Effect of ammonium supplementation on production of poly-B-hydroxybutyric acid byAlcaligenes eutrophus in batch culture

Summary Alcaligenes eutrophus H16 showed some degree of oxygen sensitivity, having the fastest specific growth rate at a dissolved oxygen tension of 60% air saturation. Growth and PHB accumulation kinetics were studied and an accelerated rate of PHB synthesis was obtained when a low concentration of NH₄Cl was supplied during the PHB synthesis stage.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

Poly(3-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> in fed-batch culture

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l⁻¹). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH₄Cl and K₂HPO₄ on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH₄Cl and 0.22% (w/v) K₂HPO₄ and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l⁻¹, respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH₄Cl, and K₂HPO₄ were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l⁻¹, PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l⁻¹ h⁻¹.     

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.     

Ammonium-induced proline and sucrose accumulation, and their significance in antioxidative activity and osmotic adjustment

Excess of ammonia generates oxidative and osmotic stress, and results in an accumulation of compatible solutes. The aim of this study was to investigate the physiological significance of excess ammonium-induced proline and sucrose accumulation on antioxidative activity and osmotic adjustment. The detached leaves of white clover (Trifolium repense L.) were fed with 0, 10, 50, 100, and 200 mM NH₄Cl, and the contribution of proline and sucrose to osmotic adjustment and their relationship with antioxidative enzymes activity were assessed. A gradual decline of relative water content and osmotic potential (Ψ_π) with increasing NH₄Cl feeding level was accompanied by an increase in ammonia concentration. Significant accumulation of proline and sucrose was observed when NH₄Cl was fed over 100 mM compared with control (0 mM NH₄Cl). The increase in enzyme activity was significant only at 200 mM for ascorbate peroxidase (APOD) and over 100 mM NH₄Cl for guaiacol peroxidase (GPOD) and catalase (CAT). The contribution of proline and sucrose to osmotic adjustment over 100 mM, where proline and sucrose accumulation was more important, maintained at control levels or significantly decreased. The content of proline and sucrose as affected by NH₄Cl feeding level was positively related with the activity of APOD, GPOD, and CAT. These results suggest that proline and sucrose accumulation induced by the excess of ammonium has a more influential role in antioxidative activity rather than osmotic adjustment.     

Growth of Azotobacter vinelandii UWD in Fish Peptone Medium and Simplified Extraction of Poly-β-Hydroxybutyrate

Azotobacter vinelandii UWD was grown in a fermentor with glucose medium with and without 0.1% fish peptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-β-hydroxybutyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH₄⁺, but PHB synthesis stopped when NH₄⁺ was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH₄⁺ depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells that demonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present in fed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from 0.43 g/g, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growth phase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphic cells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH₃ (pH 11.4) at 45°C for 10 min. This treatment removed about 10% of the non-PHB mass from the pellet, of which 60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residual mass. The polymer molecular weight (1.7 × 10⁶ to 2.0 × 10⁶) and dispersity (1.0 to 1.9) were not significantly affected (P = 0.05) by this treatment. In addition, the NH₃ extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bioplastics is discussed.     

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

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

Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations

Poly(3-hydroxybutyrate) (PHB) was produced by fed-batch cultures of Ralstonia eutropha with phosphate limitation under different glucose concentrations. When glucose was kept at 2.5 g l^–1, cell growth and PHB synthesis were limited due to the shortage of carbon source but a higher PHB content occurred in the cell-growth stage. This shows that a low glucose concentration is favorable for PHB accumulation in R. eutropha. PHB obtained with glucose at 9 g l^–1 is 1.6 times that obtained with 40 g l^–1. When glucose was in the range of 9 to 40 g l^–1, PHB concentration and productivity decreased significantly with the increase of glucose concentration. The highest PHB productivity was obtained with glucose at 9 g l^–1.     

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

The metabolic pathways of the central carbon metabolism in Saccharomyces cerevisiae are well studied and consequently S. cerevisiae has been widely evaluated as a cell factory for many industrial biological products. In this study, we investigated the effect of engineering the supply of precursor, acetyl‐CoA, and cofactor, NADPH, on the biosynthesis of the bacterial biopolymer polyhydroxybutyrate (PHB), in S. cerevisiae. Supply of acetyl‐CoA was engineered by over‐expression of genes from the ethanol degradation pathway or by heterologous expression of the phophoketolase pathway from Aspergillus nidulans. Both strategies improved the production of PHB. Integration of gapN encoding NADP⁺‐dependent glyceraldehyde‐3‐phosphate dehydrogenase from Streptococcus mutans into the genome enabled an increased supply of NADPH resulting in a decrease in glycerol production and increased production of PHB. The strategy that resulted in the highest PHB production after 100 h was with a strain harboring the phosphoketolase pathway to supply acetyl‐CoA without the need of increased NADPH production by gapN integration. The results from this study imply that during the exponential growth on glucose, the biosynthesis of PHB in S. cerevisiae is likely to be limited by the supply of NADPH whereas supply of acetyl‐CoA as precursor plays a more important role in the improvement of PHB production during growth on ethanol. Biotechnol. Bioeng. 2013; 110: 2216–2224. © 2013 Wiley Periodicals, Inc.     

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.     

Dynamics and modeling on fermentative production of poly (β-hydroxybutyric acid) from sugars via lactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus

The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly β-hydroxybutyrate (PHB) by Alcaligenes eutrophus in one fermentor. For the modeling of the effect of NH₃ concentration on the cell growth of A. eutrophus and PHB production rates, metabolic flux distributions were computed at two culture phases of cell growth and PHB production periods. It was found that the NADPH, generated through isocitrate dehydrogenate in TCA cycle, was predominantly utilized for the reaction from α-ketoglutalate to glutamate when NH₃ was abundant, while it tended to be utilized for the PHB production through acetoacetyl CoA reductase as NH₃ concentration decreased. This phenomenon was reflected in the development of mathematical model. In the mixed culture experiments, the two phases were observed, namely the lactate production phase due to L. delbrueckii and the lactate consumption phase due to A. eutrophus. The lactate concentration could be estimated on-line by the amount of NaOH solution and HCl solution supplied to keep the culture pH at constant level. Several mixed culture experiments were conducted to see the dynamics of the system. Finally, a mathematical model which can describe the dynamic behavior of the present mixed culture was developed and the model parameters were tuned for fitting the experimental data. The model may be used for several purposes such as control, optimization, and understanding process dynamics etc.     

Continuous citric acid fermentation by <Emphasis Type="Italic">Candida oleophila</Emphasis> under nitrogen limitation at constant C/N ratio

Summary The central aspect of this work was to investigate the influence of nitrogen feed rate at constant C/N ratio on continuous citric acid fermentation by Candida oleophila ATCC 20177. Medium ammonia nitrogen and glucose concentrations influenced growth and production. Space-time yield (STY) meaning volumetric productivity, biomass specific productivity (BSP), product concentration, product selectivity and citrate/isocitrate ratio increased with increasing residence time (RT). BSP increased in an exponential mode lowering nitrogen feed rates. Highest BSP for citric acid of 0.13 g/(g h) was achieved at lowest NH₄Cl concentration of 1.5 g/l and highest STY (1.2 g/l h) with 3 g NH₄Cl/l at a RT of 25 h. Citric acid 74.2 g/l were produced at 58 h RT and 6 g NH₄Cl/l. Glucose uptake rate seems to be strictly controlled by growth rate of the yeast cells. Optimum nitrogen concentration and adapted C/N ratio are essential for successful continuous citric acid fermentation. The biomass-specific nitrogen feed rate is the most important factor influencing continuous citric acid production by yeasts. Numerous chemostat experiments showed the feasibility of continuous citrate production by yeasts.     

Accumulation of ammonium ion in cadmium tolerant and sensitive cultivars of Oryza sativa

Cd-tolerant and Cd-sensitive rice cultivars were used to study the role of NH₄ ⁺ accumulation in Cd-induced toxicity. NH₄ ⁺ accumulation seems to be involved in regulating the toxicity of rice seedlings caused by CdCl₂. This conclusion was based on the observations that (a) on treatment with CdCl₂, NH₄ ⁺ content increased rapidly in the leaves of the Cd-sensitive cultivar (cv. Taichung Native 1, TN1) but not in the Cd-tolerant cultivar (cv. Tainumg 67, TNG67), (b) pretreatment with abscisic acid (ABA) enhanced Cd tolerance and reduced Cd-induced NH₄ ⁺ accumulation in TN1 seedlings, (c) exogenous application of the ABA biosynthesis inhibitor, fluridone, decreased Cd tolerance and increased NH₄ ⁺ content in leaves of TNG67, (d) exogenous application of phosphinothricin, an inhibitor of glutamine synthetase (GS), which resulted in NH₄ ⁺ accumulation in the leaves, also induced toxicity similar to Cd in TN1 seedlings. Evidence is presented to show that Cd-induced NH₄ ⁺ accumulation in TN1 leaves is attributable to a decrease in GS activity. Since Cd-treated TN1 leaves had higher glutamine and glutamate contents than control leaves, it is unlikely that glutamine (or glutamate) depletion is the mechanism which regulates Cd-induced toxicity.     

Regulatory effects of cellular nicotinamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli

Regulatory roles of nicotinamide nucleotides and three key enzymes, beta-ketothiolase (KT), NADPH-dependent acetoacetyl-CoA reductase (AAR), and citrate synthase (CS), on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli harboring a plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were examined. Cells were grown in various media and were subsequently compared for PHB concentration, PHB content, the activities of the key enzymes, and the levels of nicotinamide nucleotides. Cells of recombinant E. coli accumulated the largest amount of PHB in LB+glucose medium among those tested. PHB synthesis was not enhanced by limiting inorganic ions. The activity of CS, which competes with KT for acetyl-CoA, was lower when cells were grown in LB+glucose compared with other media. The NADPH level and the NADPH/NADP ratio were high in LB+glucose. Examination of the time profiles of the specific PHB synthesis rate, key enzyme activities, and the levels of nicotinamide nucleotides showed that PHB synthesis is most significantly affected by the NADPH level. Even though the NADH level and the NADH/NAD ratio were also high during the synthesis of PHB, no direct evidence of their positive effect on PHB synthesis was found. Low activity of CS was beneficial for PHB synthesis due to the availability of more acetyl-CoA to PHB biosynthetic pathway. In recombinant E. coli, the level of NADPH and/or the NADPH/NADP ratio seem to be the most critical factor regulating the activity of AAR and, subsequently, PHB synthesis. (c) 1996 John Wiley & Sons, Inc.     

The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH¹⁴CO₃ into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5–4-fold on incubation with 10mm-NH₄Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH¹⁴CO₃ into orotic acid was increased 3–4-fold in liver homogenate with NH₄Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH₄Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40–50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH₄Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH₄Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.     

Growth and fermentation responses of Selenomonas ruminantium to limiting and non-limiting concentrations of ammonium chloride

 The objective of this study was to assess fermentation product, growth rate and growth yield responses of Selenomonas ruminantium HD₄ to limiting and non-limiting ammonia concentrations. The ammonia half-inhibition constant for S. ruminantium in batch culture was 296 mM. Cells were grown in continuous culture with a defined ascorbate-reduced basal medium containing either 0.5, 5, 25, 50, 100 or 200 mM NH₄Cl and dilution rates were 0.07, 0.14, 0.24 or 0.40 h^-1. Ammonia was the growth-limiting nutrient when 0.5 mM NH₄Cl was provided and the half-saturation constant was 72 μM. Specific rates of glucose utilization and fermentation acid carbon formation were highest for 0.5 mM NH₄Cl. Lactate production (moles per mole of glucose disappearing) increased at the fastest dilution rate (0.40 h^-1) for 5.0 mM NH₄Cl while acetate and propionate decreased when compared to slower dilutions (0.07 and 0.14 h^-1). Lactate production remained low while acetate and propionate remained high for all dilution rates when NH₄Cl concentrations were 25 mM or greater. Yield (Y _Glc and Y _ATP) were nearly doubled when NH₄Cl was increased from 0.5 mM (25.1 g cells/mol glucose used and 13.9 g cells/mol ATP produced respectively) to the higher concentrations. Y _Glc was highest at 25 mM and 50 mM NH₄Cl (48.2 cells/mol and 43.1 cells/mol respectively) as was Y _ATP (23.2 cells/mol and 20.8 cells/mol respectively). Y _NH3 was highest at the lowest NH₄Cl concentration. The maximal fermentation product formation rate occurred at a growth-limiting ammonia concentration, while maximal glucose and ATP bacterial yields occurred at non-growth-limiting ammonia concentrations. Given the growth response of this ruminal bacterium, it is possible that maximization of ruminal bacterial yield may necessitate sacrificing the substrate degradation rate and vice versa. Received: 5 December 1995/Received revision: 2 April 1996/Accepted: 22 April 1996     

Changes of Sugar Levels in Cucumber Leaves during Ammonium Toxicity

Toxic effects of high concentrations of ammonia were studied in the cucumber plant (Cucumis sativus cv. Suisei No. 2). When the cucumber plant was cultured with 200 mg/l NH₃-N (as NH₄Cl), some characteristic symptoms, probably due to ammonium toxicity, appeared in the leaves after about 1 week, while no such symptoms were observed in the plants cultured with 20 mg/1 NH₃-N. The level of free sugars in 20 mg/l NH₃-N treated plants decreased with time and was lower than that in plants treated with 200 mg/l NH₃-N. Specially distinct differences were found as regards the levels of fructose and glucose. After 9 days' culture the content of glucose in 200 mg/l NH₃-N plants was 17 times higher than that of 20 mg/l NH₃-N plants. From the results of an incorporation of photosynthesized ¹⁴CO₂ for 3 hours into newly synthesized glucose it is evident that this accumulation of glucose can not be the degradative product of a glucose polymer such as starch. The levels of starch were also studied, and it was found that the starch level decreased due to ammonium toxicity. These results suggest that the translocation of glucose after its synthesis is inhibited by ammonium toxicity, at least up to starch synthesis.     

Synthesis of high-molecular-mass polyhydroxybutyrate from methanol in <Emphasis Type="Italic">Methyloligella halotolerans</Emphasis> C2

The influence of the concentrations of carbon and energy sources (methanol) and nitrogen (NH ₄ ⁺ ) on the yield and molecular mass (Mm) of poly-β-hydroxybutyrate (PHB) synthesized by the novel methylotroph Methyloligella halotolerans C2 was investigated. It was shown that the maximum concentrations of NH ₄ ⁺ and methanol in the medium for PHB biosynthesis were 0.15 g/L and 1 ± 0.2 mL/L, respectively. A unique high-molecular (8000–10000 kDa) PHB was obtained with NH ₄ ⁺ and methanol concentrations of 0.12 g/L and 1.25 mL/L, respectively, in a fermenter. The main physicochemical and mechanical properties of the obtained bioplastic with a molecular mass of 10000 kDa were determined.     

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

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

The Effect of Energy Substrates on PHB Accumulation of Acidiphilium cryptum DX1-1

The effect of glucose and elemental sulfur on the growth and PHB accumulation of Acidiphilium cryptum DX1-1 was investigated. Meanwhile, the differential expressions of 19 genes related with PHB accumulation, sulfur metabolism and carbon fixed in heterotrophy, phytotrophy and mixotrophy were studied by RT-qPCR. The results showed that strain DX1-1 could accumulate PHB with sulfur as the energy substance and atmospheric CO₂ as carbon resource. Glucose could improve the growth of strain DX1-1 cultured in medium with sulfur as the energy substance, and almost all the key enzyme-encoding genes related with PHB, sulfur metabolism and carbon fixed were basically up-regulated. PHB polymerase (Arcy_3030), ribulose-bisphosphate carboxylase (Acry_0825), ribulose-phosphate-epimerase (Acry_0022), and cysteine synthase A (Acry_2560) played important role in PHB accumulation, the modified expression of which could influence the PHB yield. With CO₂ as carbon resource, the main initial substance of PHB accumulation for strain DX1-1 was acetyl-CoA, instead of acetate with the glucose as the carbon resource. Because of accumulating PHB by fixed atmospheric CO₂ while independent of light, A. cryptum DX1-1 may have specifically potential in production of PHB.