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)).     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Enhanced PHB production and scale up studies using cheese whey in fed batch culture of Methylobacterium sp. ZP24

Methylobacterium sp. ZP24 produced polyhydroxybutyrate (PHB) from disaccharides like lactose and sucrose. As Methylobacterium sp. ZP24 showed growth associated PHB production, an intermittent feeding strategy having lactose and ammonium sulfate at varying concentration was used towards reaching higher yield of the polymer. About 1.5-fold increase in PHB production was obtained by this intermittent feeding strategy. Further increase in PHB production by 0.8-fold could be achieved by limiting the dissolved oxygen (DO) levels in the fermenter. The decreased DO is thought to increase flux of acetyl CO-A towards PHB accumulation over TCA cycle. Cheese whey, a dairy waste product and being a rich source of utilizable sugar and other nutrients, when used in the bioreactor as a main substrate replacing the lactose, led to further increase in the PHB production by 2.5-fold. A total of 4.58-fold increase in the PHB production was obtained using limiting DO conditions with processed cheese whey supplemented with ammonium sulfate in fed batch culture of Methylobacterium sp. ZP24. The present investigation therefore reflects on the possibility of developing a cheap biological route for production of green thermoplastics.     

Continuous polyhydroxybutyrate production from biogas in an innovative two-stage bioreactor configuration

Biogas biorefineries have opened up new horizons beyond heat and electricity production in the anaerobic digestion sector. Added-value products such as polyhydroxyalkanoates (PHAs), which are environmentally benign and potential candidates to replace conventional plastics, can be generated from biogas. This work investigated the potential of an innovative two-stage growth-accumulation system for the continuous production of biogas-based polyhydroxybutyrate (PHB) using Methylocystis hirsuta CSC1 as cell factory. The system comprised two turbulent bioreactors in series to enhance methane and oxygen mass transfer: a continuous stirred tank reactor (CSTR) and a bubble column bioreactor (BCB) with internal gas recirculation. The CSTR was devoted to methanotrophic growth under nitrogen balanced growth conditions and the BCB targeted PHB production under nitrogen limiting conditions. Two different operational approaches under different nitrogen loading rates and dilution rates were investigated. A balanced nitrogen loading rate along with a dilution rate (D) of 0.3 day⁻¹ resulted in the most stable operating conditions and a PHB productivity of ~53 g PHB m⁻³ day⁻¹. However, higher PHB productivities (~127 g PHB m⁻³ day⁻¹) were achieved using nitrogen excess at a D = 0.2 day⁻¹. Overall, the high PHB contents (up to 48% w/w) obtained in the CSTR under theoretically nutrient balanced conditions and the poor process stability challenged the hypothetical advantages conferred by multistage vs single-stage process configurations for long-term PHB production.     

Cholix toxin,an eukaryotic elongation factor 2 ADP‐ribosyltransferase,interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

Vibrio cholerae produced‐Cholix toxin (Cholix) is a cytotoxin that ADP‐ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix‐interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix‐induced poly (ADP‐ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix‐induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix‐induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB‐knockdown cells. Furthermore, Cholix induced activation of Rho‐associated coiled coil‐containing protein kinase 1 (ROCK1), which was enhanced in PHB‐knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix‐induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix‐induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.     

On-line monitoring of PHB production by mixed microbial cultures using respirometry,titrimetry and chemometric modelling

This work describes a method for on-line monitoring of biomass production, acetate consumption and intracellular polyhydroxybutyrate (PHB) storage by mixed microbial cultures (MMC). The method is based on reliable and easily available on-line measurements, namely pH, dissolved oxygen, dissolved carbon dioxide, on-line respirometry and on-line titrimetric analysis. Biomass production refers to active biomass growth and also to the synthesis of extracellular polymeric substances (EPS). The composition and kinetics of EPS synthesis has high variability depending on the culture enrichment protocol. Since the metabolism for EPS production is rather difficult to define, it was not possible to develop a reliable estimation model based on metabolic principles only. Instead, projection of latent structures (PLS) linear regression constrained by steady state carbon balance was employed. PHB concentration and biomass production rate were directly estimated by the PLS model, whereas acetate concentration was indirectly estimated through the carbon balance. The method was validated experimentally with data of four experiments carried out in a SBR. Accurate on-line estimations were obtained with regression coefficients (r²) of 0.986 and 0.980 for biomass concentration, 0.976 and 0.999 for PHB and 0.992 and 0.999 for acetate concentration in calibration and validation, respectively. These results confirm the ability of the proposed methodology for on-line monitoring of the state variables in PHB production process by MMC.     

Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.     

Development of a structured dynamic model for the production of polyhydroxybutyrate (PHB) in Azohydromonas lata cultures

The development of an accurate model representation of the fermentative production of polyhydroxybutyrate (PHB) is a prerequisite for the optimal operation and control of the microbial process. In the present work, a macroscopic model is developed to quantify the intracellular production of PHB in Azohydromonas lata bacteria. The proposed two-compartment structured model provides an accurate prediction of the metabolic and macroscopic phenomena occurring in A. lata bacterial cultures. Precisely, the proposed dynamic model accounts for biomass growth, polymer accumulation, carbon and nitrogen sources utilization and oxygen mass transfer rates. It is shown that the model can closely describe the time evolution of the bacterial culture via its direct comparison with experiments performed in flasks or/and a bioreactor. Moreover, it is shown that the model can be used as a simulation tool for process optimization and scaling-up studies of the PHB fermentative production in A. lata cultures.     

Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs), of which polyhydroxybutyrate (PHB) is the most abundant, are bacterial carbon and energy reserve materials of widespread occurrence. They are composed of 3-hydroxyacid monomer units and exist as a small number of cytoplasmic granules per cell. The properties of the C4 homopolymer PHB as a biodegradable thermoplastic first attracted industrial attention more than 20 years ago. Copolymers of C4 (3-hydroxybutyrate [3HB]) and C5 (3-hydroxyvalerate [3HV]) monomer units have modified physical properties; e.g., the plastic is less brittle than PHB, whereas PHAs containing C8 to C12 monomers behave as elastomers. This family of materials is the centre of considerable commercial interest, and 3HB-co-3HV copolymers have been marketed by ICI plc as Biopol. The known polymers exist as 2(1) helices with the fiber repeat decreasing from 0.596 nm for PHB to about 0.45 nm for C8 to C10 polymers. Novel copolymers with a backbone of 3HB and 4HB have been obtained. The native granules contain noncrystalline polymer, and water may possibly act as a plasticizer. Although the biosynthesis and regulation of PHB are generally well understood, the corresponding information for the synthesis of long-side-chain PHAs from alkanes, alcohols, and organic acids is still incomplete. The precise mechanisms of action of the polymerizing and depolymerizing enzymes also remain to be established. The structural genes for the three key enzymes of PHB synthesis from acetyl coenzyme A in Alcaligenes eutrophus have been cloned, sequenced, and expressed in Escherichia coli. Polymer molecular weights appear to be species specific. The factors influencing the commercial choice of organism, substrate, and isolation process are discussed. The physiological functions of PHB as a reserve material and in symbiotic nitrogen fixation and its presence in bacterial plasma membranes and putative role in transformability and calcium signaling are also considered.     

Selection of Type I and Type II methanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions

Type II methanotrophs produce polyhydroxybutyrate (PHB), while Type I methanotrophs do not. A laboratory-scale fluidized bed reactor was initially inoculated with a Type II Methylocystis-like dominated culture. At elevated levels of dissolved oxygen (DO, 9 mg/L), pH of 6.2–6.5 with nitrate as the N-source, a Methylobacter-like Type I methanotroph became dominant within the biofilms which did not produce PHB. A shift to biofilms capable of PHB production was achieved by re-inoculating with Type II Methylosinus culture, providing dissolved N₂ as the N-source, and maintaining a low influent DO (2.0 mg/L). The resulting biofilms contained both Types I and II methanotrophs. Batch tests indicated that biofilm samples grown with N₂ became dominated by Type II methanotrophs and produced PHB. Enrichments with nitrate or ammonium were dominated by Type I methanotrophs without PHB production capability. The key selection factors favoring Type II were N₂ as N-source and low DO.     

Proteomic analysis of placentas from cloned cat embryos identifies a set of differentially expressed proteins related to oxidative damage, senescence and apoptosis

Production of cloned mammals by somatic cell nuclear transfer is associated with functional and structural abnormalities of placentation and with abnormal fetal development. A proteomic analysis was performed in domestic cats (Felis catus) to compare cloned term placentas (CTP) obtained from cesarean section (CS) to control placentas obtained from CS or vaginal delivery. The expression of 20 proteins was altered in CTP (p<0.05) compared to control placentas. The two control groups showed that the method of delivery, vaginal delivery or CS, did not affect protein expression (p>0.05). A total of 13 proteins were up-regulated in CTP, including apoptosis-related cathepsin D (CD), annexin A1 and heat shock protein 27 (HSP 27), and seven proteins were down-regulated in CTP, including prohibitin (PHB). The expression of PHB and CD was confirmed by Western blotting and immunofluorescence staining. The abnormal expression of PHB and CD correlated with the generation of reactive oxygen species, leading to decreased mitochondrial membrane potential and telomeric DNA, which are associated with cellular senescence and apoptosis. In summary, a specific pattern of abnormal protein expression is associated with the impaired development and functions of cloned placentas and hence with decreased fetal viability. Strategies aimed at restoring normal placental protein expression may increase the efficiency of somatic cell nuclear transfer and transgenic cat production and help restore endangered species.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. I. Free cell cultures

As part of an investigation into the use of biological nitrogen fixation for fertilizer ammonia production, continuous culture studies of respiration and nitrogen fixation in the aerobic bacteria Azotobacter vinelandii under oxygen-limited conditions were conducted. Respiration and growth rates followed Monod forms with respect to dissolved oxygen concentration. However, specific nitrogen fixation rate and nitrogenase activity exhibited maximum values at dissolved oxygen concentrations of ca. 0.02 mM (10% of air saturation). These results suggest careful control of oxygen in the environment is necessary to optimize fixed nitrogen production by this organism.     

Model-based intensification of a fed-batch microbial process for the maximization of polyhydroxybutyrate (PHB) production rate

An integrated metabolic–polymerization–macroscopic model, describing the microbial production of polyhydroxybutyrate (PHB) in Azohydromonas lata bacteria, was developed and validated using a comprehensive series of experimental measurements. The model accounted for biomass growth, biopolymer accumulation, carbon and nitrogen sources utilization, oxygen mass transfer and uptake rates and average molecular weights of the accumulated PHB, produced under batch and fed-batch cultivation conditions. Model predictions were in excellent agreement with experimental measurements. The validated model was subsequently utilized to calculate optimal operating conditions and feeding policies for maximizing PHB productivity for desired PHB molecular properties. More specifically, two optimal fed-batch strategies were calculated and experimentally tested: (1) a nitrogen-limited fed-batch policy and (2) a nitrogen sufficient one. The calculated optimal operating policies resulted in a maximum PHB content (94% g/g) in the cultivated bacteria and a biopolymer productivity of 4.2 g/(l h), respectively. Moreover, it was demonstrated that different PHB grades with weight average molecular weights of up to 1513 kg/mol could be produced via the optimal selection of bioprocess operating conditions.

     

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.     

New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.     

The polyhydroxyalkanoate genes of a stress resistant Antarctic Pseudomonas are situated within a genomic island

Pseudomonas sp. 14-3 is an Antarctic bacterium that shows high stress resistance in association with high polyhydroxybutyrate (PHB) production. In this paper genes involved in PHB biosynthesis (phaRBAC) were found within a genomic island named pha-GI. Numerous mobile elements or proteins associated with them, such as an integrase, insertion sequences, a bacterial group II intron, a complete Type I protein secretion system and IncP plasmid-related proteins were detected among the 28 ORFs identified in this large genetic element (32.3kb). The G+C distribution was not homogeneous, likely reflecting a mosaic structure that contains regions from diverse origins. pha-GI has strong similarities with genomic islands found in diverse Proteobacteria, including Burkholderiales species and Azotobacter vinelandii. The G+C content, phylogeny inference and codon usage analysis showed that the phaBAC cluster itself has a complex mosaic structure and indicated that the phaB and phaC genes were acquired by horizontal transfer, probably derived from Burkholderiales. These results describe for the first time a pha cluster located within a genomic island, and suggest that horizontal transfer of pha genes is a mechanism of adaptability to stress conditions such as those found in the extreme Antarctic environment.     

A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti

During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-beta-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and beta-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.     

Increased Bioplastic Production with an RNA Polymerase Sigma Factor SigE during Nitrogen Starvation in Synechocystis sp. PCC 6803

Because cyanobacteria directly harvest CO₂ and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.