of Pseudomonas mediterranea strain CFBP 5447T, a producer of filmable medium-chain-length polyhydroxyalkanoates. Genome Announc 2(6):e01260–e01214. https://doi.org/10.1128/genomeA.01260-14 CrossRefPubMedPubMedCentralGoogle Scholar
- Mattick KL, Jorgensen F, Legan JD, Cole MB, Porter J, Lappin-Scott HM, Humphrey TJ (2000) Survival and filamentation of Salmonella entericaserovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 at low water activity. Appl Environ Microbiol 66(4):1274–1279. https://doi.org/10.1128/aem.66.4.1274-1279.2000 CrossRefPubMedPubMedCentralGoogle Scholar
- Mohanty AK, Misra M, Hinrichsen G (2000) Biofibres, biodegradable polymer and composites: an overview. Macromol Mater Eng 276/277:1–24. https://doi.org/10.1002/(SICI)1439-2054(20000301)276:1<1::AID-MAME1>3.0.CO;2-W
- Morales G, Ugidos A, Rojo F (2006) Inactivation of the Pseudomonas putida cytochrome o ubiquinol oxidase leads to a significant change in the transcriptome and to increased expression of the CIO and cbb3-1 terminal oxidases. Environ Microbiol 8(10):1764–1774. https://doi.org/10.1111/j.1462-2920.2006.01061.x CrossRefPubMedGoogle Scholar
- Nicolò MS, Franco D, Camarda V, Gullace R, Rizzo MG, Fragalà M, Licciardello G, Catara AF, Guglielmino SPP (2014) Integrated microbial process for bioconversion of crude glycerol from biodiesel into biosurfactants and PHAs. Chem Eng Trans 38:187–192. https://doi.org/10.3303/CET1438032 CrossRefGoogle Scholar
- Pappalardo F, Fragalà M, Mineo PG, Damigella A, Catara AF, Palmeri R, Rescifina A (2014) Production of filmable medium-chain-length polyhydroxyalkanoates produced from glycerol by Pseudomonas mediterranea. Int J Biol Macromol 65:89–96. https://doi.org/10.1016/j.ijbiomac.2014.01.014 CrossRefPubMedGoogle Scholar
- Sicard D, Cecioni S, Iazykov M, Chevolot Y, Matthews SE, Praly JP, Souteyrand E, Imberty A, Vidal S, Phaner-Goutorbe M (2011) AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL) filaments induced by multivalent glycoclusters. Chem Commun (Camb) 47(33):9483–9485. https://doi.org/10.1039/c1cc13097h CrossRefGoogle Scholar
- Steinbuchel A, Valentin HE (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128:219–228. https://doi.org/10.1111/j.1574-6968.1995.tb07528.x CrossRefGoogle Scholar