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.     

Nutritional and cultural conditions for production of poly-3-hydroxybutyric acid byAzotobacter chroococcum

Free-living nitrogen-fixing bacteria have been identified as a potential source of poly-3-hydroxybutyric acid (PHB). Systematic study of this ability of N₂-fixing organisms has lead to the isolation of an efficient strain, identified asAzotobacter chroococcum. Nutritional requirements and cultural conditions for optimal production of PHB by this strain under laboratory conditions were determined. In N-free liquid medium containing 2% glucose, the strain accumulated PHB up to 68% of its cell dry mass. Glucose and mannitol were found to be the best carbon sources, while organic nitrogen compounds were preferred as nitrogen source. Maximum yield (3.3 g/L) was obtained with 0.2% bactopeptone supplementation. Inorganic phosphate at a concentration suboptimal for growth had some growth-promoting effect. Under oxygen limiting conditions, biomass production was enhanced but a different response was obtained for PHB production.     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

Polyhydroxybutyrate in <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium</Emphasis>: quantification and <Emphasis Type="Italic">phbC</Emphasis> gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.     

Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge

The present study reports two bacteria, designated 87I and 112A, which were isolated from soil and activated sludge samples from Hyderabad, India, and that are capable of producing poly-3-hydroxybutyrate (PHB). Based on phenotypical features and genotypic investigations, these microorganisms were identified as Bacillus spp. Their optimal growth occurred between 28°C and 30°C and pH 7. Bacillus sp. 87I yielded a maximum of 70.04% dry cell weight (DCW) PHB in medium containing glucose as carbon source, followed by 55.5% DCW PHB in lactose-containing medium, whereas Bacillus sp. 112A produced a maximum of 67.73% PHB from glucose, 58.5% PHB from sucrose, followed by 50.5% PHB from starch as carbon substrates. The viscosity average molecular mass (M _v) of the polymers from Bacillus sp. 87I was 513 kDa and from Bacillus sp. 112A was 521 kDa. All the properties of the biopolymers produced by the two strains 87I and 112A were characterized.     

Accumulation of poly-(hydroxybutyrate) by a non-sulfur photosynthetic bacterium,Rhodobacter sphaeroides RV at different pH

Summary Effect of pH of culture media on intracellular accumulation of poly-(hydroxybutyrate) (PHB) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides strain RV was studied in pH-stat cultures. Sub-optimal pH for growth, 8.0 or 8.5 gave the higher content of PHB rather than optimal pH 7.5 for growth. These results show that growth and PHB accumulation of the bacteria can be controlled by pH of culture media.     

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.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

Accumulation of Poly (3-Hydroxybutyric Acid) by Some Soil Streptomyces

In a limited-scale survey, 55 soil streptomycetes were screened for the accumulation of poly (3-hydroxybutyrate) [PHB]. Only 18% of the isolates accumulated PHB ranging between 1.9–7.8% of the dry biomass. The promising isolate DBCC-719, identified as Streptomyces griseorubiginosus, accumulated PHB amounting to 9.5% of the mycelial dry mass in the early stationary phase when grown in chemically defined medium with 2% (wt/vol) glucose as the sole source of carbon. Nitrogen-limiting conditions were inhibitory to growth and PHB accumulation. The isolated polymer was highly soluble in chloroform, gave a sharp peak at 235 nm on digestion with concentrated H₂SO₄, and had a characteristic infrared spectrum. Received: 26 March 1999 / Accepted: 3 May 1999     

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.     

Rapid and simple analysis of poly-β-hydroxybutyrate content by capillary isotachophoresis

Summary We present a rapid method for the direct analysis of poly--hydroxybutyrate (PHB) content in the soil bacteria Alcaligenes eutrophus. PHB from the fresh cells was converted by sulfuric acid to the crotonic acid and measured by capillary isotachophoresis after the neutralization by CaCO₃. The method can be used for rapid and routine monitoring of the fermentation processes in samples containing 0.001 to 20 mg of PHB.     

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,optimization and characterization of polyhydroxybutyrate,a biodegradable plastic by Bacillus spp.

Poly-β-hydroxybutyrate (PHB) is the intracellular lipid reserve accumulated by many bacteria. The most potent terrestrial bacterium Bacillus cereus SE-1 showed more PHB accumulating cells (22.1 and 40% after 48 and 72 h) than that of the marine Bacillus sp. CS-605 (5 and 33% after 48 and 72 h). Both the isolates harbored phbB gene and the characteristics C=O peak was observed in the extracted PHB by Fourier transformed infrared spectroscopy analysis. Maltose was found to be the most suitable carbon source for the accumulation of PHB in B. cereus SE-1. The extracted PHB sample from B. cereus SE-1 was blended with a thermoplastic starch (TS) and an increased thermoplasticity and decreased crystallinity were observed after blending in comparison to the standard PHB. The melting temperature (T_m), melting enthalpy (?Hf), and crystallinity (X_c) of the blended PHB sample were found to be 109.4 °C, 64.58 J/g, and 44.23%, respectively.     

The lipopolysaccharide of the phototrophic bacterium Ectothiorhodospira vacuolata

The lipopolysaccharide of Ectothiorhodospira vacuolata was obtained by the phenol-water procedure. It contained a 3-O-methyl-hexose, glucose, galacturonic and glucuronic acids. The finding of d-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate (tentatively identified) suggested a core-structure. The lipid fraction of the lipopolysaccharide contained phosphate and both, 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The major fatty acids were amine-bound 3-OH-10:0 and 3-OH-12:0 and esterbound 14:0 and 16:0 Sodium deoxycholate gel-electrophoresis, showing a single band only, indicated R-type character of the lipopolysaccharide of Ectothiorhodospira vacuolata.Abbreviations DOC sodium deoxycholate - GC/MS combined gasliquid chromatography - PAGE polyacrylamide gel-electrophoresis     

Kinetics and effect of nitrogen source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture

Summary A kinetic study of the production of poly--hydroxybutyric acid (PHB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the PHB production phase. The effect of ammonia feeding on PHB production was consequently investigated. The nitrogen source (ammonia water) was supplied at a low constant feeding rate after the growth phase in which cell mass concentration reached 60 g/l. Feeding with a small quantity of ammonia resulted in a more rapid increase in intracellular PHB content than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated PHB but also reduction of microbial PHB synthetic activity.     

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.     

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.     

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g⁻¹ and PHB productivity (P_PHB) 0.10 and 0.06 g l⁻¹ h⁻¹. This 40% P_PHB difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol⁻¹ for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol⁻¹). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.     

Microbial degradation of poly-β-hydroxybutyrate using landfill soils

The population of poly-β-hydroxybutyrate-degrading microorganisms and the biodegradation of PHB in local landfill soils were examined in vitro and in vivo. Forty-two PHB-degraders consisting of 12 bacteria, 25 actinomycetes and 5 moulds were isolated. The total PHB-degraders averaged 4.7 × 10⁷ and 20 × 10⁴ colony forming units (cfu)/g for San Mateo wet and dry soils, respectively, and 2.3 × 10⁷ and 8.5 × 10⁴ cfu/g for Carmona wet and dry samples, respectively. The PHB-degraders formed 0–59% of the total microbial population in San Mateo and 8–42% in Carmona. Complete (100%) degradation of PHB powder was observed for Chryseomonas-27 and Aspergillus-39 on day 5 in shake flask culture and for Streptomyces-4 on day 7. Burial test in landfill soils showed a 90–91% weight loss of PHB film strips within four weeks; the weight loss of polypropylene film strips was up to 0.12% only. Scanning electron micrographs of degraded films revealed the attachment of microbial cells and fungal mycelium and spores on the surfaces. Holes and cavities were also noted due to the microbial degradation processes.