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.     

Effect of total nutrient feed on production of poly-3-hydroxybutyrate by Methylobacterium sp. ZP24 grown on sugars

Methylobacterium sp. ZP24 is able to produce poly-3-hydroxybutyrate (PHB) from sucrose and lactose. As the production of PHB is growth-associated, a strategy of intermittent feeding of sugars and other nutrients was assessed for obtaining high yields of the polymer. Higher PHB synthesis was obtained at increased sugar feed rates. Cellular PHB contents of 63% and 71%, with productivities of up to 0.354 and 0.645 g PHB/l h were obtained from sucrose and lactose, respectively. A short-duration semicontinuous production level of up to 2.4 g PHB/l h was achieved in the lactose fermentation. Journal of Industrial Microbiology & Biotechnology (2000) 25, 276–279. Received 06 June 2000/ Accepted in revised form 31 August 2000     

Increased PHB productivity by high-cell-density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer

Alcaligenes latus, a growth-associated PHB producer, was cultivated by a pH-stat modal fed-batch culture technique to attain high PHB productivity. Both sucrose solution and inorganic medium were fed in conjunction with the supply of ammonia solution which serves as a nitrogen source and as a means of pH control. Compositions of the inorganic medium were formulated by elemental analysis of A. latus cell mass. The effect on inoculum size was examined to reduce culture time. High concentrations of cell (142 g/L) and PHB (68.4 g/L) were obtained in a short culture time (18 h) with an inoculum size of 13.7 g/L. The PHB content and the PHB productivity at the end of the fed-batch culture were 50% of dry cell weight and 4.0 g PHB/(L . h), respectively. (c) 1996 John Wiley & Sons, Inc.     

Kinetic studies and biochemical pathway analysis of anaerobic poly-(R)-3-hydroxybutyric acid synthesis in Escherichia coli

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 +/- 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.     

Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans

A sheathed bacterium Sphaerotilus natans could not survive at 4°C for 2 months, and mutants that exhibited different colony phenotypes were obtained only by repeating the short period of storage at 4 °C. The ability of these mutants and the parent strain to produce poly-3-hydroxybutyrate (PHB) was compared in batch cultures. The parent strain accumulated 30% (w/w) PHB, while one of the mutants defective in sheath formation, designated as T2, accumulated over 50% PHB. Because T2 did not require strict air or nitrogen limitation for polymer accumulation, its production was growth-associated, allowing one-stage fermentation. In a pH-controlled fermentation using a jar fermentor, 10 g/l glucose was converted into 2.0 g/l PHB in 24 h.     

Kinetic Studies and Biochemical Pathway Analysis of Anaerobic Poly-(R)-3-Hydroxybutyric Acid Synthesis in Escherichia coli

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 ± 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.     

Growth-associated production of poly-β-hydroxybutyrate from glucose or alcoholic distillery wastewater by Actinobacillus sp. EL-9

Summary The characteristics of poly--hydroxybutyrate (PHB) production from glucose or alcoholic distillery wastewater by isolated Actinobacillus sp. EL-9 were investigated. PHB production was not dependent on nutrients limitation in Actinobacillus sp. EL-9. The PHB accumulation of Actibobacillus sp. EL-9 followed a growth-associated type where the cell growth and PHB accumulation were carried out simultaneously. The Actinobacillus sp. EL-9 was shown to synthesize and accumulate PHB from alcoholic distillery wastewater during growth. The best growth and PHB production were obtained with enzyme-hydrolyzed alcoholic distillery wastewater.     

Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.     

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.     

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.     

Growth-associated production of poly(hydroxybutyric acid) by Azotobacter beijerinckii from organic nitrogen substrates

Poly(hydroxybutyric acid) (PHB) was produced by a selectant of Azotobacter beijerinckii in media containing only organic nitrogen sources such as N substrates. The chosen compounds were casein peptone, yeast extract, casamino acids and urea, each combined with carbon substrates glucose or sucrose. The PHB was synthesized under growth-associated conditions. The concentrations amounted to more than 50% of cell dry mass on casein peptone/glucose as well as urea/glucose medium within 45 h fermentation time. Corresponding to these yields, productivities of about 0.8 g PHB l⁻¹ h⁻¹ were discovered. The highest values increased to 1.06 g PHB l⁻¹ h⁻¹ on casein peptone/glucose medium and 1.1 g PHB l⁻¹ h⁻¹ on yeast extract/glucose medium after a period of 20 h. It was found that oxygen limitation was essential for successful product formation, as demonstrated earlier. These data from basic research may support further investigations into the use of technical proteins from renewable sources as substrates for PHB production by a strain of A. beijerinckii. Received: 3 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

Mathematical modelling and process optimization of a continuous 5-stage bioreactor cascade for production of poly[-(R)-3-hydroxybutyrate] by Cupriavidus necator

A multistage system for poly(hydroxyalkanoate) (PHA) production consisting of five continuous stirred tank reactors in series (5-CSTR) with Cupriavidus necator DSM 545 as production strain was modelled using formal kinetic relations. Partially growth-associated production of PHA under nitrogen limited growth was chosen as modelling strategy, thus the Luedeking-Piret’s model of partial growth-associated product synthesis was applied as working hypothesis. Specific growth rate relations adjusted for double substrate (C and N source) limited growth according to Megee et al. and Mankad-Bungay relation were tested. The first stage of the reactor cascade was modelled according to the principle of nutrient balanced continuous biomass production system, the second one as two substrate controlled process, while the three subsequent reactors were adjusted to produce PHB under continuous C source fed and nitrogen deficiency. Simulated results of production obtained by the applied mathematical models and computational optimization indicate that PHB productivity of the whole system could be significantly increased (from experimentally achieved 2.14 g L^?1 h^?1 to simulated 9.95 g L^?1 h^?1) if certain experimental conditions would have been applied (overall dilution rate, C and N source feed concentration). Additionally, supplemental feeding strategy for switching from batch to continuous mode of cultivation was proposed to avoid substrate inhibition.     

Comparative study on the production of poly(3-hydroxybutyrate) by thermophilic Chelatococcus daeguensis TAD1: a good candidate for large-scale production

In spite of numerous advantages on operating fermentation at elevated temperatures, very few thermophilic bacteria with polyhydroxyalkanoates (PHAs)-accumulating ability have yet been found in contrast to the tremendous mesophiles with the same ability. In this study, a thermophilic poly(3-hydroxybutyrate) (PHB)-accumulating bacteria (Chelatococcus daeguensis TAD1), isolated from the biofilm of a biotrickling filter used for NOx removal, was extensively investigated and compared to other PHB-accumulating bacteria. The results demonstrate that C. daeguensis TAD1 is a growth-associated PHB-accumulating bacterium without obvious nutrient limitation, which was capable of accumulating PHB up to 83.6 % of cell dry weight (CDW, w/w) within just 24 h at 45 °C from glucose. Surprisingly, the PHB production of C. daeguensis TAD1 exhibited strong tolerance to high heat stress as well as nitrogen loads compared to that of other PHB-accumulating bacterium, while the optimal PHB amount (3.44?±?0.3 g l^?1) occurred at 50 °C and C/N?=?30 (molar) with glucose as the sole carbon source. In addition, C. daeguensis TAD1 could effectively utilize various cheap substrates (starch or glycerol) for PHB production without pre-hydrolyzed, particularly the glycerol, exhibiting the highest product yield (Y _P/S, 0.26 g PHB per gram substrate used) as well as PHB content (80.4 % of CDW, w/w) compared to other carbon sources. Consequently, C. daeguensis TAD1 is a viable candidate for large-scale production of PHB via utilizing starch or glycerol as the raw materials.     

Growth-associated production of poly-3-hydroxybutyrate byBacillus mycoides

Bacillus mycoides strain RIJ B-017, a growth-associated poly-3-hydroxybutyrate (PHB) producer was grown on sucrose-containing media. PHB accumulated in cells up to 72% of dry cell mass. The overall maximum value of PHB yield (Y _p/s) and productivities (Q _p andq _p) 250 mg_p/g_s, 120 mg_p L⁻¹ h⁻¹ and 30 mg_p g_x ⁻¹ h⁻¹, respectively, were obtained at 15 g/L sucrose. Differential scanning calorimeter heating curve showed two peaks, one at 95.9 °C and another at 165.4°C with a shoulder around 154.6 °C. The viscosity-average molar mass in chloroform at 27°C was 505 kDa. The carbon content of PHB was 55.4% of the mass.     

富养罗尔斯通氏菌菌株PHB"泄漏"机理的初探

建立了一种分离纯化聚羟基丁酸(Polyhydroxybutyrate,PHB)颗粒的改良方法。采用这种方法从Ralstonia eutropha菌株H16(野生型)、SK1489(Tn5诱变的PHB泄漏菌株)、JMP222(野生的PHB泄漏菌株)分离了PHB颗粒。进一步比较研究了不同菌株的PHB解聚酶和3-羟基丁酸脱氢酶的活性。研究结果表明,菌株SK1489的PHB解聚酶活性(48h培养后达1．82U／mg)明显高于野生型菌株H16(48h培养后达0．37U／mg),菌株JMP222的3-羟基丁酸脱氢酶活性(培养96h后达165．9U／mg)比菌株H16培养(96h后达64．0U／mg)高许多。这些结果显示,不同菌株PHB的泄漏有不同的原因,突变株SK1489导致PHB泄漏的原因是解聚酶活性高,而野生型JMP222PHB泄漏的原因主要是3-羟基丁酸脱氢酶活性高。     

Model of the anaerobic metabolism of the biological phosphorus removal process: Stoichiometry and pH influence

In the anaerobic phase of a biological phosphorus removal process, acetate is taken up and converted to PHB utilizing both energy generated in the degradation of polyphosphate to phosphate, which is released, and energy generated in the conversion of glycogen to poly-beta-hydroxy butyrate (PHB). The phosphate/acetate ratio cannot be considered a metabolic constant, because the energy requirement for the uptake of acetate is strongly influenced by the pH value. The observed phosphate/acetate ratio shows a variation of 0.25 to 0.75 P-mol/C-mol in a pH range of 5.5 to 8.5. It is shown that stored glycogen takes part in the metabolism to provide reduction equivalents and energy for the conversion of acetate to PHB. A structured metabolic model, based on glycogen as the source of the reduction equivalents in the anaerobic phase and the effect of the pH on the energy requirement of the uptake of acetate, is developed. The model explains the experimental results satisfactorily. (c) 1994 John Wiley & Sons, Inc.     

Salinity-induced accumulation of poly-β-hydroxybutyrate in rhizobia indicating its role in cell protection

Summary Poly β-hydroxybutyrate (PHB) is an energy and carbon storage material accumulated in response to the limitation of an essential nutrient. The effect of different salt concentrations on growth and PHB accumulation of four different Sinorhizobium strains was examined. Irrespective of the strain, a defined trend in the accumulation of PHB inside the cells was observed. While minimum PHB content was accumulated at low or zero salinity, maximum was observed by the salt-tolerant strains at higher salt concentrations. This suggests a definite role for PHB in cell protection in saline conditions.     

Poly(3‐hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation

Numerous bacteria accumulate poly(3‐hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species.