title: Optimization of intracellular product release from Neisseria denitrificans using microfluidizer.
abstract: Disruption of Neisseria denitrificans cells by microfluidizer was optimized using a factorial experiments design. The pH, pretreatment time, cell concentration, NaCl, ethylenediamine tetraacetic acid (EDTA) and Triton X-100 concentrations showed significant impact on disruption process and the process was optimized using central composite design and response surface methodology (RSM). Investigation revealed optimum conditions: 90 min pretreatment at pH 9.0 containing 110 g L(-1) cells (dry cell weight), 50 mM NaCl, 10 mM EDTA, and 0.2% Triton X-100. At optimized conditions, the disruption rate increased twofold, up to 5.62 ± 0.27 × 10(-3) MPa(-a); meanwhile, yield of intracellular content was increased by 26%, with 1 g of cells resulting in 113.2 ± 8.2 mg proteins, 12.1 ± 0.7 mg nucleic acids, 21.0 ± 1.2 mg polysaccharides, 0.99 ± 0.08 kU glucose-6-phosphate dehydrogenase (G6PD), and 10,100 ± 110 kU restriction endonuclease NdeI endonuclease. Particle size distribution analysis revealed nearly twofold larger cell lysate particles with diameter of 120 nm. For optimal release of intracellular content, 9200 J/g of energy was needed (95% confidence), yielding 6900 J/g energy savings. Model equations generated from RSM on cell disruption of N. denitrificans were found adequate to determine significant factors and its interaction. The results showed that optimized combination of known pretreatment and disruption methods could considerably improve cell disruption efficiency.
authors.affiliation: a Department of Chemical Technology , Kaunas University of Technology , Kaunas , Lithuania.
authors.forename: Robert
authors.initials: R
authors.lastname: Stupak
authors.forename: Nerijus
authors.initials: N
authors.lastname: Makauskas
authors.forename: Kostas
authors.initials: K
authors.lastname: Radzevičius
authors.forename: Zenonas
authors.initials: Z
authors.lastname: Valančius
mesh_terms: D003470:Culture Media / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D044085:Microfluidics; D009343:Neisseria / Q000737:chemistry*; D017830:Octoxynol; D013696:Temperature
chemical_list: D003470:Culture Media; D017830:Octoxynol
keywords: Neisseria denitrificans; cell disruption; central composite design; high-pressure homogenizer; microfluidizer; response-surface methodology