Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by Pseudomonas putida KT2440 (original) (raw)
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Applied Microbiology and Biotechnology, 2007
Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h −1 but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ=0.15 h −1 ) produced 70 g l −1 biomass containing 75% PHA. At a higher exponential feed rate (μ=0.25 h −1 ), the overall productivity was increased but less biomass (56 g l −1 ) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbonlimited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.
Journal of Industrial Microbiology and Biotechnology, 2015
A medium-chain-length poly-3-hydroxyalkanote (MCL-PHA) depolymerase knockout mutant of Pseudomonas putida KT2440 was produced by double homologous recombination. A carbon-limited shake-flask study confirmed that depolymerase activity was eliminated. Lysis of both mutant and wild-type strains occurred under these conditions. In carbon-limited, fed-batch culture, the yield of unsaturated monomers from unsaturated substrate averaged only 0.62 mol mol−1 for the phaZ minus strain compared to 0.72 mol mol−1 for the wild type. The mutant strain also produced more CO2 and less residual biomass from the same amount of carbon substrate. However, most results indicated that elimination of PHA depolymerase activity had little impact on the overall yield of biomass and PHA.
Polyhydroxyalkanoate production in Pseudomonas putida from alkanoic acids of varying lengths
PLOS ONE, 2023
Many studies have been conducted to produce microbial polyhydroxyalkanoates (PHA), a biopolymer, from Pseudomonas sp. fed with various alkanoic acids. Because this previous data was collected using methodologies that varied in critical aspects, such as culture media and size range of alkanoic acids, it has been difficult to compare the results for a thorough understanding of the relationship between feedstock and PHA production. Therefore, this study utilized consistent culture media with a wide range of alkanoic acids (C7-C14) to produce medium chain length PHAs. Three strains of Pseudomonas putida (NRRL B-14875, KT2440, and GN112) were used, and growth, cell dry weight, PHA titer, monomer distribution, and molecular weights were all examined. It was determined that although all the strains produced similar PHA titers using C7-C9 alkanoic acids, significant differences were observed with the use of longer chain feedstocks. Specifically, KT2440 and its derivative GN112 produced higher PHA titers compared to B-14875 when fed longer chain alkanoates. We also compared several analytical techniques for determining amounts of PHA and found they produced different results. In addition, the use of an internal standard had a higher risk of calculating inaccurate concentrations compared to an external standard. These observations highlight the importance of considering this aspect of analysis when evaluating different studies.
Journal of Industrial Microbiology & Biotechnology, 2015
commercial interest due to their biocompatibility and biodegradability [14, 18]. PHAs are classified as shortchain-length PHAs (SCL-PHAs) when the pendant group of monomer varies from 0 to 2 carbons, medium-chainlength PHAs (MCL-PHAs) when there are three or more carbons on the pendant group and SCL-MCL-PHAs when polymers consist of both SCL and MCL monomers [19, 22]. MCL-PHAs are of increasing interest because of their low crystallinity, high elasticity [13, 25] and the possibility of having different functional groups, such as alkenes [21], aromatic groups [12], halogen [9, 16, 20], esters [30] and phenoxy groups [27] on their side chains. Since more than 100 different MCL-PHA monomers can be incorporated, MCL-PHAs can exhibit a wide variety of properties with many possible applications including coatings, medical implants, drug delivery, water-based latex paints and others [39, 40]. PHAs are more expensive to produce than conventional plastics with expenditures almost evenly divided between carbon source, fermentation process and separation process [37]. Metabolic engineering may be used to achieve higher PHA cellular content, more effective carbon source usage, and to obtain novel PHAs with valuable properties, thus increasing commercially viability. One such approach is the elimination of PhaZ activity. The phaZ gene encoding PhaZ is located between phaC1 and phaC2 genes of the MCL-PHA metabolism gene cluster which consists of phaC1, phaZ, phaC2, phaD, phaF and phaI genes in Pseudomonas putida KT2440 whose entire genome has been mapped [17, 24]. The PhaZ of P. putida KT2442, a spontaneous rif r mutant of P. putida KT2440 [2], is an intracellular MCL-PHA depolymerase and an MCL-PHA granule surface-associated protein [7, 8]. PhaZ is reported to play a crucial role in the turnover of MCL-PHAs under carbon starvation in P. putida KT2442 [5].
Studies on polyhydroxyalkanoates biosynthesis by some Pseudomonas spp. strains
Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are naturally produced by bacteria and accumulates in cytoplasm in the form of granules, in particular culture broth conditions. PHAs are biodegradable, biocompatible and have useful mechanical properties that recommend them for divers applications in various fields. In order to obtain mcl-PHAs of microbial origin we used two Pseudomonas spp. strains, namely Pseudomonas putida ICCF 391 and Pseudomonas fluorescens ICCF 392. Researches have focused the ability of these two strains to use structurally related or not related substrates, to obtain biopolymers with controlled composition, and growth the amount of PHAs in reproducible conditions. Moreover, bioprocess conditions for mcl-PHAs biosynthesis, fermentation broth processing, and polymers composition-were reproducible. As the results achieved with the two strains were similar, researches continued with Pseudomonas fluorescens strain, which is less studied regarding the potential of PHA biosynthesis. Were carried out (co) polymer films containing more than 85% PHO, as determined by GC-FID.
Bioengineering, 2019
High cell density (HCD) fed-batch cultures are widely perceived as a requisite for high-productivity polyhydroxyalkanoate (PHA) cultivation processes. In this work, a reactive pulse feed strategy (based on real-time CO2 or dissolved oxygen (DO) measurements as feedback variables) was used to control an oxygen-limited fed-batch process for improved productivity of medium chain length (mcl-) PHAs synthesized by Pseudomonas putida LS46. Despite the onset of oxygen limitation half-way through the process (14 h post inoculation), 28.8 ± 3.9 g L−1 total biomass (with PHA content up to 61 ± 8% cell dry mass) was reliably achieved within 27 h using octanoic acid as the carbon source in a bench-scale (7 L) bioreactor operated under atmospheric conditions. This resulted in a final volumetric productivity of 0.66 ± 0.14 g L−1 h−1. Delivering carbon to the bioreactor as a continuous drip feed process (a proactive feeding strategy compared to pulse feeding) made little difference on the final vo...
BMC Biotechnology, 2012
Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. The genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h(-1) and a final cell dry weight (CDW) of 2.5 g L(-1) with a maximal yield of 0.5 g CDW g(-1) xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w(-1) of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.