Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803

Poly-beta-hydroxybutyrate (PHB) accumulation in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, was studied under various cultural and nutritional conditions. Under controlled condition, cells harvested at the stationary phase of growth depicted maximum accumulation of PHB, i.e., 4.5% (w/w of dry cells) as compared to lag (1.8%) or logarithmic (2.9%) phases of cultures. A temperature range of 28-32 degrees C and pH between 7.5 and 8.5 were preferred for PHB accumulation. Cells cultivated under regular light-dark cycles accumulated more PHB (4.5%) than those grown under continuous illumination (2.4%). Nitrogen and phosphorus starvation stimulated PHB accumulation up to the tune of 9.5 and 11% (w/w of dry cells), respectively. Synechocystis cells pre-grown in glucose (0.1%)-supplemented BG-11 medium when subjected to P-deficiency in presence of acetate (0.4%), PHB accumulation was boosted up to 29% (w/w of dry cells), the value almost 6-fold higher with respect to photoautotrophic condition. Fishpond discharges were found as suitable media for PHB accumulation in the test cyanobacterium.     

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: effects of metabolic inhibitors

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.     

Process optimization for poly-beta-hydroxybutyrate production in a nitrogen fixing cyanobacterium, Nostoc muscorum using response surface methodology

A five-level-four-factor central composite rotary design was employed to find out the interactive effects of four variables, viz. concentrations of acetate, glucose and K2HPO4, and dark incubation period on poly-beta-hydroxybutyrate (PHB) production in a N2-fixing cyanobacterium, Nostoc muscorum. Acetate, glucose and dark incubation period exhibited positive impacts on PHB yield. Using response surface methodology (RSM), a second order polynomial equation was obtained by multiple regression analysis. A yield of 45.6% of dry cell weight (dcw) was achieved at reduced level of nutrients, i.e. 0.17% acetate, 0.16% glucose and 5 mg l(-1) K2HPO4 at a dark incubation period of 95 h as compared to 41.6% PHB yield in 0.4% acetate, 0.4% glucose and 40 mg l(-1) K2HPO4 at a dark incubation period of 168 h under single factor optimization strategy.     

Poly(β-hydroxybutyrate) production by a moderate halophile,Halomonas boliviensis LC1

The moderate halophile Halomonas boliviensis, isolated from a Bolivian saline soil sample, was able to accumulate poly(β-hydroxybutyrate) (PHB) when grown under conditions of nutrient limitation and excess carbon source. The concentration of sodium chloride in the medium influenced the cell-growth, -size, and rate of PHB accumulation. Cultivation in shake flasks led to a PHB accumulation of about 54 wt.% with respect to cell dry weight at 4.5% (w/v) NaCl in a medium with butyric acid and sodium acetate as carbon sources. The production of PHB was substantially improved to a maximum value of 88 wt.% during cultivation under controlled conditions of pH and oxygen concentration in a fermentor. The use of glucose and sucrose, respectively, as carbon source could also lead to the production of PHB at an average level of 55 wt.%.     

Batch cultivation of Methylosinus trichosporium OB3b: V. Characterization of poly-beta-hydroxybutyrate production under methane-dependent growth conditions

Methanotrophs have promising applications in the bioremediation of chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (PHB). Batch bioreactor culture conditions were studied for the accumulation of PHB by methane-grown Methylosinus trichosporium OB3b, and to evaluate the effect of PHB on the bacterial capacity to degrade trichloroethylene (TCE), a common groundwater contaminant. The PHB content of the washed and lyophilized cells was measured by gas chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the polymer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mixtures, respectively. During bioreactor growth in a Cu-deficient modified Higgins' medium, the cells accumulated PHB upon depletion of nitrate. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of approximately 10% (w/w) were reached after 140 to 160 h, without adversely affecting the propene or TCE epoxidation specific rate given by whole cells containing soluble methane monooxygenase (sMMO). The TCE biotransformation capacity ( approximately 0.25 mg TCE oxidized/mg dry cell wt) of resting cells containing approximately 10% PHB was consistently approximately 1.6-fold greater than that of cells containing only approximately 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotransformation capacity further. By replacing the bioreactor inlet air + CO(2) mixture with pure O(2) at approximately 85 h of batch operation, a PHB accumulation of approximately 45% was achieved after 160 h, but the whole-cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 muM Cu-supplemented Higgins' nitrate minimal salts medium (particulate MMO formation) accumulated up to 50% PHB in only 120 h, coupled with a very high biomass yield of 18 g dry cell wt/L. High PHB accumulations above approximately 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the absorbance at 660 nm, which is commonly used to monitor bacterial growth. (c) 1996 John Wiley & Sons, Inc.     

Enhanced poly-beta-hydroxybutyrate accumulation in a unicellular cyanobacterium, Synechocystis sp. PCC 6803

AIM: To stimulate poly-beta-hydroxybutyrate (PHB) accumulation in Synechocystis sp. PCC 6803 by manipulating culture conditions. METHODS AND RESULTS: Stationary phase cultures of Synechocystis sp. PCC 6803 were subjected to N- and P-deficiency, chemoheterotrophy and limitations of gas-exchange. Enhanced PHB accumulation was observed under all the above conditions. However, interaction of P-deficiency with gas-exchange limitation (GEL) in the presence of exogenous carbon boosted PHB accumulation maximally. CONCLUSIONS: Combined effects of P-deficiency and GEL boosted PHB accumulation up to 38% (w/w) of dry cell weight (dcw) in Synechocystis sp. PCC 6803 in the presence of fructose and acetate. This value is about eightfold higher as compared with the accumulation under photoautotrophic growth condition. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: These results showed a good potential of Synechocystis sp. PCC 6803 in accumulating poly-beta-hydroxybutyrate, an appropriate raw material for biodegradable and biocompatible plastic. Poly-beta-hydroxybutyrate could be an important material for plastic and pharmaceutical industries.     

Enhancement of poly-β-hydroxybutyrate accumulation in <Emphasis Type="Italic">Nostoc muscorum</Emphasis> under mixotrophy,chemoheterotrophy and limitations of gas-exchange

Nostoc muscorum, a heterocystous cyanobacterium, produced poly--hydroxybutyrate ( PHB) up to 8% (w/w) dry cells when grown photoautotrophically but 35% when grown mixotrophically with 0.4% (w/v) glucose and acetate after 21 d. Gas-exchange limitations under mixotrophy and chemoheterotrophy with 0.4% (w/v) acetate enhanced the accumulation up to 40–43% (w/w) dry cells, the value almost 5-fold higher with respect to photoautotrophic condition.Revisions requested/10 September 2004; Revisions received 9 September 2004/11 November 2004     

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.     

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.     

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.     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in Bacillus mycoides RLJ B-017

AIMS: To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. METHODS AND RESULTS: An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Yp/s) and productivities (Qp and qp) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 gp gS(-1), 0.104 +/- 0.012 gp l(-1) h(-1) and 0.03 gp gx(-1) h(-1), respectively when grown in a medium containing 20 gs l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0,3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2% , respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0 .1, temperature 30 +/- 0.5 degrees C, agitation 650 rev min(-1) and oxygen transfer rate 3.8 mmol O(2) l(-1) h(-1). CONCLUSIONS: Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. SIGNIFICANCE AND IMPACT OF THE STUDY: Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/- 0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.     

PHB accumulation in Nostoc muscorum under different carbon stress situations

Use of algae for intracellular poly-β-hydroxybutyrate (PHB) accumulation for bioplastic production offers an opportunity in economic efficiency by reduced costs. The cyanobacterium Nostoc muscorum is a PHB accumulator which presents a great potential as raw material supplier because of short generation cycles. Here, we examined a range of experimental conditions including different growth conditions of phosphate-starved cells with the addition of external carbon sources. The highest, absolute PHB accumulation was measured in a phosphate-starved medium with 1% (w/w) glucose and 1% (w/w) acetate. PHB accumulated inside algae cells. After 23 days of growth in phosphate-starved medium, 1 L of culture contained up to 145.1 mg PHB. The highest PHB accumulation based on the cell dry weight was in an experiment with aeration and CO₂ addition. The intracellular level of PHB was up to 21.5% cell dry weight after 8 days.     

Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions

Activated sludge submitted to aerobic dynamic feeding conditions showed a good and stable capacity to store polyhydroxybutyrate (PHB). The system, working for 2 years, selected a microbial population with a high PHB storage capacity. The influence of carbon and nitrogen concentrations on the PHB accumulation yield was studied in a range of 15-180 Cmmol/l for acetate and 0-2.8 Nmmol/l for ammonia. Low ammonia concentrations favored PHB accumulation. The maximum PHB content, 67.5%, was obtained for 180 Cmmol/l of acetate supplied in one pulse. However, such high substrate concentration proved to be inhibitory for the storage mechanism, causing a slowdown of the specific PHB storage rate. In order to avoid substrate inhibition, 180 Cmmol/l of acetate was supplied in different ways: continuously fed and in three pulses of 60 Cmmol/l each. In both cases the specific PHB storage rate increased and the PHB content obtained were 56.2% and 78.5%, respectively. The latter value of PHB content is similar to that obtained by pure cultures and was never reported for mixed cultures. Addition of acetate by pulses controlled by the oxygen concentration was kept for 16 days, the PHB content being always above 70% of cell dry weight.     

Occurrence of poly-beta-hydroxybutyrate in Spirulina species.

Several strains of photoautotrophically grown Spirulina spp. contained poly-beta-hydroxybutyrate (PHB) at concentrations never exceeding a few milligrams per gram of dry weight. Under mixotrophic growth conditions in the presence of acetate, PHB reached values greater than 2.5% of dry weight. With pyruvate, no significant effect on PHB accumulation was obtained.     

Poly-3-hydroxybutyrate production byAzotobacter chroococcum

Thirty-seven soil isolates and mutants ofAzotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2 %) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 μg/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 μg/g.     

Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop

Polyhydroxyalkanoate bio-based plastics made from renewable resources can reduce petroleum consumption and decrease plastic waste disposal issues as they are inherently biodegradable in soil, compost and marine environments. In this paper, the successful engineering of the biomass crop switchgrass ( Panicum virgatum L.) for the synthesis of polyhydroxybutyrate (PHB) is reported. Polymer production was monitored in more than 400 primary transformants grown under in vitro and glasshouse conditions. Plants containing up to 3.72% dry weight of PHB in leaf tissues and 1.23% dry weight of PHB in whole tillers were obtained. Results from the analysis of the polymer distribution at the cellular and whole plant levels are presented, and target areas for the improvement of PHB production are highlighted. Polymer accumulation was also analysed in the T₁ generation obtained from controlled crosses of transgenic plants. This study presents the first successful expression of a functional multigene pathway in switchgrass, and demonstrates that this high-yielding biomass crop is amenable to the complex metabolic engineering strategies necessary to produce high-value biomaterials with lignocellulose-derived biofuels.     

Production of poly-\-hydroxybutyrate in Acinetobacter spp. isolated from activated sludge

Two strains of Acinetobacter sp. isolated from activated sludge actively removing phosphate were examined for their abilities to produce poly-\-hydroxybutyrate (PHB). When yield-limited by phosphate, strain RA3117 contained material that stained with Sudan Black, but contained only 0.9% PHB on a dry weight basis. This strain contained no sudanophilic material or PHB when limited by ammonia or sulphate. When strain RA3757 was limited by phosphate, ammonia or sulphate it produced 2.0, 7.8 and 11.5% PHB, respectively, on a dry weight basis. \-Ketothiolase and acetoacetyl-coenzyme A (CoA) reductase were only observed in RA3757 cell-free extracts. \-Ketothiolase was produced both in cells with and without PHB whereas acetoacetyl-CoA reductase was found only in cells accumulating PHB. When RA3757 was grown in ammonia-limiting medium with acetate, butyrate, caproate or ethanol as carbon source, similar levels of PHB were produced. When cells were grown on valerate, RA3757 produced 5.6 poly-\-hydroxyvalerate and 0.9% PHB on a dry weight basis. Correspondence to: J. W. May     

The production of polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in media containing glycerol and casein hydrolysates

Methylobacterium rhodesianum and Ralstonia eutropha were cultivated to produce polyhydroxybutyrate (PHB) using media which contained glycerol and casein hydrolysates as C/N-substrates. In these media the pH had not to be regulated during the fermentations. The first strain accumulated an average of 39% PHB during 92 h of cultivation in flasks and 50% PHB during 45 h of cultivation in fermenters. The second one yielded an average of 47% PHB during 67 h of cultivation using casein peptone and 65% PHB during 45 h of cultivation using Casamino acids in the medium. Calculated N-balances showed that about 65% of the supplied nitrogen was used for growth of non-PHB cell dry mass. The conversion of glycerol to PHB was 17% (w/w).     

Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.