Production of poly-β-hydroxybutyrate by fed-batch culture of recombinantEscherichia coli

Summary A recombinantEscherichia coli strain harboring the PHB biosynthesis genes fromAlcaligenes eutrophus was used to produce poly--hydroxybutyrate (PHB) by pH-stat fedbatch culture. Initial glucose concentration for optimal growth was found to be 20g/L from a series of flask cultures. A final PHB concentration of 88.8 g/L could be obtained after 42 hrs of cultivation.     

Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax

The bacterial storage polymer poly-β-hydroxybutyrate (PHB) has the potential to be used as an alternative anti-infective strategy for aquaculture rearing. In this research, the effects of (partially) replacing the feed of European sea bass juveniles with PHB were investigated. During a 6-week trial period, the PHB showed the ability to act as an energy source for the fish. This indicated that PHB was degraded and used during gastrointestinal passage. The gut pH decreased from 7.7 to 7.2 suggesting that the presence of PHB in the gut led to the increased production of (short-chain fatty) acids. The diets supplemented with 2% and 5% PHB (w/w) induced a gain of the initial fish weight with a factor 2.4 and 2.7, respectively, relative to a factor 2.2 in the normal feed treatment. Simultaneously, these treatments showed the highest bacterial range-weighted richness in the fish intestine. Based on molecular analysis, higher dietary PHB levels induced larger changes in the bacterial community composition. From our results, it seems that PHB can have a beneficial effect on fish growth performance and that the intestinal bacterial community structure may be closely related to this phenomenon.     

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly--hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content. © Rapid Science Ltd. 1998     

Rapid and simple analysis of poly-β-hydroxybutyrate content by capillary isotachophoresis

Summary We present a rapid method for the direct analysis of poly--hydroxybutyrate (PHB) content in the soil bacteria Alcaligenes eutrophus. PHB from the fresh cells was converted by sulfuric acid to the crotonic acid and measured by capillary isotachophoresis after the neutralization by CaCO₃. The method can be used for rapid and routine monitoring of the fermentation processes in samples containing 0.001 to 20 mg of PHB.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Transport of poly-β-hydroxybutyrate in human plasma

Poly-β-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20–30% of total plasma PHB; 6–14% in the very low density lipoproteins (VLDL), 8–16% in the low density lipoproteins (LDL), and < 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70–80%) was found in protein fractions of density > 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')₂ identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of ¹⁴C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus on different carbon sources

Poly--hydroxybutyrate was produced in shake cultures by Alcaligenes eutrophus H16 on fructose, xylose, and fumaric, itaconic, lactic and propionic acids in a three-stage process. The maximum polymer concentration of 6.9 g l^–1 (69% of cell dry matter) was obtained with 20g l^–1 of fructose with a volumetric productivity of about 0.22 g l^–1 h^–1 at 24h. Up to about 3 g l^–1 (about 50% of cell dry matter) of polymer was also produced on lactic and propionic acids as the sole carbon source during the production phase. In multivatiate optimization employing an orthogonal 2³-factorial central composite experimental design with fructose as the substrate in a single-stage process, the optimal initial fructose concentration decreased from 35 g l^–1 to 24 g l^–1 when the incubation time was increased from about 35 h to 96 h. The optimal shaking speed range was 90–113 rpm. Correspondence to: S. Linko     

Pathway of anaerobic poly-β-hydroxybutyrate degradation byIlyobacter delafieldii

Ilyobacter delafieldii produced an extracellular poly--hydroxybutyrate (PHB) depolymerase when grown on PHB; activity was not detected in cultures grown on 3-hydroxybutyrate, crotonate, pyruvate or lactate. PHB depolymerase activity was largely associated with the PHB granules (supplied as growth substrate), and only 16% was detected free in the culture supernatant. Monomeric 3-hydroxybutyrate was detectable as a product of depolymerase activity. The monomer was fermented to acetate, butyrate and H₂. After activation by coenzyme A transfer from acetyl-CoA or butyryl-CoA, the resultant 3-hydroxybutyryl-CoA was oxidized to acetoacetyl-CoA (producing NADH), followed by thiolytic cleavage to yield acetyl-CoA which was further metabolized to acetyl-phosphate, then to acetate with concomitant ATP production. The reducing equivalents (NADH) could be disposed of by the evolution of H₂, or by a reductive pathway in which 3-hydroxybutyryl-CoA was dehydrated to crotonyl-CoA and reduced to butyryl-CoA. In cocultures ofI. delafieldii withDesulfovibrio vulgaris on PHB, the H₂ partial pressure was much lower than in the pure cultures, and sulfide was produced. Thus interspecies hydrogen transfer caused a shift to increased acetate and H₂ production at the expense of butyrate.     

Stimulation of poly-β-hydroxybutyrate oxidation in Sphaerotilus discophorus by manganese and magnesium

Summary When grown with glucose, S. discophorus synthesized large amounts of poly--hydroxybutyrate which accumulated intracellularly as sudanophilic granules. The rate of endogenous oxygen consumption by such cells was markedly increased by Mn⁺⁺ and even more by Mg⁺⁺. It has been shown that these inorganic ions stimulate the oxidation of the intracellular poly--hydroxybutyrate.Dedicated by the senior author to Prof. C. B. van Niel on the occasion of his 70th birthday with gratitude for many unforgettable years of association, instruction and stimulation.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Summary Three transformants of Alcaligenes eutrophus harbouring the recombinant plasmids containing phbCAB, phbAB, and phbC genes, were cultivated to investigate the effect of cloned genes on cell growth and poly--hydroxybutyrate accumulation. Both in the nutrient-rich and minimal media, the increased PHB accumulation in the transformants was observed compared to the parent strain, and this was the result of the increased enzyme activities in the transformants. Low carbon concentration and high C/N molar ratio favored higher PHB accumulations in the transformants. The transformant harbouring the phbC gene showed the highest PHB accumulation, which indicated that PHB synthase was the most critical enzyme for PHB biosynthesis in the transformant.     

Microbial production of poly-β-hydroxybutyrate by marine microbes isolated from various marine environments

Considering the industrial interest of Poly-β-hydroxybutyrate (PHB), bacteria isolated from the various marine arenas were screened for their ability to accumulate PHB and were compared with Wausteria eutropha (MTCC-1285). Among the 42 isolates, four strains showed the accumulation of PHB. The maximum PHB producer Vibrio sp. (MK4) was further studied in detail. To increase the productivity, steps were taken to evaluate the effect of carbon sources, nitrogen sources, pH and sodium chloride concentration on PHB productivity by MK4. The optimized conditions were further used for the batch fermentation over a period of 72 h. Significantly higher maximum biomass of 9.1 g/L with a PHB content of 4.223 g/L was obtained in a laboratory-scale bioreactor at 64 h, thus giving a productivity of 0.065 g/L/h. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis and ¹H NMR analysis. Thus, the study highlights the potential of the use of Vibrio sp (MK4) in the commercial production of PHB.     

The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii

1. The enzymes beta-ketothiolase, acetoacetyl-CoA reductase, acetoacetate-succinate CoA-transferase (;thiophorase') and d(-)-3-hydroxybutyrate dehydrogenase have been partially purified from crude extracts of glucose-grown nitrogen-fixing batch cultures of Azotobacter beijerinckii. The condensation of acetyl-CoA to acetoacetyl-CoA catalysed by beta-ketothiolase is inhibited by CoASH, and the reverse reaction is inhibited by acetoacetyl-CoA. Acetoacetyl-CoA reductase has K(m) for acetoacetyl-CoA of 1.8mum and is inhibited by acetoacetyl-CoA above 10mum. The enzyme utilizes either NADH or NADPH as electron donor. The second enzyme of poly-beta-hydroxybutyrate degradation, d(-)-3-hydroxybutyrate dehydrogenase, is NAD(+)-specific and is inhibited by NADH, pyruvate and alpha-oxoglutarate. CoA transferase is inhibited by acetoacetate, the product of hydroxybutyrate oxidation. In continuous cultures poly-beta-hydroxybutyrate biosynthesis ceased on relaxation of oxygen-limitation and the rates in situ of oxygen consumption and carbon dioxide evolution of such cultures increased without a concomitant increase in glucose uptake. 2. On the basis of these and other findings a cyclic mechanism for the biosynthesis and degradation of poly-beta-hydroxybutyrate is proposed, together with a regulatory scheme suggesting that poly-beta-hydroxybutyrate metabolism is controlled by the redox state of the cell and the availability of CoASH, pyruvate and alpha-oxoglutarate. beta-Ketothiolase plays a key role in the regulatory process. Similarities to the pathways of poly-beta-hydroxybutyrate biosynthesis and degradation in Hydrogenomonas are discussed.     

Quantitation of poly-β-hydroxybutyrate by fluorescence of bacteria and granules stained with Nile blue A

Summary Fluorescence from poly--hydroxybutyrate (PHB) inclusions inside Azotobacter vinelandii UWD cells stained with Nile blue A was shown to be proportional to PHB concentration. The intensity of the fluorescence was greatest in native, fluid inclusions and was the least in extracted, crystallized granules. However, isolated air-dried PHB granules also were proportionally stained with Nile blue A. The results show that Nile blue A can be used in the quantitative determination of PHB in a variety of cells.     

Improvement in cell yield of Methylobacterium sp. by reducing the inhibition of medium components for poly-β-hydroxybutyrate production

The inhibitory effect of the concentrations of medium components on the growth of Methylobacterium sp. for poly--hydroxybutyrate production was investigated by measuring the specific growth rates for various concentrations of each medium component. When the methanol concentration was increased, the cell growth decreased and was strongly inhibited above 6% (v/v) methanol. Ammonia, calcium and iron ion did not significantly inhibit the cell growth while there were some inhibitory effects at high concentrations of sodium, potassium, and magnesium. In particular, phosphate gave most significant inhibition at concentrations higher than 75 mM. By using an automatic feeding control system of methanol, ammonia, phosphate, and minerals, their concentrations were maintained within the level necessary to reduce the inhibition of medium components. The finial dry cell weight of Methylobacterium sp. in such a system was 172 g/l at 84 h.     

Production of poly-(β-hydroxybutyrate) from saponified Vernonia galamensis oil by Alcaligenes eutrophus

Saponified vernonia oil was converted exclusively to poly(β-hydroxybutyrate) (PHB) by Alcaligenes eutrophus in a single-stage batch culture. After harvesting, centrifugation followed by lyophilization, the resulting dried cells contained up to 42.8 wt% PHB having a peak molecular mass of 381 863 Da, weight-average molecular mass of 308 390 Da, and a polydispersity of 1.1. The PHB had a melting point (T_m) range of 163–174°C with a maximum at 172°C (lit. T_m, 175°C), and heat of fusion of 18.43 cal g⁻¹. Fermentation performed under varying conditions of nitrogen limitation indicated that there was no significant effect of nitrogen concentration on the molecular mass of PHB produced from vernonia oil by A. eutrophus. Received 27 March 1998/ Accepted in revised form 17 July 1998     

Suppression of inflammation by helminths: a role for the gut microbiota?

Multiple recent investigations have highlighted the promise of helminth-based therapies for the treatment of inflammatory disorders of the intestinal tract of humans, including inflammatory bowel disease and coeliac disease. However, the mechanisms by which helminths regulate immune responses, leading to the amelioration of symptoms of chronic inflammation are unknown. Given the pivotal roles of the intestinal microbiota in the pathogenesis of these disorders, it has been hypothesized that helminth-induced modifications of the gut commensal flora may be responsible for the therapeutic properties of gastrointestinal parasites. In this article, we review recent progress in the elucidation of host–parasite–microbiota interactions in both animal models of chronic inflammation and humans, and provide a working hypothesis of the role of the gut microbiota in helminth-induced suppression of inflammation.     

On-line culture fluorescence measurement during the batch cultivation of poly-β-hydroxybutyrate producing Alcaligenes eutrophus

Estimation of biomass concentration by on-line fluorimetry was examined during batch cultivations of Alcaligenes eutrophus ATCC 17697, a poly-β-hydroxybutyric acid (PHB) producer. The results obtained revealed a linear correlation between total culture fluorescence and total biomass concentration. The total fluorescence level was attributed to cellular fluorescence and to the fluorescence of compounds presumably produced by the cells and secreted into the culture medium. An increase in the specific cellular fluorescence during the course of the experiments indicated a shift in metabolism that favored the production of PHB.     

Do experimental units of different scale affect the biological performance of European sea bass Dicentrarchus labrax larvae?

The effects of different tank volumes (2000, 500 and 40 l) on European sea bass Dicentrarchus labrax larval rearing, relating to growth, survival, quality and stress variables, were investigated. A dynamic energy budget (DEB) model was used to analyse the results. The hydrodynamics of the tanks exhibited differences, with the water currents in the 2000 l tanks to be almost one order of magnitude stronger than those in the 40 l ones. Important differences in fish growth were observed between small and large tank‐rearing volumes, with the smallest tank resulting in the slowest growth. Based on the DEB model analysis, growth differences were related to feeding rates, with growth in the smaller tank limited by food availability. Differences in survival rates were not statistically significant among the tank‐rearing volumes. The quality evaluation of the fry (in terms of swimbladder, jaw and skeletal abnormalities) showed differences, with the smallest tank having the highest percentage of deformed individuals. This could be attributed to both the feeding variances and the hydrodynamics in the tanks. No differences were observed in terms of whole‐body cortisol at the two developmental stages; flexion, and when the larvae body was fully covered by melanophores; when analysis was performed. This indicates that the allostatic load exerted on fish of different groups was similar and inside the fish‐coping abilities range, in terms of the cortisol response axis. The selection of the experimental scale is of importance, especially when the results are to be transferred and applied on an industrial scale.     

Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli

A second promoter (P1) was inserted to the PHA (poly--hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5 with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly--hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 10⁶ Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 10⁷ Da was observed without IPTG being added.