Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans.

Pseudomonas resinovorans produced poly-beta-hydroxyalkanoates (PHAs) when grown on hydrocarbons but not on glucose. In a chemostat culture, the PHA composition was beta-hydroxybutyrate (C4)-beta-hydroxyhexanoate (C6)-beta-hydroxyoctanoate (C8)-beta-hydroxydecanoate (C10) (1:15:75:9) on octanoate and C4-C6-C8-C10 (8:62:23:7) on hexanoic acid. Contrary to the reported behavior of Pseudomonas oleovorans, the PHA accumulation rate increased under ammonium limitation on octanoate.     

Continuous Production of Long-Side-Chain Poly-beta-Hydroxyalkanoates by Pseudomonas oleovorans

Shake flask experiments showed that Pseudomonas oleovorans began to be growth inhibited at 4.65 g of sodium octanoate liter, with total inhibition at 6 g liter. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter in the feed, the maximum specific growth rate was 0.51 h, and the maximum specific rate of poly-beta-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein h at a dilution rate (D) of 0.25 h. When the specific growth rate (mu) was <0.3 h, the PHA composition was relatively constant with a C(4)/C(6)/C(8)/C(10) ratio of 0.1:1.7:20.7:1.0. At mu > 0.3 h, a decrease in the percentage of C(8) with a concomitant increase in C(10) monomers as mu increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At D = 0.24 h and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate consumed, the yield of PHA was 0.085 g of PHA g of octanoate used, and 7.9 g of octanoate was consumed for each gram of NH(4) supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass h. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.     

Kinetics of medium-chain-length polyhydroxyalkanoate production by a novel isolate of Pseudomonas putida LS46

Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P.?putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P.?putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12?h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48?h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas?putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C(6)-C(14) saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20?mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1-68.8?mol%) in P.?putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88?mol%).     

Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseudomonas putida

The ability of Pseudomonas putida to synthesize polyhydroxyalkanoate (PHA) from 36 different carboxylic acids containing various functional groups was examined. This bacterium did not utilize short carboxylic acids (C(4)-C(6)) containing bromine, methoxy, ethoxy, cyclohexyl, phenoxy, and olefin groups as the sole carbon substrate. No polymer was isolated from the cells grown with carboxylic acids bearing hydroxyl, amino, para-methoxyphenoxy, and para-ethoxyphenoxy groups regardless of the carbon substrate chain lengths used even when they were cofed with nonanoic acid. Of all the carbon substrates evaluated, only 6-para-methylphenoxyhexanoic acid, 8-para-methylphenoxyoctanoic acid, 8-meta-methylphenoxyoctanoic acid, 10-undecenoic acid, and 10-undecynoic acid supported both growth and the production of PHA containing the corresponding functional groups by P. putida. The present results indicate that the carbon availability of P. putida for growth and PHA production is significantly different from that of P. oleovorans.     

Continuous Production of Long-Side-Chain Poly-β-Hydroxyalkanoates by Pseudomonas oleovorans

Shake flask experiments showed that Pseudomonas oleovorans began to be growth inhibited at 4.65 g of sodium octanoate liter^-1, with total inhibition at 6 g liter^-1. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter^-1 in the feed, the maximum specific growth rate was 0.51 h^-1, and the maximum specific rate of poly-β-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein^-1 h^-1 at a dilution rate (D) of 0.25 h^-1. When the specific growth rate (μ) was <0.3 h^-1, the PHA composition was relatively constant with a C₄/C₆/C₈/C₁₀ ratio of 0.1:1.7:20.7:1.0. At μ > 0.3 h^-1, a decrease in the percentage of C₈ with a concomitant increase in C₁₀ monomers as μ increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At D = 0.24 h^-1 and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate^-1 consumed, the yield of PHA was 0.085 g of PHA g of octanoate^-1 used, and 7.9 g of octanoate was consumed for each gram of NH₄⁺ supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass^-1 h^-1. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.     

Production of polyhydroxyalkanoates by Pseudomonas nitroreducens

A strain coded AS 1.2343 was isolated from oil-contaminated soil in an oil-field in North China Tianjian City and it was identified as Pseudomonas nitroreducens. The strain demonstrated some unusual ability to synthesize polyhydroxybutyrate (PHB) homopolymer from medium-chain-length (mcl) fatty acids including hexanoate and octanoate. While polyhydroxyalkanoates (PHA) consisting of mcl hydroxyalkanoate (HA) monomers such as hydroxyoctanoate (HO) and hydroxydecanoate (HD) were the major compositions when butyrate, decanoate, lauric acid and tetradecanoic acid were used as substrates for the cell growth, respectively. PHA was accumulated up to 77% of the cell dry weight when growth was conducted in lauric acid, it appeared that the HA contents in the PHA would not be much affected by the changing of the lauric acid concentration. Varying the concentration ratio of butyrate to octanoate could change the composition of PHA accumulated by the strain. Yet PHB homopolymer was always the only polyester synthesized by the strain, regardless of the octanoate concentration change. Additionally, the ratio of carbon to nitrogen (C/N) in butyrate media was found to have effects on the PHA monomer content, as C/N increased from 2 to 100, content of HB decreased from 100% to 7%. PHA polyester synthesized by cells of Pseudomonas nitroreducens AS 1.2343 was a blend polymers consisting of acetone-insoluble HB and acetone-soluble mcl HA monomers.     

Chemical characterization and physical and biological activities of rhamnolipids produced by Pseudomonas aeruginosa BN10

Pseudomonas aeruginosa BN10 isolated from hydrocarbon-polluted soil was found to produce rhamnolipids when cultivated on 2% glycerol, glucose, n-hexadecane, and n-alkanes. The rhamnolipids were partially purified on silica gel columns and their chemical structures elucidated by combination of one- and two-dimensional 1H and 13C NMR techniques and ESI-MS analysis. Eight structural rhamnolipid homologues were identified: Rha-C10-C8, Rha-C10-C10, Rha-C10-C12:1, Rha-C10-C12, Rha2-C10-C8, Rha2-C10-C10, Rha2-C10-C12:1, and Rha2-C10-C12. The chemical composition of the rhamnolipid mixtures produced on different carbon sources did not vary with the type of carbon source used. The rhamnolipid mixture produced by Pseudomonas aeruginosa BN10 on glycerol reduced the surface tension of pure water from 72 to 29 mN m(-1) at a critical micellar concentration of 40 mg 1(-1), and the interfacial tension was 0.9 mN m(-1). The new surfactant product formed stable emulsions with hydrocarbons and showed high antimicrobial activity against Gram-positive bacteria. The present study shows that the new strain Pseudomonas aeruginosa BN10 demonstrates enhanced production of the di-rhamnolipid Rha2-C10-C10 on all carbon sources used. Due to its excellent surface and good antimicrobial activities the rhamnolipid homologue mixture from Pseudomonas aeruginosa BN10 can be exploited for use in bioremediation, petroleum and pharmaceutical industries.     

Structure and polymer form of poly-3-hydroxyalkanoates produced by Pseudomonas oleovorans grown with mixture of sodium octanoate/undecylenic acid and sodium octanoate/5-phenylvaleric acid

PHAs (poly-3-hydroxyalkanoates) obtained by Pseudomonas oleovorans grown with mixed carbon sources were investigated. Mixed carbon sources were sodium octanoate/undecylenic acid and sodium octanoate/5-phenylvaleric acid. Effect of carbon source in pre-culture on PHAs structure was investigated. Main fermentation was conducted with mixture of sodium octanoate/undecylenic acid, and PHA contained both saturated and unsaturated units. When more undecylenic acid was used in the medium, the ratio of unsaturated unit increased and the T(g) of the products also changed. The PHA grown with mixture of sodium octanoate and undecylenic acid was a random copolymer, which was determined by DSC analysis. Using mixed carbon sources of sodium octanoate and 5-phenylvaleric acid, highest dry cell weight and PHA concentration were obtained when 0.02g or 0.04g of 5-phenylvaleric acid were added in 50mL medium. Cultured with sodium octanoate and 5-phenylvaleric acid, PHA containing HO (3-hydroxyoctanoate) unit and HPV (3-hydroxy-5-phenylvalerate) unit was produced. T(g) of the products fell between those of pure PHO and pure PHPV. By means of DSC analysis and fractionation method, the PHA obtained was regarded as a random copolymer.     

Role of (R)-specific enoyl coenzyme A hydratases of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp in the production of polyhydroxyalkanoates

Four (R)-specific enoyl CoA hydratases (PhaJ) interconnect the β-oxidation pathway with PHA biosynthesis in Pseudomonas aeruginosa. The use of antisense technique and over-expression to delineate the role of two of these enzymes, PhaJ1 and PhaJ4 forms the basis of this study. It has been observed that P. aeruginosa recombinant with phaJ1 antisense construct, fed with different fatty acids, produces PHA with less hydroxy octanoate (7–11% reduction) and a proportionate increase in other monomer fractions, compared to that of the control. Recombinants bearing phaJ4 antisense construct are found to contain less hydroxy decanoate (10–11% reduction) and more or less equal amount of hydroxy octanoate, compared to that of the control. P. aeruginosa has produced PHA with more hydroxy octanoate and decanoate (6–17% increase), respectively, when PhaJ1 and PhaJ4 have been over-expressed individually or along with PhaC1. PhaJ1 and PhaJ4 are found to be involved mainly in the production of hydroxy octanoyl CoA and hydroxy decanoyl CoA, respectively, in P. aeruginosa. The strongest accumulation of hydroxy octanoate and hydroxy decanoate has been observed along with hydroxy butyrate, in PHA, produced by E. coli, when PhaC1 has been co-expressed with PhaJ1 and PhaJ4, respectively. We have demonstrated, for the first time, the polymerization of hydroxy butyryl CoA monomers in recombinant E. coli by PhaC1 of P. aeruginosa.     

High cell density cultivation ofPseudomonas oleovorans for the production of poly(3-hydroxyalkanoates)

Fed-batch culture ofPseudomonas oleovorans was carried out for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) using octanoate as a carbon source. Octanoate and the salt solution containing ammonium sulfate and magnesium sulfate were intermittently fed in the course of fermentation. Cell mass and PHA concentrations of 42.8 and 16.8 g/L, respectively, could be obtained in 40 h. The PHA content and the PHA productivity were 39.2% and 0.42 g PHA/L-h, respectively. The yields of cell mass and PHA were 0.71 g dry cell mass/goctanoate and 0.28 g PHA/g octanoate, respectively. Therefore, octanoate can be used for the production of MCL-PHAs to a high, concentration with high productvity.     

Altering the substrate specificity of polyhydroxyalkanoate synthase 1 derived from Pseudomonas putida GPo1 by localized semirandom mutagenesis

The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Pp), class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA- as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB(-)4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB(-)4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB(-)4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.     

Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of PHAMCL from waste vegetable oil. The proportion of 3- hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.     

Sequence and transcriptional studies of five clustered flagellin genes from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The Escherichia coli 3-ketoacyl-ACP reductase gene (fabGEc) was cloned using a PCR technique to investigate the metabolic link between fatty acid metabolism and polyhydroxyalkanoate (PHA) production. Three plasmids respectively harboring fabGEc and the poly-3-hydroxyalkanoate synthesis genes phaCAc and phaC1Ps from Aeromonas caviae and Pseudomonas sp. 61-3 respectively were constructed and introduced into E. coli HB101 strain. On a two-stage cultivation using dodecanoate as the sole carbon source, recombinant E. coli HB101 strains harboring fabGEc and phaC genes accumulated PHA copolymers (about 8 wt% of dry cell weight) consisting of several (R)-3-hydroxyalkanoate units of C4, C6, C8, and C10. It has been suggested that overexpression of the fabGEc gene leads to the supply of (R)-3-hydroxyacyl-CoA for PHA synthesis via fatty acid degradation.     

Molecular and structural characterization of the biosurfactant produced by Pseudomonas aeruginosa DAUPE 614

Pseudomonas aeruginosa DAUPE 614 produced rhamnolipids (3.9gL(-1)) when cultivated on a medium containing glycerol and ammonium nitrate. These rhamnolipids reduced the surface tension of water to 27.3mNm(-1), with a critical micelle concentration of 13.9mgL(-1). The maximum emulsification index against toluene was 86.4%. The structure of the carbohydrate moiety of the glycolipid was determined by gas chromatography-mass spectroscopy (GC-MS) analysis allied to electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) 1D, 2D (13)C, (1)H spectroscopy. The hydroxyl fatty acids were analyzed by GC-MS as hydroxy-acetylated fatty acid methyl ester derivatives. The positions of the fatty acids in the lipid moiety were variable, with 6 mono-rhamnolipid homologues (Rha-C(10)-C(10); Rha-C(10)-C(8); Rha-C(8)-C(10); Rha-C(10)-C(12:1); Rha-C(12)-C(10); Rha-C(10)-C(12)) and 6 di-rhamnolipid homologues (Rha(2)-C(10)-C(10); Rha(2)-C(10)-C(8); Rha(2)-C(8)-C(10); Rha(2)-C(10)-C(12:1); Rha(2)-C(12)-C(10); Rha(2)-C(10)-C(12)). The ratio of Rha(2)-C(10)-C(10) to Rha-C(10)-C(10) was higher than has been reported in previous studies. Our methodology allowed us to distinguish between the isomeric pairs Rha-C(10)-C(8)/Rha-C(8)-C(10), Rha-C(10)-C(12)/Rha-C(12)-C(10), Rha(2)-C(10)-C(8)/Rha(2)-C(8)-C(10) and Rha(2)-C(12)-C(10)/Rha(2)-C(10)-C(12). For each isomeric pair, the congener with the shorter chain adjacent to the sugar was always more abundant than the congener with longer chain.     

Polyhydroxyalkanoate production by antarctic soil bacteria isolated from Casey Station and Signy Island

Polyhydroxyalkanoate (PHA) is a family of biopolymers produced by some bacteria and is accumulated intracellularly as carbon and energy storage material. Fifteen PHA-producing bacterial strains were identified from bacteria isolated from Antarctic soils collected around Casey Station (66°17'S, 110°32'E) and Signy Island (60°45'S, 45°36'W). Screening for PHA production was carried out by incubating the isolates in PHA production medium supplemented with 0.5% (w/v) sodium octanoate or glucose. 16S rRNA gene sequence analysis revealed that the isolated PHA-producing strains were mainly Pseudomonas spp. and a few were Janthinobacterium spp. All the isolated Pseudomonas strains were able to produce medium-chain-length (mcl) PHA using fatty acids as carbon source, while some could also produce mcl-PHA by using glucose. The Janthinobacterium strains could only utilize glucose to produce polyhydroxybutyrate (PHB). A Pseudomonas isolate, UMAB-40, accumulated PHA up to 48% cell dry mass when utilizing fatty acids as carbon source. This high accumulation occurred at between 5°C and 20°C, then decreased with increasing temperatures. Highly unsaturated mcl-PHA was produced by UMAB-40 from glucose. Such characteristics may be associated with the ability of UMAB-40 to survive in the cold.     

Cloning and Molecular Analysis of Poly(3-Hydroxyalkanoate) Biosynthesis Genes in Pseudomonas aureofaciens

Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6. Received: 10 January 2001 / Accepted: 7 June 2001     

Parvibaculum lavamentivorans DS-1T degrades centrally substituted congeners of commercial linear alkylbenzenesulfonate to sulfophenyl carboxylates and sulfophenyl dicarboxylates

Commercial linear alkylbenzenesulfonate (LAS) contains 20 congeners of linear alkanes (C(10) to C(13)) substituted subterminally with the 4-sulfophenyl moiety in any position from lateral to central. Parvibaculum lavamentivorans DS-1(T) degrades each of eight laterally substituted congeners [e.g., 2-(4-sulfophenyl)decane (2-C10-LAS); herein, compounds are named systematically by chain length (e.g., C(10)) and by the position of the substituent on the chain (e.g., position 2)] to a major sulfophenyl carboxylate [SPC; here 3-(4-sulfophenyl)butyrate (3-C4-SPC)] and two minor products, namely, the alpha,beta-unsaturated SPC (SPC-2H, here 3-C4-SPC-2H) and the SPC+2C (here 5-C6-SPC) species (D. Schleheck, T. P. Knepper, K. Fischer, and A. M. Cook, Appl. Environ. Microbiol. 70:4053-4063). The degradation of centrally substituted congeners by strain DS-1 was examined in this work. 5-C10-LAS yielded not only the predicted 4-C8-SPC, 4-C8-SPC-2H, and 6-C10-SPC (about 70% of products) but also sulfophenyl dicarboxylates (SPdC), i.e., C6-, C8-, and C10-SPdC. These were identified by electrospray ionization-mass spectrometry (ESI-MS) after separation by high-pressure liquid chromatography (HPLC). ESI ion-trap MS and ESI-time of flight-MS were used to confirm the identities of key intermediates. Different mixtures of congeners obtained by separation of commercial LAS by HPLC were degraded, and the degradative products were compared. If a congener carried the sulfophenyl substituent on the 5, 6, or 7 position, SPdCs were formed as well as SPC, SPC-2H, and SPC+2C, whereas the substituent on the 2, 3, or 4 position yielded only SPC, SPC-2H, and SPC+2C. Some 50 products were generated from the 20 LAS congeners: 11 major SPCs, each with an SPC-2H and an SPC+2C (i.e., 33 SPC and SPC-2H species), and about 17 SPdC species. A large array of compounds, many in low quantities, is thus generated by P. lavamentivorans DS-1 during the degradation of commercial LAS.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.