Accumulation of Poly[( R )-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Abstract

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C 0 /N 0 ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH 4 ؉ in excess at low C 0 /N 0 ratios, (ii) purely nitrogen-limited growth conditions at high C 0 /N 0 ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C 0 /N 0 ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C 0 /N 0 ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C 0 /N 0 ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C 0 /N 0 ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.Pseudomonas oleovorans is able to accumulate mediumchain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs) as intracellular carbon and energy storage compounds when grown with alkanes (9, 24), mcl-alkanoates (3, 21), n-alkanols (16), or many derivatives thereof (27,38). mcl-PHAs are hydrophobic polyesters and are attracting increasing interest for their possible application as biologically produced and inherently biodegradable substitutes for conventional plastic materials (6,8,10,20,32). Particularly, when subjected to nitrogen limitation in batch (22, 24) and chemostat culture (17,33,34), P. oleovorans has been reported to accumulate high cellular mcl-PHA contents.The nature and availability of essential nutrients are important parameters in determining the extent of storage compound accumulation in P. oleovorans (23,24). Generally, the production of microbial cell mass is limited by the restricted availability of a particular nutrient. In batch culture, the exhaustion of a specific nutrient terminates the exponentialgrowth phase, whereas in chemostat culture the biomass concentration is usually controlled by the permanent limitation of a single defined nutrient, typically the carbon and energy source. However, several authors have shown that two or more nutrients can simultaneously limit the production of biomass (for a summary, see reference 14). This phenomenon has also been observed for P. oleovorans, where we have found that under defined chemostat steady-state cultivation c...

Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] byPseudomonas putida GPo1: Batch versus chemostat production

“…MclPHA can be accumulated by P. putida GPo1 in chemostat cultures during dual nutrient (C,N) limited growth, i.e., during simultaneous nitrogen (N) and carbon (C) limitation (Durner et al, 2000;Hartmann et al, 2004;Zinn et al, 2003). Durner et al (2000) defined a so called dual nutrient (C,N) limited growth regime (DNLGR) and showed it to be a function of the dilution rate and the carbon to nitrogen ratio (C 0 /N 0 ) of the medium feed (see Fig.…”

“…Such conditions can be achieved in batch or fed-batch cultures (Brandl et al, 1988;Gross et al, 1989;Preusting et al, 1993). Recently, it has also been shown in chemostat cultures of P. putida GPo1 that PHA production can take place when nitrogen and carbon are limiting growth simultaneously (dual nutrient (C,N) limited growth) (Durner et al, 2000;Zinn et al, 2003). Various mclPHAs with side chains containing functional groups, for example carboncarbon double (Lageveen et al, 1988) and triple bonds (Kim et al, 1998), acetoxy and ketone (Jung et al, 2000), or aromatic groups (Curley et al, 1996;Fritzsche et al, 1990;Kim et al, 1999) have been produced.…”

Production of medium-chain-length polyhydroxyalkanoate by Pseudomonas oleovorans grown in sugary cassava extract supplemented with andiroba oil

“…In general, the biomass yield coefficient (Y X/S ) were low compared to the values found by Durner et al (2000) (0.62 to 0.97 g X /g S ), who studied poly[(R)-3-hydroxyalkanoates] accumulation in P. oleovorans using octanoate as a substrate. The performance observed here can be attributed to the metabolic flux that is related to PHA production with respect to the use of cellular energy or as a result of physical impediments by the intracellular granules formed preventing cell division, which contributed to low biomass production.…”

“…Bacteria of the genus Pseudomonas, which belongs to ribosomal RNA homology group I, are able to synthesize PHA using a variety of carbon sources, including glucose, fructose and sucrose (Diniz et al, 2004;Santhanam & Sasidharan, 2010;Davis et al, 2013), n-octane and octanoate (Smet et al, 1983;Durner et al, 2000), and fatty acids (Du & Yu, 2002;Allen et al, 2010), among others.…”

Optimization and Characterization of Poly[R]hydroxyalkanoates of Pseudomonas aeruginosa

“…The organism was found to accumulate higher percentage of PHA in glucose containing medium. PHA producing bacteria can be divided into two groups based on the culture media .In the first group ,the bacteria accumulates PHA at exponential phase only if there is a limited amount of nutrients (eg, Pseudomonas oleovorans) 19 .The second group, accumulates PHA during growth and it does not requires nutrient limitation(e.g. recombinant E.coli) 20 .…”