Poly-beta-hydroxybutyrate Research Articles

Enhancing Phosphorus Release from Sewage Sludge in Anaerobic Digestion via Thermal Hydrolysis Pretreatment: Insights from Phosphorus Speciation and Molecular Biological Pathways.

This study explores the mechanisms enhancing phosphorus (P) release from sludge in anaerobic digestion (AD) with thermal hydrolysis pretreatment (THP) using sequential chemical extraction, X-ray absorption near-edge structure spectroscopy (XANES), 31P NMR, and multiomics. THP-treated sludge notably increased liquid-phase P by 53.8% over 3 days compared to sewage sludge (SS), identifying solid-phase Fe-P as the primary P source. The THP+AD also provided a higher abundance of bacteria that contributed to P release through multiple pathways (MPRPB), whereas SS+AD enriched some microbial species with single P release pathway. Moreover, species co-occurrence network analysis underlined the pivotal role of P-releasing bacteria in THP+AD, with 8 out of 16 keystones being P-releasers. Among the 63 screened genes that were related to P transformations and release, the poly beta-hydroxybutyrate (PHB) synthesis genes associated with polyphosphate bacteria-mediated P release were more abundant in THP+AD than in SS+AD. Furthermore, the upregulation of genes involved in methyl phosphonate metabolism in the THP-treated sludge enhanced the methane production potential of the AD process. These findings suggested that MPRPB were indeed the main contributors to P release, and enrichment in the THP+AD process enhanced their capability for P liberation.

Non-targeted metabonomics and transcriptomics revealed the mechanism of mulberry branch extracts promoting the growth of Sanghuangporus vaninii mycelium.

Sanghuangprous vaninii is a wood-inhabiting fungus, and its mycelium and fruiting body show excellent medicinal values. Mulberry is one of the major hosts of S. vaninii, however, the mechanism of mulberry affecting the growth of S. vaninii has not been reported. In the present study, a mulberry-inhabiting strain of S. vaninii was selected to explore the effects of mulberry branch extracts (MBE) on the growth of the strain. Results showed that MBE could significantly promote the growth of S. vaninii mycelium at the concentration of 0.2 g/l. After 16 days of liquid culture, the dry weight of mycelium in 0.2 g/l MBE medium was higher by three times compared with that in the control. The non-targeted metabonomic analysis of the culture medium at different culture times and concentrations was conducted to find the key components in MBE that promoted the growth of S. vaninii mycelium. Under the different concentrations of MBE culture for 10 and 16 days, 22 shared differential metabolites were identified. Next, in accordance with the peak value trend of these metabolites, HPLC–MS and liquid culture validation, four components derived from MBE (i.e., scopoletin, kynurenic acid, 3,5-dihydroxybenzoic acid and 2,4-dihydroxybenzoic acid) could significantly increase the growth rate of mycelium at the concentration of 2 mg/l. Transcriptomic and qRT-PCR analyzes showed that MBE could upregulate hydrolase-related genes, such as serine–glycine–asparaginate–histidine (SGNH) hydrolase, alpha-amylase, poly-beta-hydroxybutyrate (PHB) depolymerase, glycosyl hydrolase family 61, cerato-platanin protein and Fet3, which might enhance the nutrient absorption ability of S. vaninii. Importantly, MBE could significantly increase the content of harmine, androstenedione and vesamicol, which have been reported to possess various medicinal effects. Results suggested that MBE could be an excellent additive for liquid culture of S. vaninii mycelium, and these hydrolase-related genes also provided candidate genes for improving the nutrient absorption capacity of S. vaninii.

The direct and interactive effects of elevated CO2 and additional nitrate on relative costs and benefits of legume-rhizobia symbiosis

Rising concentrations of carbon dioxide (CO2) is likely to have important effects on growth and development of plants and on their relationship with symbiotic microbes. A rise in CO2 could increase demand by plant hosts for nutrient resources, which may increase host investments in beneficial symbionts. In the legume-rhizobia mutualism, while elevated CO2 is often associated with increased nodule growth and investment in N2-fixing rhizobia, it is yet unclear if this response depends on the mutualistic quality of the rhizobia. To test if host carbon allocation towards more-beneficial nodules are similar to less-beneficial (but still effective) nodules when plant N demand changes, we manipulated plant C and N status with elevated CO2 and additional nitrate. We used two isogenic Rhizobium etli strains that differ in their ability to synthesize an energy reserve compound, poly-beta-hydroxybutyrate (PHB), as well as their efficiencies for nitrogen fixation and nodulation rates, resulting in two Phaseolus vulgaris host groups with either large number of small nodules or small number of large nodules. The addition of nitrate negatively affected carbon allocation towards nodules, and elevated CO2 reversed this effect, as expected. However, this alleviation of nodule inhibition was greater on plants that started with greater numbers of smaller nodules. If smaller nodules indicate less-efficient or low-fixing rhizobia, this study suggests that increased demand for nitrogen in the face of elevated CO2 has the potential to disproportionately favor less-beneficial strains and increase variation of nitrogen fixation quality among rhizobia.

Signs of a phyllospheric lifestyle in the genome of the stress-tolerant strain Azospirillum brasilense Az19

Azospirillum brasilense Az19 is a plant-beneficial bacterium capable of protecting plants from the negative effects of drought. The objective of this study was to determine and analyze the genomic sequence of strain Az19 as a means of identifying putative stress-adaptation mechanisms. A high-quality draft genome of ca. 7 Mb with a predicted coding potential of 6710 genes was obtained. Phylogenomic analyses confirmed that Az19 belongs to the brasilense clade and is closely related to strains Az39 and REC3. Functional genomics revealed that the denitrification pathway of Az19 is incomplete, which was in agreement with a reduced growth on nitrate under low O2 concentrations. Putative genes of the general stress response and oxidative stress-tolerance, as well as synthesis of exopolysaccharides, carotenoids, polyamines and several osmolytes, were detected. An additional poly-beta-hydroxybutyrate (PHB) synthase coding gene was found in Az19 genome, but the accumulation of PHB did not increase under salinity. The detection of exclusive genes related to DNA repair led to discover that strain Az19 also has improved UV-tolerance, both in vitro and in planta. Finally, the analysis revealed the presence of multiple kaiC-like genes, which could be involved in stress-tolerance and, possibly, light responsiveness. Although A. brasilense has been a model for the study of beneficial plant-associated rhizobacteria, the evidence collected in this current study suggests, for the first time in this bacterial group, an unexpected possibility of adaptation to the phyllosphere.

Asymmetric Open-Closed Dimer Mechanism of Polyhydroxyalkanoate Synthase PhaC.

SummaryBiodegradable polyester polyhydroxyalkanoate (PHA) is a promising bioplastic material for industrial use as a replacement for petroleum-based plastics. PHA synthase PhaC forms an active dimer to polymerize acyl moieties from the substrate acyl-coenzyme A (CoA) into PHA polymers. Here we present the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, bound to CoA. The structure reveals an asymmetric dimer, in which one protomer adopts an open conformation bound to CoA, whereas the other adopts a closed conformation in a CoA-free form. The open conformation is stabilized by the asymmetric dimerization and enables PhaC to accommodate CoA and also to create the product egress path. The bound CoA molecule has its β-mercaptoethanolamine moiety extended into the active site with the terminal SH group close to active center Cys291, enabling formation of the reaction intermediate by acylation of Cys291.

Effects of static magnetic field on the performances of anoxic/oxic sequencing batch reactor

Two anoxic/oxic (A/O) sequencing batch reactor (SBR) processes were utilized to study the effects of static magnetic field (SMF) on biological wastewater treatment process. Except for conventional indices, the reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+) ratio and electron transport system activity (ETSA), as well as poly-beta-hydroxybutyrate (PHB) and extracellular polymetric substance (EPS) contents in two reactors which were with and without SMF under two cyclic times (12 h and 8 h) were monitored. When the process was enhanced by SMF, the total nitrogen removal efficiency can be improved (>80%), and the NADN/NAD+ ratio, ESTA, the maximum EPS content and the maximum PHB content in the reactor with SMF were higher. Besides, SMF can reduce the microorganism community diversity and make species distribute more even and abundant. SMF can promote the performance of A/O SBR process via improving electron transport and microbial community.

High amorphous poly-beta-hydroxybutyrate (PHB) content in a probiotic Bacillus strain displays better protective effects in Vibrio-challenged gnotobiotic Artemia

In this study, the Bacillus sp. JL47, a superior PHB-accumulating Bacillus strain identified from the previous work, was tested for its protective effects in gnotobiotic Artemia franciscana during a pathogenic Vibrio campbellii challenge. The utilization of gnotobiotic Artemia is important in this experiment because any possible microbial interference (which are naturally present in conventional culture system) are eliminated in this model culture system, hence the interpretation of the results in this mechanistic study can be more conclusive. The survival of the Artemia fed the Bacillus sp. JL47 at 1×107cellsmL−1 was significantly higher as compared with the challenged control and the survival was almost doubled when the dose was increased to 5×107cellsmL−1. However, feeding the Artemia at 106cellsmL−1 or lower showed no significant protective effects. Based on these densities, the estimated concentration of amorphous PHB showing a significant protection in Artemia was c. 2.44mgL−1 and the effects were even better when the amorphous PHB level was increased to 12.19mgL−1. Furthermore, feeding Bacillus sp. JL47 containing 55% amorphous PHB (on CDW) to Artemia showed a significantly higher survival in a Vibrio challenge relative to Bacillus containing 29% PHB. The data suggest that the amorphous PHB accumulated in the Bacillus sp. JL47 strain is an important determinant for the increased survival of challenged Artemia.

Nutrient Recovery From Cyanobacteria Biomasses Using Purple Nonsulfur Bacterium Rhodopseudomonas palustris

Occurrences of harmful cyanobacterial blooms are a worldwide environmental problem in most eutrophic lake ecosystems. But what should be noticed is that cyanobacteria can be used as a useful resource due to the wide range of metabolites they produce. Nutrient partitioning using purple nonsulfur bacteria (PNSB) has the potential to biologically concentrate nutrients. The present study evaluated the kinetics of nutrients released from decomposed field blue green algae (BGA) biomasses. The potential of nutrient acquisition from decomposed BGA biomasses for culturing Rhodopseudomonas palustris (R. palustris) was investigated via fed-batch experiments. Results indicated that R. palustris stimulated in algae substrates with algae biomasses ranging from 3.33 to 10 g/L. Removal efficiencies of N and P in the stationary phase of growth were at least 40% and 95%, respectively, of all the nitrogen (N) and phosphorus (P) released. Additionally, the cellular contents like total lipid and poly-beta-hydroxybutyrate (PHB), as well as the fatty acids produced by R. palustris, were consistent. Hence, practice based on the bacterial production for the nutrient recovery from BGA biomasses provides a new insight in field algae disposal. It will lower the chances of secondary pollution due to algae decay and produce giant cells of R. palustris and surely will prosper the industries applying R. palustris.

A probiotic Bacillus strain containing amorphous poly-beta-hydroxybutyrate (PHB) stimulates the innate immune response of Penaeus monodon postlarvae

In this study, the PHB-accumulating Bacillus sp. JL47 strain (capable of accumulating 55% PHB on cell dry weight) was investigated for its effects on the immune response of giant tiger shrimp (Penaeus monodon) postlarvae (PL) before and after the Vibrio campbellii challenge. Briefly, shrimp PL were cultured and fed with Artemia nauplii enriched with Bacillus sp. JL47. Shrimp receiving the Artemia nauplii without JL47 enrichment were used as control. After 15 days of feeding, the shrimp were challenged with pathogenic V. campbellii LMG 21363 at 106 cells mL−1 by immersion. Relative expression of the immune related genes encoding for prophenoloxidase (proPO), transglutaminase (TGase) and heat shock protein 70 (Hsp70) in the shrimp were measured before (0 h) and after (3, 6, 9, 12, 24 h) the Vibrio challenge by quantitative real-time PCR using β-actin as the reference gene. The expressions of TGase and proPO were significantly up-regulated (p < 0.05) within 9 h and 12 h, respectively after challenge in shrimp receiving the Bacillus sp. JL47 as compared to the challenged and non-challenged controls. Hsp70 expression was significantly increased (p < 0.05) at 3 h post-challenge in all challenged shrimp. Interestingly, proPO and TGase genes were significantly up-regulated (p < 0.05) in Bacillus sp. JL47 treated shrimp even before the Vibrio challenge was applied. No up-regulation in the Hsp70 gene, however, was observed under these conditions. The data suggest that the protective effect of the PHB-accumulating Bacillus sp. JL47 in shrimp was due to its capacity to stimulate the innate immune related genes of the shrimp, specifically the proPO and TGase genes. The application of probiotic Bacillus species, capable of accumulating a significant amount of PHB, is suggested as potential immunostimulatory strategy for aquaculture.

Probing the Kinetic Anabolism of Poly-Beta-Hydroxybutyrate in Cupriavidus necator H16 Using Single-Cell Raman Spectroscopy.

Poly-beta-hydroxybutyrate (PHB) can be formed in large amounts in Cupriavidus necator and is important for the industrial production of biodegradable plastics. In this investigation, laser tweezers Raman spectroscopy (LTRS) was used to characterize dynamic changes in PHB content—as well as in the contents of other common biomolecule—in C. necator during batch growth at both the population and single-cell levels. PHB accumulation began in the early stages of bacterial growth, and the maximum PHB production rate occurred in the early and middle exponential phases. The active biosynthesis of DNA, RNA, and proteins occurred in the lag and early exponential phases, whereas the levels of these molecules decreased continuously during the remaining fermentation process until the minimum values were reached. The PHB content inside single cells was relatively homogenous in the middle stage of fermentation; during the late growth stage, the variation in PHB levels between cells increased. In addition, bacterial cells in various growth phases could be clearly discriminated when principle component analysis was performed on the spectral data. These results suggest that LTRS is a valuable single-cell analysis tool that can provide more comprehensive information about the physiological state of a growing microbial population.

Formation of Polyphosphate-Poly-Beta-Hydroxybutyrate Granule-Like Complexes in Heart Failure Myocytes

Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates linked together by phosphoanhydride bonds. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of rabbit ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca2+-dependent activation of the mitochondrial permeability transition pore (mPTP). Using DAPI as a probe for polyP, we showed that polyP levels depend on the activity of the mitochondrial respiratory chain. Here, we visualized intracellular polyP distribution in cardiomyocytes using the affinity of the Xpress epitope-tagged recombinant polyphosphate-binding domain (PPBD) of E. coli exopolyphosphatase and immunocytochemical approach. Primarily mitochondrial location of polyP was demonstrated in both control and heart failure (HF) myocytes from two different HF models: (1), non-ischemic rabbit HF model induced by combined aortic insufficiency and stenosis and (2), targeted microbead embolization of the first diagonal branch of left anterior descending coronary artery in Yucatan mini-pigs. However, enhanced formation of polyP dense spots was observed in HF conditions. Furthermore, polyP dense spots overlapped with poly-beta-hydroxybutyrate (PHB) accumulation areas in HF myocytes as detected with specific PHB antibodies (anti-PHB IgG) raised against a synthetic 8-mer of PHB. It is known that polyP can form channels with Ca2+ and PHB, a polymerized form of the ketone body beta-hydroxybutyrate, with characteristics similar to mPTP. These data suggest that polyP-PHB complex formation in HF myocytes could contribute to the enhanced propensity of mPTP opening despite decreased mitochondrial Ca2+ uptake.

Draft genome sequence of Thalassobius mediterraneus CECT 5383(T), a poly-beta-hydroxybutyrate producer.

Thalassobius mediterraneus is the type species of the genus Thalassobius and a member of the Roseobacter clade, an abundant representative of marine bacteria. T. mediterraneus XSM19T (= CECT 5383T) was isolated from the Western Mediterranean coast near Valencia (Spain) in 1989. We present here the draft genome sequence and annotation of this strain (ENA/DDBJ/NCBI accession number CYSF00000000), which is comprised of 3,431,658 bp distributed in 19 contigs and encodes 10 rRNA genes, 51 tRNA genes and 3276 protein coding genes. Relevant findings are commented, including the complete set of genes required for poly-beta-hydroxybutyrate (PHB) synthesis and genes related to degradation of aromatic compounds.

Encapsulation of PHB (Poly Beta Hydroxy Butyrate) as Nanoparticle with Simvastastin for Drug Delivery

The recent advances in the field of nanotechnologies have made nanoparticles to be very promising in the delivery of bioactive agents, drug discovery and diagnostics. The present study deals with extraction of PHB (Poly beta hydroxy butyrate) from soil samples. The development of PHB nanoparticle and Simvastatin for drug delivery process. Simvastatin is a drug with white, non-hygroscopic, crystalline powder that is insoluble in water and freely soluble in chloroform and ethanol. The statins plays an important role in reducing cardiovascular related morbidity and mortality in patients with or without coronary artery disease and with or without high cholesterol levels. The drug can be efficiently encapsulated into the polymer matrix as a nanoparticle that release their payload slowly over time. The studies on physical and chemical characterization of developed nanoparticle revealed its efficiency of drug delivery.

Enantioselective Physiological Effects of the Herbicide Diclofop on Cyanobacterium Microcystis aeruginosa

Water blooms caused by cyanobacteria are currently major global environmental issues. The outbreaks induced by nutrient elements have attracted much attention; however, the effects of environmental pollutants on the cyanobacteria are themselves poorly understood, especially those due to chiral chemicals. To explore the enantioselective eco-effects of the chiral herbicide diclofop-methyl (DM) and its major metabolite diclofop acid (DA), the physiological characteristics of Microcystis aeruginosa were investigated. The results showed that using both biomass and protein content as growth parameters is necessary to access the impact of the herbicides, that stimulation biomass production by R-DA and S-DA was apparent (nonessential), and that the concentration of 5 mg/L is worth noting. Ultrastructure changes in gas vacuoles, thylakoids, glycogen, cyanophycin granules, poly beta-hydroxybutyrate, polyhedral body, and lipids indicated different toxicity modes among the four chemicals. The different effects between R-DA and S-DA demonstrated that R-DA probably acts as a proton ionophore shuttling protons across the plasmalemma, whereas S-DA did not demonstrate such action. The toxicity order in the present study is S-DA < R-DA < DM < DA. Stimulation of the growth of M. aeruginosa during the first 3 days by herbicidally inactive S-DA was greater than that due to R-DA, which is adverse to water quality in water bodies. Therefore, using the herbicidally active R-enantiomer is recommended. These results are helpful in understanding the enantioselective effects of chiral pesticides on cyanobacteria, which is important for environmental assessment and protection. It is also helpful for guiding the application of chiral pesticides in agricultural settings.

Formation of polyhydroxyalkanoate in aerobic anoxygenic phototrophic bacteria and its relationship to carbon source and light availability.

Aerobic anoxygenic phototrophic bacteria (AAPB) are unique players in carbon cycling in the ocean. Cellular carbon storage is an important mechanism regulating the nutrition status of AAPB but is not yet well understood. In this paper, six AAPB species (Dinoroseobacter sp. JL1447, Roseobacter denitrificans OCh 114, Roseobacter litoralis OCh 149, Dinoroseobacter shibae DFL 12(T), Labrenzia alexandrii DFL 11(T), and Erythrobacter longus DSMZ 6997) were examined, and all of them demonstrated the ability to form the carbon polymer polyhydroxyalkanoate (PHA) in the cell. The PHA in Dinoroseobacter sp. JL1447 was identified as poly-beta-hydroxybutyrate (PHB) according to evidence from Fourier transform infrared spectroscopy, differential scanning calorimetry, and (1)H nuclear magnetic resonance spectroscopy examinations. Carbon sources turned out to be critical for PHA production in AAPB. Among the eight media tested with Dinoroseobacter sp. JL1447, sodium acetate, giving a PHA production rate of 72%, was the most productive carbon source, followed by glucose, with a 68% PHA production rate. Such PHA production rates are among the highest recorded for all bacteria. The C/N ratio of substrates was verified by the experiments as another key factor in PHA production. In the case of R. denitrificans OCh 114, PHA was not detected when the organism was cultured at C/N ratios of <2 but became apparent at C/N ratios of >3. Light is also important for the formation of PHA in AAPB. In the case of Dinoroseobacter sp. JL1447, up to a one-quarter increase in PHB production was observed when the culture underwent growth in a light-dark cycle compared to growth completely in the dark.

Analyses of binding sequences of the PhaR protein of Rhodobacter sphaeroides FJ1

The phaC, phaP, phaR, and phaZ genes are involved in the synthesis, accumulation, and degradation of poly-beta-hydroxybutyrate (PHB). These genes encode the PHB synthase, phasin, regulatory protein, and PHB depolymerase, respectively, and are located in the same locus in the genome of Rhodobacter sphaeroides FJ1, a purple nonsulfur bacterium capable of producing PHB. We have previously found that the PhaR protein binds to the promoter regions of phaP, phaR, and phaZ and represses their expression. In this study, we determined that PhaR binds to an 11-bp palindromic sequence, 5'-CTGCN(3)GCAG-3', located at nucleotides -69 to -59 and -97 to -87 relative to the translation start site of phaP. Substitution of the three spacer nucleotides with any three or four nucleotides in this sequence had no effect on PhaR binding, but all other base deletions or substitutions in this sequence abolished its ability to bind PhaR both in vitro and in vivo. These results suggest that PhaR regulates the expression of phaP in R. sphaeroides FJ1.

Biochemical, mechanical, and spectroscopic analyses of genetically engineered flax fibers producing bioplastic (poly‐β‐hydroxybutyrate)

The interest in biofibers has grown in recent years due to their expanding range of applications in fields as diverse as biomedical science and the automotive industry. Their low production costs, biodegradability, physical properties, and perceived eco-friendliness allow for their extensive use as composite components, a role in which they could replace petroleum-based synthetic polymers. We performed biochemical, mechanical, and structural analyses of flax stems and fibers derived from field-grown transgenic flax enriched with PHB (poly-beta-hydroxybutyrate). The analyses of the plant stems revealed an increase in the cellulose content and a decrease in the lignin and pectin contents relative to the control plants. However, the contents of the fibers' major components (cellulose, lignin, pectin) remain unchanged. An FT-IR study confirmed the results of the biochemical analyses of the flax fibers. However, the arrangement of the cellulose polymer in the transgenic fibers differed from that in the control, and a significant increase in the number of hydrogen bonds was detected. The mechanical properties of the transgenic flax stems were significantly improved, reflecting the cellulose content increase. However, the mechanical properties of the fibers did not change in comparison with the control, with the exception of the fibers from transgenic line M13. The generated transgenic flax plants, which produce both components of the flax/PHB composites (i.e., fibers and thermoplastic matrix in the same plant organ) are a source of an attractive and ecologically safe material for industry and medicine.

Effects of indole-3-acetic acid on Sinorhizobium meliloti survival and on symbiotic nitrogen fixation and stem dry weight production

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and alpha-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-beta-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.

Overproduction of poly-β-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-beta-hydroxybutyrate (PHB), a polymer of industrial importance. This arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated approximately 61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also approximately 11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine-Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.

Potassium deficiency results in accumulation of ultra-high molecular weight poly-β-hydroxybutyrate in a methane-utilizing mixed culture

To investigate the effect of various single nutrient deficiencies on poly-beta-hydroxybutyrate (PHB) biosynthesis in a methane-utilizing mixed culture (dominant species Methylocystis sp. GB 25 DSM 7674). Poly-beta-hydroxybutyrate accumulation experiments were performed in 7 and 70 l bioreactors and initiated by potassium, sulfur or iron deficiency. After 24 h the PHB content reached levels of 33.6%, 32.6% and 10.4% respectively. Interestingly a polymer with an ultra-high average-weight molecular weight (M(w)) of 3.1 MDa was accumulated under potassium-limited conditions. When sulfur and iron were lacking M(w) were lower by 20.6 and 41.6%. Potassium-deficiency experiments were furthermore characterized by a maximum specific PHB formation rate 0.08 g g(-1)residual biomass (R) h(-1) and a yield coefficient of 0.45 g PHB g(-1) CH(4). Biosynthesis of an ultra-high M(w) PHB in a methane-utilizing mixed culture can be induced by potassium deficiency. This study greatly extends the knowledge in the field of bacterial biopolymer formation with gaseous substrates. The special system used here combines the use of methane a low-cost substrate available from natural and renewable sources with the possibility of employing a mixed-culture in an open system for the synthesis of a high-value product.