Calorimetrically recognized maximum yield of poly-3-hydroxybutyrate (PHB) continuously synthesized from toxic substrates

The broader usage of poly-beta-hydroxybutyrate (PHB), for instance as bulk plastics, calls for cheap raw materials and greater overall process efficiency. The bacterial synthesis is generally induced and promoted by the limitation of growth via nitrogen, oxygen or phosphate depletion with the simultaneous excess and higher concentration of the carbon substrate. Consequently, toxic substrates have been considered unsuitable for PHB synthesis. Nevertheless, a single-stage continuous process for producing PHB from toxic substrates using microorganisms was developed and is reported here. The maximum heat flux during continuous growth and the maximum yield of PHB versus the substrate consumption rate were found to coincide. This suggests the possibility of controlling the conversion of a growth-inhibiting substrate into PHB and maximizing the process efficiency. The observed correlation occurred irrespective of the substrates investigated (phenol or sodium benzoate), the PHB-producing strain (Ralstonia eutropha JMP 134 or Variovorax paradoxus JMP 116), or the type of limitation imposed. The maximum PHB yields obtained comprised up to 50% of cell dry mass.     

Fed-batch cultivation of Wautersia eutropha

Batch kinetics of polyhydroxybutyrate (PHB) synthesis in a bioreactor under controlled conditions of pH and dissolved oxygen gave a biomass of 14 g l(-1) with a PHB concentration of 6.1 g l(-1) in 60 h. The data of the batch kinetics was used to develop a mathematical model, which was then extrapolated to fed-batch by incorporating the dilution due to substrate feeding. Offline computer simulation of the fed-batch model was done to develop the nutrient feeding strategies in the fed-batch cultivation. Fed-batch strategies with constant feeding of only nitrogen and constant feeding of both nitrogen and fructose were tried. Constant feeding strategy for nitrogen and fructose gave a better PHB production rate of 0.56 g h(-1) over the value obtained in batch cultivation (PHB production rate - 0.4 g h(-1)).     

Biodegradation of phenolic industrial wastewater in a fluidized bed bioreactor with immobilized cells of Pseudomonas putida

The paper presents the main results obtained from the study of the biodegradation of phenolic industrial wastewaters by a pure culture of immobilized cells of Pseudomonas putida ATCC 17484. The experiments were carried out in batch and continuous mode. The maximum degradation capacity and the influence of the adaptation of the microorganism to the substrate were studied in batch mode. Industrial wastewater with a phenol concentration of 1000 mg/l was degraded when the microorganism was adapted to the toxic chemical. The presence in the wastewater of compounds other than phenol was noted and it was found that Pseudomonas putida was able to degrade these compounds. In continuous mode, a fluidized-bed bioreactor was operated and the influence of the organic loading rate on the removal efficiency of phenol was studied. The bioreactor showed phenol degradation efficiencies higher than 90%, even for a phenol loading rate of 0.5 g phenol/ld (corresponding to 0.54 g TOC/ld).     

Exopolysaccharide and Poly-(beta)-Hydroxybutyrate Coproduction in Two Rhizobium meliloti Strains

The effects of different nitrogen and carbon sources on cell growth, pH, and exopolysaccharide (EPS) and poly-(beta)-hydroxybutyrate (PHB) production by two strains of Rhizobium meliloti (M5N1 and Su47) are reported. Differences in the behavior of glucose- and fructose-grown cells were shown, in particular with the M5N1 strain. Growth in a glucose-containing medium was accompanied by acidification of the culture medium, which leads to cell death. On fructose, acidification was detected only in the medium with a mineral nitrogen supply. A lag phase in EPS production was observed with cells grown with glucose, probably related to an initial extracellular conversion of the carbohydrate into an acid. No lag phase was observed in EPS production from fructose or in PHB synthesis whatever the carbon source. A decrease in PHB content was noticed for both strains under conditions where acidification of media occurred. The extent of production, emphasized by the use of a coproduction index, indicates that the M5N1 strain is a more promising organism than is the Su47 strain for polymer production. Such a strain, put in rich medium (containing yeast extract) supplemented with fructose, accumulated PHB up to 85% of dry cell weight and excreted about 1.5 g of EPS per liter in the medium. Regulation of the coproduction of EPS and PHB by these cells is suggested.     

Producing poly-3-hydroxybutyrate with a high molecular mass from methane

Poly-3-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could provide a suitable substrate for the production of PHB. Methane is cheap and plentiful - not only as natural gas, but also as biogas. The methanotrophic strain Methylocystis sp. GB 25 DSMZ 7674 is able to accumulate PHB in a brief non-sterile process. The studies were carried out using a 7-l and a 70-l pressure bioreactor. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (ammonium, phosphorus or magnesium) in batch culture. The PHB content of biomass was as high as 51%; efficiency was highest during the first 5 h of the product formation process. The maximum PHB yield relative to the methane consumed was estimated to be 0.55 g g(-1). The PHB produced is of very high quality, having a high molecular mass of up to 2.5x10(6) Da.     

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.     

Poly(3-hydroxybutyrate) as an endogeneous substrate for H 2 evolution in Rhodovulum sulfidophilum

In the absence of an external substrate, H 2 was evolved in Rhodovulum sulfidophilum under light-anaerobic conditions, along with degradation of poly(3-hydroxybutyrate) (PHB). Cells grown with succinate as a sole carbon source accumulated only a small amount of PHB compared with that in cells grown with a multiple substrate consisting of a mixture of four organic acids. Unlike PHB-containing cells, PHB-deficient cells did not evolve H in the absence of an external substrate. Nitrogenase activity was expressed while no hydrogenase activity was detected during the incubation of PHB-containing cells. These results suggest that intracellular PHB serves as a substrate for the H evolution catalyzed by nitrogenase when an external substrate is lacking.     

Temporal change in the spatial genetic structure of a sika deer population with an expanding distribution range over a 15-year period

Since the 1980s, the sika deer (Cervus nippon Temminck, 1838) population of Hokkaido, Japan, has grown, resulting in range expansion. To assess the effects of this range expansion on the spatial genetic structure of the population, we compared subpopulation structures during 2 different periods (168 samples for 1991–1996, and 169 samples for 2008–2010), using mitochondrial DNA (mtDNA; D-loop) and microsatellites (9 loci). The number of gene-based subpopulations decreased across the 15-year period; specifically from four to three subpopulations based on mtDNA, and from two to one subpopulation based on microsatellite DNA. The fusion of the two northern subpopulations caused the change to the mtDNA-based structure, which might be explained by the dispersal of females from higher to lower density subpopulations. In comparison, the reason for the change in the microsatellite DNA-based structure was unclear, because no significant genetic differentiation was observed between the two study periods. A stable mtDNA-based structure was maintained in the north and central population separated by a west-to-east boundary, while a north-to-south boundary in eastern Hokkaido maintained stability in the eastern subpopulation versus all other subpopulations. The findings of this study demonstrate the importance of understanding gene flow within a structured population to implement effective management efforts; for instance, the culling of one subpopulation might not affect an adjacent subpopulation, because deer movement is limited between the subpopulations.     

Flow calorimetry and dielectric spectroscopy to control the bacterial conversion of toxic substrates into polyhydroxyalcanoates

The microbial conversion of toxic substrates into valuable products in continuous culture requires the equivalent of a tight rope walk between formation of the desired product and intoxication of the microbial catalyst. The condition of the latter is reflected immediately by changes in heat flow rate and beta-dispersion in an electrical RF field. Therefore, these were applied to the example of the continuous growth-associated synthesis of polyhydroxyalcanoates (PHA) from phenol by the bacterial strain Variovorax paradoxus DSM 4065. By controlling the supply of phenol to the chemostat, the rates of degradation, biomass formation, and synthesis of target product, respectively, were increasingly elevated until the onset of poisoning the organisms. The boundary between the maximum rates and the initiation of intoxication coincided with a sudden change in the heat flux. Using this occurrence, it was possible to develop a control strategy and test it successfully for a time period of 80 h. After 40 h the process stabilized at mean values, i.e., at rates of 92% phenol degradation, 100% biomass formation, and 70 - 75% of PHA formation compared with the situation shortly before poisoning the organisms. Using a moving-average technique to filter the raw dielectric spectroscope data, changes were followed in biomass concentration of approximately 100 mg/L. However, this technique was not sensitive or rapid enough to control the process.     

Dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in Ralstonia eutropha

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation. The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.     

Accumulation of poly-beta-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources

Accumulation of poly-beta-hydroxybutyrate (PHB) in Nostoc muscorum was studied. Cells harvested at stationary phase of growth depicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. In contrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such as NH(4)Cl and KNO(3), were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbon sources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accumulation in N. muscorum was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected to dark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniques to improve the expression level of PHB photoproduction in cyanobacteria.     

Substrate consumption and biomass growth of Ralstonia eutropha at various S0/X0 levels in batch cultures

The biomass growth, substrate consumption and polyhydrobutyrate (PHB) production of Ralstonia eutropha with butyric acid and fructose as the carbon and energy sources at various ratios of initial substrate concentration (S0) to initial biomass concentration (X0) were investigated in this study. Results indicated that the PHB content increased with the increasing S0/X0 ratio. Different substrates exhibited a similar trend for cell growth and substrates consumption with the changing S0/X0 ratio. The specific consumption rates of both butyric acid and fructose increased with the increasing S0/X0 ratio. An S0/X0-dependent kinetic model was modified to describe the kinetics of biomass growth and substrate consumption for R. eutropha. This model was verified with the experimental results from this work and in literature.     

Diffusion of phenol through a biofilm grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor

Diffusion of phenol through a biofilm attached to activated carbon particles was investigated. The biofilm was grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor operating in a fed-batch mode. It was found that phenol did not adsorb on the biofilm and that the diffusion coefficient of phenol within the biofilm varied from 13 to 39% of its corresponding value in water. The diffusion coefficient of phenol within the biofilm was reduced by increasing the biofilm density. An extensive literature review of diffusion of substrates through biofilms indicated that this conclusion could be extended to biofilms grown on flat surfaces, rotating cylinders, and even bioflocs.     

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.     

Inhibition Kinetics of Phenol Degradation by Pseudomonas aeruginosa from Continuous Culture and Washout Data

Steady states of a continuous culture with an inhibitory substrate were used to estimate kinetic parameters under substrate limitation (chemostat operation). Pure cultures of an indigenous Pseudomonas aeruginosa were grown in continuous culture on phenol, the sole source of carbon and energy, at dilution rates of 0.010 to 0.20 h^{- 1}. Using different dilution rates, several steady states were investigated and the specific phenol consumption rates were calculated. In addition, phenol degradation was investigated by increasing the dilution rate above the critical dilution rate (washout cultivation). The results showed that the specific phenol consumption rate increased with increased dilution rate at steady state and that the degradation by Pseudomonas aeruginosa can be described by simple substrate inhibition kinetics under substrate limitation but cannot be described by simple substrate inhibition kinetics under washout cultivation. Fitting of the steady-state data from continuous cultivation to various inhibition models resulted in the best fit for the Yano and Koga kinetic inhibition model. The r_{s max} value of 0.278 mg/mg/h obtained from the Yano and Koga equation was comparable to the experimentally calculated r_{s max} value of 0.283 mg/mg/h obtained under washout cultivation.     

Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase

Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34mg PHB/g glucose in MS medium with glucose and acetate as carbon source.     

Synchronized human diploid fibroblasts: progression capabilities of a subpopulation that fails to keep pace with the predominant, rapidly dividing cohort of cells

Highly synchronized cultures of HSF-55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA-specific stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S-phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early- and late-replicating species of human DNA.     

Effect of Aromatic Compounds on Cellular Fatty Acid Composition of Rhodococcus opacus

In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds.     

Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain

The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC 1.14.13.7] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.