A fundamental dual regulatory role of citrate on the biosyntheses of thuringiensin and poly-<Emphasis Type="Italic">β</Emphasis>-hydroxybutyrate in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> YBT-032

The production of α-ketoglutarate, adenine, thuringiensin production rate and thuringiensin yield on glucose consumed increased by 22%, 36%, 40% and 40%, respectively, in presence of 2 g citrate/l. However, citrate decreased pyruvate production, poly-β-hydroxybutyrate (PHB) production rate and PHB yield by 62%, 31% and 45%, respectively. The activities of pyruvate kinase and glucose-6-phosphate dehydrogenase were 36%–45% lower and 50%–120% higher than those of the control, respectively. The results suggest that citrate regulated the carbon flux to synthesis of adenine present in thuringiensin with a higher efficiency of utilization of glucose by decreasing PHB synthesis.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Production of poly(β-hydroxybutyric acid) and exopolysaccharide by Azotobacter beijerinckii WDN-01

The physico-chemical factors influencing the production of poly(-hydroxybutyric acid) [PHB] and exopolysaccharide (EPS) by a yellow pigmented Azotobacter beijerinckii strain WDN-01 were investigated. Under N-free condition with excess carbon, PHB accumulation attained its maximum at the late exponential phase followed by a sharp decline while EPS production was more or less parallel with growth. Polymer synthesis, however, was carbon-source-specific, the highest yield of PHB (2.73 g/l) and EPS (1.5 g/l) was obtained with 3% (w/v) glucose and mannitol respectively. Organic N-sources enhanced PHB production significantly, but inorganic nitrogenous compounds were inhibitory to both PHB and EPS synthesis. At optimum K₂HPO₄ concentration, the polymer yield was attributed to biomass yield. Oxygen-limiting conditions, irrespective of carbon sources favoured production of PHB and EPS.     

Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Thiolysis of poly(3-hydroxybutyrate) with polyhydroxyalkanoate synthase from Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) is synthesized from 3-hydroxybutyryl-CoA by polyhydroxyalkanoate synthase and hydrolyzed by PHB depolymerase. In this study, we focused on the reverse reaction of polyhydroxyalkanoate synthase, and propose the possibility that PHB can be degraded through a novel process, that is thiolysis of PHB with CoASH. Polyhydroxyalkanoate synthase of Ralstonia eutropha was incubated with 14C-labeled PHB and CoASH. The reaction mixture was fractionated by HPLC and then analyzed with a scintillation counter. The analysis revealed 3-hydroxybutyryl-CoA to be a product of the reaction. When NADP+ and acetoacetyl-CoA reductase were added to the reaction mixture, an increase in absorbance at 340 nm was observed. Native PHB inclusion bodies from R. eutropha also showed thiolytic activity. This is the first indication that polyhydroxyalkanoate synthase catalyzes both the synthesis and degradation of PHB, and that native PHB inclusion bodies has thiolytic activity.     

Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli

In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M _r∼55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca²⁺- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Wastewater utilization for poly-β-hydroxybutyrate production by the cyanobacterium Aulosira fertilissima in a recirculatory aquaculture system

Intensive aquaculture releases large quantities of nutrients into aquatic bodies, which can lead to eutrophication. The objective of this study was the development of a biological recirculatory wastewater treatment system with a diazotrophic cyanobacterium, Aulosira fertilissima, and simultaneous production of valuable product in the form of poly-β-hydroxybutyrate (PHB). To investigate this possible synergy, batch scale tests were conducted under a recirculatory aquaculture system in fiber-reinforced plastic tanks enhanced by several manageable parameters (e.g., sedimentation, inoculum size, depth, turbulence, and light intensity), an adequate combination of which showed better productivity. The dissolved-oxygen level increased in the range of 3.2 to 6.9 mg liter⁻¹ during the culture period. Nutrients such as ammonia, nitrite, and phosphate decreased to as low as zero within 15 days of incubation, indicating the system''s bioremediation capability while yielding valuable cyanobacterial biomass for PHB production. Maximum PHB accumulation in A. fertilissima was found in sedimented fish pond discharge at 20-cm culture depth with stirring and an initial inoculum size of 80 mg dry cell weight (dcw) liter⁻¹. Under optimized conditions, the PHB yield was boosted to 92, 89, and 80 g m⁻², respectively for the summer, rainy, and winter seasons. Extrapolation of the result showed that a hectare of A. fertilissima cultivation in fish pond discharge would give an annual harvest of ∼17 tons dry biomass, consisting of 14 tons of PHB with material properties comparable to those of the bacterial polymer, with simultaneous treatment of 32,640 m³ water discharge.     

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis> with proteome analysis

RecombinantEscherichia coli strain harboring the λp _R-p _L promotor and heterologus poly-β-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression ofphb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lactate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Overexpression of NAD kinase in recombinant <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> harboring the <Emphasis Type="BoldItalic">phbCAB</Emphasis> operon improves poly(3-hydroxybutyrate) production

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y _PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.     

植株密度和氮素互作对垂穗披碱草生物量分配的影响

垂穗披碱草（Elymus nutans）是高寒地区建植和改良栽培草地的首选草种。虽然合理植株密度和氮素添加量是垂穗披碱草栽培草地稳产的关键因子,但两者之间是否存在最佳互作组合仍不清楚。采用盆栽试验,通过分析不同植株密度（58、102、146株/m²）和氮素添加量（0、200、400 mg/kg）组合状态下垂穗披碱草株高、单株分蘖数、地上生物量、地下生物量、根系体积和地上地下生物量比,以确定理论上是否存在植株密度和氮素添加量的最佳组合。结果表明：随植株密度增加,垂穗披碱草株高、地上生物量和地上地下生物量比值均先增加后降低,而单株分蘖数逐渐减小,根系体积和地下生物量先增加后保持相对稳定;随氮素添加量增加,垂穗披碱草单株分蘖数、地上生物量和地上地下生物量比值均表现为先增加后降低,地下生物量逐渐降低。植株密度与氮素添加量互作虽然对垂穗披碱草的根系体积和单株分蘖数没有显著影响,但两者互作显著影响了垂穗披碱草株高、地上生物量、地下生物量、地上地下生物量比（P<0.01）,这些指标与植株密度和氮素添加量的关系均表现为一个开口向下的抛物面。当植株密度为102株/m²和氮素添加量为200 mg/kg时,垂穗披碱草栽培草地产量最大,生物量分配最优。垂穗披碱草植株密度和氮素添加互作时理论上存在最佳组合,这为垂穗披碱草栽培草地的田间管理提供了理论依据。     

Recovery of Poly-β-Hydroxybutyrate from Estuarine Microflora

Poly-β-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-¹⁴C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Poly-beta-hydroxybutyrate production by Pseudomonas sp. RZS 1 under aerobic and semi-aerobic condition

Pseudomonas sp. RZS1 was isolated from distillery effluent and identified based on phenotypic characters and 16s rRNA sequencing. It accumulated optimum amount (703.79 microg/mg of biomass) of poly-beta-hydroxybutyrate (PHB) under aerobic process of fermentation and 75 microg/mg of biomass under the anaerobic process of fermentation. Aerobic fermentation yielded 9.3-fold more PHB than semi-aerobic fermentation. Acetone alcohol method proved to be the best suitable recovery method as it gave 703.79 microg PHB per mg of biomass with a percentage recovery yield of 70.37. It started to accumulate PHB at the end of lag phase (from 6 h of incubation). Optimum amount of PHB (20 microg/ml) was reported during early stationary phase (30 h of incubation). Extracted PHB showed two peaks, minor one at 248 nm and major one at 365 nm. IR spectra revealed the presence of functional groups characteristics of PHB.     

Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by solvent extraction with 1,2‐propylene carbonate

An integrated procedure for the recovery of polyhydroxybutyrate (PHB) produced by Cupriavidus necator based on the extraction with 1,2‐propylene carbonate was evaluated. The effect of temperature (100–145°C) and contact time (15–45 min), precipitation period, and biomass pretreatments (pH shock and/or thermal treatments) on PHB extraction efficiency and polymer properties was evaluated. The highest yield (95%) and purity (84%) were obtained with the combination of a temperature of 130°C and a contact time of 30 min, with a precipitation period of 48 h. Under these conditions, PHB had a molecular weight of 7.4×10⁵, which was the highest value obtained. Lower values (2.2×10⁵) were obtained for higher temperatures (145°C), while lower temperatures resulted in incomplete extraction yields (45–54%). No further yield improvement was achieved with the pH/heat pretreatments, but the polymer's molecular weight was increased to 1.3×10⁶. The PHB physical properties were not significantly affected by any of the tested procedures, as shown by the narrow ranges obtained for the glass transition temperature (4.8–5.0°C), melting temperature (170.1–180.1°C), melting enthalpy (77.8–88.5 J/g) and crystallinity (55–62%). 1,2‐Propylene carbonate was shown to be an efficient solvent for the extraction of PHB from biomass. The precipitation procedure was found to highly influence the polymer recovery and its molecular weight. Although polymer molecular weight and purity were improved by applying pH/heat pretreatment to the biomass, the procedure involves the use of large amounts of chemicals, which increases the recovery costs and makes the process environmentally unfriendly.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome‐targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β‐oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.     

Structural and metabolic correlation for <Emphasis Type="Italic">Bacillus megaterium</Emphasis> ACBT03 in response to colchicine biotransformation

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.     

Hypochlorite digestion method for efficient recovery of PHB from <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We reported the optimum amount of PHB accumulated by Alcaligenes faecalis during its 24 h growth under nitrogen deficient conditions. After 24 h incubation decrease in the amount of PHB was recorded. Hypochlorite digestion of biomass of organism followed by extraction with a solvent system consisting of 1:1 mixture of ethanol and acetone resulted in efficient recovery of PHB vis-à-vis earlier methods. This solvent system gave a high recovery yield, i.e. 5.6 gL⁻¹ vis-à-vis earlier reported yield, 1.34 gL⁻¹ (by same method), 0.63 gL⁻¹ (by chloroform extraction method) and 1.1 gL⁻¹ (by dispersion method).     

Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and Nostoc PCC7936

Two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested with regard to their metal removal capability by using copper as model metal. The experiments, carried out with the sole cyanobacterial biomass suspended in distilled water and confined into small dialysis tubings, showed that C. capsulata biomass is characterized by the best efficiency in metal removal, with a q_max (maximum amount of copper removed per biomass unit) of 96 ± 2 mg Cu(II) removed per g of protein in comparison with the value of 79 ± 3 of Nostoc PCC7936 biomass. The experimental data obtained with both cyanobacterial biomass best fit the Langmuir sorption isotherm. The sorption of copper started from the first minutes of contact with the metal and attained the equilibrium state, when no more copper removal was evident, after 5 and 6 hours, for C. capsulata and Nostoc PCC7936, respectively. The best efficiency in Cu(II) removal was obtained at pH 6.1–6.2, while the presence of Mg²⁺ or Ca²⁺ reduced copper removal capability of both species to 60–70% of their q_max. The results showed that the biomass of C. capsulata and Nostoc PCC7936 possesses a high affinity and a high specific uptake for copper, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use the capsulated trichomes of the two cyanobacteria for the bioremoval of heavy metals from polluted water bodies.     

Production of poly-β-hydroxybutyrate (PHB) by <Emphasis Type="Italic">Alcaligenes latus</Emphasis> from maple sap

Maple sap, an abundant natural product especially in Canada, is rich in sucrose and thus may represent an ideal renewable feedstock for the production of a wide variety of value-added products. In the present study, maple sap or sucrose was employed as a carbon source to Alcaligenes latus for the production of poly-β-hydroxybutyrate (PHB). In shake flasks, the biomass obtained from both the sap and sucrose were 4.4 ± 0.5 and 2.9 ± 0.3 g/L, and the PHB contents were 77.6 ± 1.5 and 74.1 ± 2.0%, respectively. Subsequent batch fermentation (10 L sap) resulted in the formation of 4.2 ± 0.3 g/L biomass and a PHB content of 77.0 ± 2.6%. The number average molecular weights of the PHB produced by A. latus from maple sap and pure sucrose media were 300 ± 66 × 10³ and 313 ± 104 × 10³ g/mol, respectively. Near-infrared, ¹H magnetic resonance imaging (MRI), and ¹³C-MRI spectra of the microbially produced PHB completely matched those obtained with a reference material of poly[(R)-3-hydroxybutyric acid]. The polymer was found to be optically active with [α]²⁵ _D equaled to −7.87 in chloroform. The melting point (177.0°C) and enthalpy of fusion (77.2 J/g) of the polymer were also in line with those reported, i.e., 177°C and 81 J/g, respectively.