Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Synthesis and accumulation of poly(3-hydroxybutyric acid) by Rhizobium sp

Forty-two Rhizobium strains obtained from different culture collections were evaluated quantitatively for poly(3-hydroxy-butyric acid) [PHB] production in shake flask culture. The majority of the strains produced the maximum amount of PHB during the late exponential or stationary phase of growth. Synthesis and accumulation of PHB in different species of Rhizobium were found to vary between 1-38% of their dry biomass. Growth and PHB production by the Rhizobium strain TAL-640 were greatly influenced by the C-source and D-mannitol was fundamental to both processes. The identity and purity of PHB isolated from TAL-640 have also been confirmed by UV-, IR- and 1H-NMR spectroscopic analyses.     

Effects of Culture Conditions on Poly(beta-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(beta-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH(2)PO(4), and 0.2% NH(4)Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.     

Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium

Hybridization experiments were carried out between high molecular weight, denatured, agar-embedded deoxyribonucleic acid (DNA) and homologous, nonembedded, sheared, denatured (14)C-labeled DNA from a strain of Agrobacterium tumefaciens and Rhizobium leguminosarum (the reference strains) in the presence of sheared, nonembedded, nonlabeled DNA (competing DNA) from the same or different nomen-species of Agrobacterium, Rhizobium, Chromobacterium, and several other organisms. Percentage of DNA homology was calculated from the results. The findings are discussed in relation to previous taximetric studies, present classification schemes, and guanine-cytosine content of the DNA. Strains of A. tumefaciens, A. radiobacter, A. rubi, A. rhizogenes, R. leguminosarum, and R. meliloti exhibited a mean percentage of DNA homology greater than 50 with the two reference strains. A. tumefaciens, A. radiobacter, and A. rubi were indistinguishable on the basis of DNA homology, with strain variations for this group involving up to 30% of their base sequences. The remainder of the organisms studied fall into at least six distinct genetic groups: (i) R. (Agrobacterium) rhizogenes, which is more homologous to R. leguminosarum than to the A. tumefaciens-A. radiobacter group; (ii) R. leguminosarum; (iii) R. meliloti; (iv) R. japonicum, which has a mean DNA homology of some 38 to 45% with the reference strains; (v) Chromobacterium, which is as genetically remote from the reference strains as, for example, Pseudomonas; and (vi) A. pseudotsugae strain 180, which has a DNA homology with A. tumefaciens and R. leguminosarum of only about 10%. Since this latter homology value is similar to what was found after hybridizations between the reference strains and organisms such as Escherichia coli and Bacillus subtilis, A. pseudotsugae should definitely be removed from the genus.     

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame.

By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment.     

The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors.

The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region IIb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.     

Effects of Culture Conditions on Poly(β-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(β-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH₂PO₄, and 0.2% NH₄Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.     

Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living nitrogen-fixing bacterium accumulates poly(3-hydroxybutyric acid) [PHB, 69% of cell dry weight (CDW)] when grown on glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV with 19.2 mol% 3HV] when grown on glucose and valerate. Use of ethylene glycol (EG) and/or polyethylene glycols (PEGs) of low molecular weight as sole carbon source were detrimental to A. chroococcum growth and polymer yields. PEG-200, however, in the presence of glucose was incorporated into the polyhydroxyalkanoate (PHA) polymer. Addition of PEG-200 (150 mM) to culture medium during mid-log phase growth favored increased incorporation of EG units (12.48 mol%) into the PHB polymer. In two-step culture experiments, where valerate and PEG simultaneously were used in fresh medium, EG was incorporated most effectively in the absence of glucose, leading to the formation of a copolymer containing 18.05 mol% 3HV and 14.78 mol% EG. The physico-mechanical properties of PEG-containing copolymer (PHBV–PEG) were compared with those of the PHB homopolymer and the PHBV copolymer. The PHBV–PEG copolymer appeared to have less crystallinity and greater flexibility than the short-chain-length (SCL) PHA polymers.     

Polyhydroxyalkanoate production in recombinant Escherichia coli

Abstract The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-β-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate- co -3-hydroxyvalerate (PHB- co -3-).     

Biological activity of rhizobial siderophore.

Non-nodulating mutant of Rhizobium leguminosarum biovar trifolli produces the phenolate type of siderophore consisting of 2,3-dihydroxybenzoic acid and threonine. The activity of this compound against the various bacteria was tested. Only, the growth of R. leguminosarum strains was stimulated by siderophore. The other species of Rhizobium, especially R. meliloti, were sensitive to this agent. The growth of R. meliloti was also inhibited by agrobactin and pseudobactin. This effect was reversed by ferric iron.     

Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments.

The gram-negative soil bacteria Rhizobium spp. infect and establish a nitrogen-fixing symbiosis with legume crops which involves the mutual exchange of diffusable signal molecules. In this study, Rhizobium leguminosarum containing a nod-lacZ gene fusion was used to screen the most effective plant-to-bacteria signal molecules for pea and lentil and the induction conditions. Out of a number of signal compounds including apigenin, daidzein, genistein, hesperetin, kaempferol, luteolin, naringenin, and rutin, hesperetin and naringenin were found to be the most effective plant-to-bacteria signal molecules. The induction of nod genes was temperature-dependent, where nod gene induction was decreased with dropping incubation temperature. The combination of hesperetin at 7 microM and naringenin at 3 microM resulted in better induction of nod gene activities compared to either hesperetin or naringenin alone. Nodulation and plant dry matter accumulation of pea and lentil plants receiving preinduced R. leguminosarum were higher than those of plants receiving uninduced R. leguminosarum cells in controlled environment growth chamber conditions. Preinduced Rhizobium with hesperetin at a concentration of 10 microM increased nodule number on average by 60.5% and dry matter accumulation by 14% in field pea at 17 degrees C, while it was 32% and 9% at 24 degrees C, respectively. Similarly, averaged over two rhizobial strains, a 59% and 6% increase in nodule number and biomass production at 17 degrees C, and a 39% and 27% at 24 degrees C, were obtained from lentil inoculated with hesperetin-induced R. leguminosarum, respectively.     

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Application of self-cycling fermentation technique to the production of poly-beta-hydroxybutyrate

The production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 in a cyclone bioreactor was compared using various culture methods: batch, fed-batch, and self-cycling fermentation (SCF) with and without extended periods of nutrient deprivation. SCF is a semi-continuous method that results in a nutrient limitation for every successive generation of cells and, therefore, may have advantages for products whose formation follow secondary metabolite kinetics. Use of the SCF technique without extended nutrient deprivation produced a PHB concentration of 1.2 g L(-1) as 40% of the biomass dry weight. With nitrogen deprivation for 4 or 6 h, the concentration of PHB decreased when compared to the standard SCF technique. However, nitrogen deprivation periods of 8 h resulted in an increase in PHB concentration to 2.7 g L(-1) or 59% of the biomass dry weight. The nutrient cycling may act to repress PHB accumulation during periods of nitrogen deprivation, unless a time threshold has been reached, after which PHB accumulation occurs as in normal batch culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 815-820, 1997.     

Production of Poly-beta-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-beta-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-beta-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter h (from a constant value of 1.6 g of cellular protein liter). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 x 10 g mol and a polydispersivity index of 3.9. Poly(beta-hydroxybutyric acid-beta-hydroxyvaleric acid) copolymer was produced with a poly-beta-hydroxybutyric acid-poly-beta-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Binary conjugational transfer system of vectors pRK290 and pLAFRI is proficient both ways between Escherichia coli and Rhizobium

Wide host range vector plasmids pRK290 and pLAFRI carrying genomic fragments of Rhizobium are transferable both ways between R. meliloti and R. leguminosarum cells on the one hand and to E. coli cells on the other, in triparental matings involving E. coli cells carrying pRK2013, the helper for Tra functions to the vector plasmids. The vector plasmids pRK290 and pLAFRI can be employed for recovering clones harbored by R. leguminosarum and R. meliloti by transfer to Rhizobium cells by direct matings of the library with them.     

Characterization and primary structures of DNA-binding HU-type proteins from Rhizobiaceae

The DNA-binding HU-type proteins from several species of Rhizobiaceae including Rhizobium meliloti, two strains of Rhizobium leguminosarum with highly different phenotypic characters and Agrobacterium tumefaciens, were characterized and their amino acid sequences were determined. HU-type proteins isolated from R. leguminosarum L18 and A. tumefaciens are identical and show slight differences with the R. meliloti HU-type protein. On the other hand the R. leguminosarum L53 HU-type protein is quite different from the proteins cited above; several amino acid substitutions encountered in this protein result in significant changes in the folding of the polypeptide chain. The biochemical characteristics of these proteins are in good agreement with the respective position of these bacteria in the phylogeny determined by numerical taxonomy.