Growth of Pseudomonas oleovorans GPol in continuous culture containing a bulk n-octane phase resulted in changes of the fatty acid composition of the membrane lipids. Compared to citrate-grown cells, the ratio of C18 to C16 fatty acids and the ratio of unsaturated to saturated fatty acids increased as a result of growth on octane. Trans-unsaturated fatty acids, which are rarely found in bacteria, were formed during continuous growth of P. oleovorans on octane. Moreover, the mean acyl chain length and unsaturated fatty acids also increased as the growth rates increased both in octane-grown and citrate-grown cells. Differential scanning calorimetry measurements of extracted lipids showed the transition temperature of membrane lipids from octane-grown cells increased from about 24°C to 32°C as the growth rate increased, whereas cells grown on citrate showed a constant transition temperature of about 6°C at all growth rates tested, indicating a decrease of membrane lipid fluidity in octane-grown cells. Because alkanes are known to increase bilayer fluidity by intercalating between lipid fatty acyl chains, the increased transition temperature of the lipids of cells grown on octane may be a physiological response of P. oleovorans to compensate for the direct effects of octane on its cellular membranes.

Poly((R)-3-hydroxyalkanoate)s (PHAs) are bacterial storage polyesters, currently receiving much attention because of their potential application as biodegradable and biocompatible plastics. Among them are the PHAs from Pseudomonas oleovorans, which are semicrystalline elastomers. Their applicability is seriously limited by their low melting temperature as well as by their low crystallization rate. Both problems were overcome by crosslinking of unsaturated pendent groups, which were incorporated in the polymer by tailoring the carbon source for biosynthesis. Crosslinking was established by electron-beam irradiation and resulted in a true rubber with constant properties over a large temperature range from -20 to +170-degrees-C. Even after crosslinking, the material was still biodegradable. To our knowledge this is the first microbially produced biodegradable rubber.

When Pseudomonas oleovorans is continuously cultured on a two-phase medium consisting of an aqueous minimal salts medium phase with growth-limiting amounts of ammonium (16.7 mm) and an n-octane phase as carbon and energy source, the cells reach a steady-state density of about 2–3 g l−1 and accumulate a storage compound, poly(3-hydroxyalkanoate) (PHA), the amount of which depends on the dilution rate used. The PHA productivity is maximal at a dilution rate of 0.20 h−1 and yields 0.17 g PHA l−1 h−1. To improve the PHA productivity of this two-liquid-phase chemostat, the density of PHA-accumulating cells growing at a rate of 0.20 h−1 was raised by increasing the concentration of the limiting nutrient ammonium in the medium feed in discrete steps from 16.7 to 116.6 mm. After medium optimization with retention of ammonium-limiting conditions and an increase of oxygen transfer rates (the stirrer speed was increased from 1000 to 1800 rev min−1 and the airflow from 200 to 550 ml min−1), the cell density could be raised to 11.6 g l−1 with a PHA productivity of 0.58 g PHA l−1 h−1. P. oleovorans remained stable with respect to PHA formation in these two-liquid phase continuous cultures for at least 1 month: cells that were isolated after 760 h of continuous growth (about 150 doublings) were phenotypically similar to the cells that were used to inoculate the long-term cultivation.

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour.

Escherichia coli is able to grow on sugars in the presence of a bulk n-alkane phase. When E. coli is equipped with the alk genes from Pseudomonas oleovorans, the resulting recombinant strain converts n-alkanes into the corresponding alkanoic acids. To study the effects of growth rate and exposure to a bulk apolar phase on the physiology and the productivity of E. coli, we have grown this microorganism in two-liquid-phase continuous cultures containing 5% (v/v) n-octane.In contrast to batch cultures of wild-tape E. coli grown in the presence of n-octane, cells remained viable during the entire continuous culture, which lasted 200 h. Bioconversion of n-octane to n-octanoic acid by a recombinant E. coli (alk(+)) in a two-liquid-phase continuous culture was made possible by optimizing both the recombinant host strain and the conditions of culturing the organism. Continuous production in such two-phase systems has been maintained for the least 125 h without any changes in the product concentration in the fermentation medium. The volumetric productivity was determined as a function of growth rate and showed a maximum at a dilution rate D = 0.32 h(-1), reaching a continuous production rate of 0.5 g octanoate/L . h (4 tons/m(3) . year).

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHAm). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHAm and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHAm and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHAm and PHB accumulating strains have shown that PHAm and PHB granules can be distinguished from each other: PHAm granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHAm and PHB chains were stored in separate granules. To be able to determine whether the separation of PHAm and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHAm granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHAm and PHB, the resulting polymer chains are segregated completely and stored in separate granules.

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (Mw), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA− mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the Mw of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.

In this paper we describe the biosynthesis of a series of 1-alkanols from the corresponding n-alkanes. To this end, we introduced the alkane hydroxylation system of Pseudomonas oleovorans into Pseudomonas putida PpS81 (alcA81). The resulting recombinant strain PpS8141 can oxidize n-alkanes to the corresponding 1-alkanols but is unable to utilize the alkanols, which therefore accumulate in the medium.PpS8141 was grown in two-liquid-phase bioreactors. The different medium-chain-length alkanes formed a bulk organic phase, which constituted 20% of the total volume. The aqueous phase contained the growth substrate citrate, octanoate, or pyruvate. PpS8141 was able to grow well in the presence of a bulk apolar phase only when pyruvate was used as the growth substrate. Hexane was toxic to this strain and did not allow growth. The strain oxidized C7-C-11 linear alkanes to 1-alkanols with production rates superior to the wild-type P. oleovorans rates. With n-octane and n-nonane as organic phase and substrate, we found the highest production rates of 1-alkanol. The growth rate in the exponential phase and the maximal 1-alkanol production rate showed an inverse relationship, which we interpret to be the result of the metabolic burden of the conversion.The results show that the recombinant strain PpS8141 may allow the biotechnological production of primary aliphatic alcohols.

Pseudomonas oleovorans is able to grow on linear aliphatic hydrocarbons of medium chain length as sole energy and carbon source. When nitrogen, sulfur, or magnesium is limiting, P. oleovorans produces an intracellular polyester poly(beta-hydroxyalkanoate) (PHA) from the excess alkanoic acid formed from the alkanes supplied in the medium. To study the effect of growth rate and exposure to bulk amounts of n-octane on the physiology and morphology of P. oleovorans, we have established continuous cultures of this organism in two-liquid phase media containing about 15% (v/v) n-octane. P. oleovorans was grown in an ammonium-limited single-stage chemostat at growth rates varying from D = 0.05 to D = 0.46 h-1.In contrast to batch cultures of P. oleovorans grown on n-octane, both rapidly and slowly growing cells remained fully viable during the entire continuous culture experiments, which typically lasted 200-300 h. The cellular morphology of these cells was studied as a function of time by freeze-fracture electron microscopy, which provided information on changes in membrane ultrastructure and revealed large and small PHA granules in slowly and rapidly growing cells, respectively.The cell density, cellular protein content, and PHA content were determined as a function of growth rate. The cell density decreased from 2.25 to 1.32 mg ml-1, while the PHA content of the cells decreased from 46.7% to 8.3% of the total cell dry weight when the dilution rate (= growth rate) increased from 0.09 to 0.46 h-1. The rest biomass concentration, defined as the difference between total biomass and PHA, was almost independent of the cellular growth rate. The cellular protein content relative to the rest biomass increased from 24% to 46% when the growth rate increased from 0.09 to 0.46 h-1, indicating that rapidly growing cells contain more protein than slowly growing cells, which correlates well with the qualitative data of the electron micrographs.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPhysical characteristics of poly(3-hydroxyalkanoates) and poly(3-hydroxyalkenoates) produced by Pseudomonas oleovorans grown on aliphatic hydrocarbonsHans Preusting, Atze Nijenhuis, and Bernard WitholtCite this: Macromolecules 1990, 23, 19, 4220–4224Publication Date (Print):September 1, 1990Publication History Published online1 May 2002Published inissue 1 September 1990https://pubs.acs.org/doi/10.1021/ma00221a007https://doi.org/10.1021/ma00221a007research-articleACS PublicationsRequest reuse permissionsArticle Views376Altmetric-Citations129LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Pseudomonas oleovorans grows on C 6 to C 12 n -alkanes and 1-alkenes. These substrates are oxidized to the corresponding fatty acids, which are oxidized further via the β-oxidation pathway, yielding shorter fatty acids which have lost one or more C 2 units. P. oleovorans normally utilizes β-oxidation pathway intermediates for growth, but in this paper we show that the intermediate 3-hydroxy fatty acids can also be polymerized to intracellular poly-( R )-3-hydroxyalkanoates (PHAs) when the medium contains limiting amounts of essential elements, such as nitrogen. The monomer composition of these polyesters is a reflection of the substrates used for growth of P. oleovorans . The largest monomer found in PHAs always contained as many C atoms as did the n -alkane used as a substrate. Monomers which were shorter by one or more C 2 units were also observed. Thus, for C-even substrates, only C-even monomers were found, the smallest being ( R )-3-hydroxyhexanoate. For C-odd substrates, only C-odd monomers were found, with ( R )-3-hydroxyheptanoate as the smallest monomer. 1-Alkenes were also incorporated into PHAs, albeit less efficiently and with lower yields than n -alkanes. These PHAs contained both saturated and unsaturated monomers, apparently because the 1-alkene substrates could be oxidized to carboxylic acids at either the saturated or the unsaturated ends. Up to 55% of the PHA monomers contained terminal double bonds when P. oleovorans was grown on 1-alkenes. The degree of unsaturation of PHAs could be modulated by varying the ratio of alkenes to alkanes in the growth medium. Since 1-alkenes were also shortened before being polymerized, as was the case for n -alkanes, copolymers which varied with respect to both monomer chain length and the percentage of terminal double bonds were formed during nitrogen-limited growth of P. oleovorans on 1-alkenes. Such polymers are expected to be useful for future chemical modifications.

Pseudomonas oleovorans grows on C6toC12n-alkanes and1-alkenes. Thesesubstrates areoxidized tothe corresponding fatty acids, which areoxidized further viathe,8-oxidation pathway, yielding shorter fatty acids whichhavelost one or more C2units. P.oleovorans normally utilizes ,8-oxidation pathway intermediates for growth, butinthis paper we showthattheintermediate 3-hydroxy fatty acids can alsobepolymerized to intracellular poly-(R)-3-hydroxyalkanoates (PHAs)whenthemediumcontains limiting amountsofessential elements, suchasnitrogen. Themonomer composition ofthese polyesters isa reflection ofthesubstrates used forgrowth ofP.oleovorans. Thelargest monomer foundinPHAsalways contained asmany Catomsasdidthe n-alkane used asasubstrate. Monomerswhich wereshorter byoneormore C2units werealso observed. Thus, forC-evensubstrates, onlyC-even monomers were found, thesmallest being(R)-3-hydroxyhexanoate. For C-oddsubstrates, onlyC-oddmonomers werefound, with(R)-3-hydroxyheptanoate asthesmallest monomer. 1-Alkenes werealso incorporated into PHAs,albeit less efficiently andwithloweryields thann-alkanes. These PHAscontained bothsaturated andunsaturated monomers,apparently because the1-alkene substrates could beoxidized tocarboxylic acids ateither thesaturated or theunsaturated ends.Up to55% ofthePHA monomers contained terminal double bondswhenP.oleovorans was grown on 1-alkenes. Thedegree of unsaturation ofPHAscouldbemodulated byvarying theratio ofalkenes toalkanes inthegrowth medium. Since 1-alkenes were also shortened before being polymerized, aswasthecaseforn-alkanes, copolymers which varied withrespect toboth monomer chain length andthepercentage ofterminal double bondswere formed during nitrogen-limited growth ofP.oleovorans on 1-alkenes. Suchpolymers areexpected tobeuseful for future chemical modifications. Manybacteria areable toaccumulate intracellular reserve materials, varying frominternal n-alkane pools topolyphos- phate (20, 22). Mostcommonispoly-3-hydroxybutyric acid