Polyhydroxybutyrate Degradation Research Articles

Unveiling the potential of bacterial isolates from plastic-polluted environments: enhancement of polyhydroxybutyrate biodegradation

This study explores the biodegradation potential of microbial isolates focusing on their ability to utilize biopolymers as sole carbon source. Previously described isolates have been investigated through agar-based screen for the ability to degrade plastic-related substrates in powder form, and four strains have been selected for further assessment. Polyhydroxybutyrate (PHB) films degradation was examined through liquid culture, soil burial, and respirometry assays. Structural and chemical alterations in PHB were analysed using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The most successful strains were tested for the ability to degrade PHB/bacterial nanocellulose (BNC) blends. Bacillus sp. DG90 excelled in PHB degradation, achieving 60% weight loss in liquid culture, while Streptomyces sp. DG19 exhibited a notable degradation rate of 51 ± 1.7%. Soil burial assays underscored the impact of environmental factors on degradation rates, emphasizing the role of soil composition and nitrogen availability. In respirometry assay, PHB films were severely defragmented by Streptomyces sp. DG19 with overall weight loss of 83%, while for Bacillus sp. DG90, this percentage reached 39%. FTIR and DSC analyses suggested potential hydrolysis and structural alterations in treated samples. This study observed rapid PHB degradation (83% in 3 weeks) while, considering the complex composition of modern biomaterials, also showcased the potential of examined strains to degrade PHB-BNC blends up to 85%.

Bacterial diversity of biofilms on polyhydroxybutyrate exposed to marine conditions: Ex-situ vs. in-situ tests

Biofilms form on any available surface and, depending on the characteristics of the material and the environmental conditions, biodegradation can take place. We compared the bacterial composition of polyhydroxybutyrate (PHB)-related biofilm communities from marine ex-situ and in-situ tests to assess the differences in diversity and abundance between these two biofilms. This comparison will help to better assess the transferability of tank tests to real-life scenarios. The in-situ tests were set up in the Mediterranean Sea on the Island of Elba, Italy where PHB-tensile bars were lodged in the sediments. This created a water-exposed aerobic and mud-planted anaerobic scenario. The ex-situ tests were modeled after in-situ tests and performed in temperature-controlled tanks. The PHB-related biofilms were harvested after 240 days of exposure along with planktonic bacteria, and particle- and sediment-related biofilm. The bacterial composition was elucidated using 16S rDNA sequencing. Biofilms harvested from the in-situ test were more diverse, less even, and contained more rare species compared to biofilms from the ex-situ test. The PHB-related biofilm was characterized by a higher abundance of the bacterial order Desulfobacterales. The composition of PHB-related biofilm varied significantly between the two tests and between aerobic and anaerobic conditions. The composition of PHB-related biofilm was significantly different from planktonic bacteria, particle, and sediment-related biofilm, showing the influence of PHB on the biofilm composition. Thus, the ex-situ tank test for PHB degradation cannot, in terms of bacterial composition, simulate the in-situ conditions to their full extent.

A strategy to promote the convenient storage and direct use of polyhydroxybutyrate-degrading Bacillus sp. JY14 by lyophilization with protective reagents

BackgroundBioplastics are attracting considerable attention, owing to the increase in non-degradable waste. Using microorganisms to degrade bioplastics is a promising strategy for reducing non-degradable plastic waste. However, maintaining bacterial viability and activity during culture and storage remains challenging. With the use of conventional methods, cell viability and activity was lost; therefore, these conditions need to be optimized for the practical application of microorganisms in bioplastic degradation. Therefore, we aimed to optimize the feasibility of the lyophilization method for convenient storage and direct use. In addition, we incoporated protective reagents to increase the viability and activity of lyophilized microorganisms. By selecting and applying the best protective reagents for the lyophilization process and the effects of additives on the growth and PHB-degrading activity of strains were analyzed after lyophilization. For developing the lyophilization method for protecting degradation activity, it may promote practical applications of bioplastic-degrading bacteria.ResultsIn this study, the polyhydroxybutyrate (PHB)-degrading strain, Bacillus sp. JY14 was lyophilized with the use of various sugars as protective reagents. Among the carbon sources tested, raffinose was associated with the highest cell survival rate (12.1%). Moreover, 7% of raffionose showed the highest PHB degradation yield (92.1%). Therefore, raffinose was selected as the most effective protective reagent. Also, bacterial activity was successfully maintained, with raffinose, under different storage temperatures and period.ConclusionsThis study highlights lyophilization as an efficient microorganism storage method to enhance the applicability of bioplastic-degrading bacterial strains. The approach developed herein can be further studied and used to promote the application of microorganisms in bioplastic degradation.

Expression, thermal stability modification and application in PHB degradation of polyhydroxyalkanoate depolymerase from Thermomonospora umbrina

Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.

Selected Simple Natural Antimicrobial Terpenoids as Additives to Control Biodegradation of Polyhydroxy Butyrate.

In this experimental research, different types of essential oils (EOs) were blended with polyhydroxybutyrate (PHB) to study the influence of these additives on PHB degradation. The blends were developed by incorporating three terpenoids at two concentrations (1 and 3%). The mineralization rate obtained from CO2 released from each sample was the factor that defined biodegradation. Furthermore, scanning electron microscope (SEM), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA) were used in this research. The biodegradation percentages of PHB blended with 3% of eucalyptol, limonene, and thymol after 226 days were reached 66.4%, 73.3%, and 76.9%, respectively, while the rate for pure PHB was 100% after 198 days, and SEM images proved these results. Mechanical analysis of the samples showed that eucalyptol had the highest resistance level, even before the burial test. The other additives showed excellent mechanical properties although they had less mechanical strength than pure PHB after extrusion. The samples' mechanical properties improved due to their crystallinity and decreased glass transition temperature (Tg). DSC results showed that blending terpenoids caused a reduction in Tg, which is evident in the DMA results, and a negligible reduction in melting point (Tm).

Finding a Benign Plasticizer to Enhance the Microbial Degradation of Polyhydroxybutyrate (PHB) Evaluated by PHB Degrader Microbulbifer sp. SOL66

As a biodegradable plastic, polyhydroxybutyrate (PHB) has relatively poor mechanical properties, preventing its wider use. Various plasticizers have been studied to improve the mechanical properties of PHB; however, due to the slow degradation speed in the soil environment and lack of evaluation methods, studies on the degradation of PHB with plasticizers are rarely reported. In this study, by applying Microbulbifer sp. SOL66, which is able to degrade PHB very quickly, a benign plasticizer was evaluated with good properties and good degradability, not inhibiting microbial activities. Eight different plasticizers were applied with PHB and Microbulbifer sp. SOL66, PHB film containing 10% and 20% tributyl citrate showed significant biodegradability of PHB. It was confirmed that tributyl citrate could increase the speed of PHB degradation by Microbulbifer sp. SOL66 by 88% at 1 day, although the degree of degradation was similar after 3 days with and without tributyl citrate. By the analysis of microbial degradation, physical, chemical, and mechanical properties, tributyl citrate was shown not only to improve physical, chemical, and mechanical properties but also the speed of microbial degradation.

Polyhydroxyalkanoates (PHAs) degradation by the newly isolated marine Bacillus sp. JY14

Polyhydroxyalkanoates (PHAs) are bioplastic substitutes for petroleum-derived plastics that may help to reduce the increasing environmental impact of plastic pollution. Among them, polyhydroxybutyrate (PHB) is a promising biopolymer, incentivizing many researchers to search for PHB-producing and PHB-degrading bacteria for improved PHB utilization. Many novel PHB-producing microorganisms have been discovered; however, relatively few PHB-degrading bacteria have been identified. Six PHB-degrading bacteria were found in marine soil and investigated their PHB-degrading abilities under various temperature and salinity conditions using solid-media based culture. Finally, thermotolerant and halotolerant PHB-degrader Bacillus sp. JY14 was selected. PHB degradation was confirmed by monitoring changes in the physical and chemical properties of PHB films incubated with Bacillus sp. JY14 using scanning electron microscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography. Further, PHB degradation ability of Bacillus sp. JY14 was measured in liquid culture by gas chromatography. After 14 days of cultivation with PHB film, Bacillus sp. JY14 achieved approximately 98% PHB degradation. Applying various bioplastics to assess the bacteria's biodegradation capabilities, the result showed that Bacillus sp. JY14 could also degrade P(3HB-co-4HB) and P(3HB-co-3HV). Overall, this study identified a thermotolerant and halotolerant bacteria capable of PHB degradation under solid and liquid conditions. These results suggest that this bacteria could be utilized to degrade various PHAs.

Improvement of polyhydroxybutyrate (PHB) plate-based screening method for PHB degrading bacteria using cell-grown amorphous PHB and recovered by sodium dodecyl sulfate (SDS)

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable plastic. Considering the environmental issues of petroleum-based plastics, PHB is promising as it can be degraded in a relatively short time by bacteria to water and carbon dioxide. Substantial efforts have been made to identify PHB-degrading bacteria. To identify PHB-degrading bacteria, solid-based growth or clear zone assays using PHB as the sole carbon source are the easiest methods; however, PHB is difficult to dissolve and distribute evenly, and bacteria grow slowly on PHB plates. Here, we suggest an improved PHB plate assay using cell-grown PHB produced by Halomonas sp. and recovered by sodium dodecyl sulfate (SDS). Preparation using SDS resulted in evenly distributed PHB plates that could be used for sensitive depolymerase activity screening in less time compared with solvent-melted pellet or cell-grown PHB. With this method, we identified 15 new strains. One strain, Cutibacterium sp. SOL05 (98.4% 16S rRNA similarity to Cutibacterium acne), showed high PHB depolymerase activity in solid and liquid conditions. PHB degradation was confirmed by clear zone size, liquid culture, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The results indicate this method can be used to easily identify PHB-degrading bacteria from various sources to strengthen the benefits of bioplastics.

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His‐tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate‐based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

Comparative genomics of Sphingopyxis spp. unravelled functional attributes

Members of genus Sphingopyxis are known to thrive in diverse environments. Genomes of 21 Sphingopyxis strains were selected. Phylogenetic analysis was performed using GGDC, AAI and core-SNP showed agreement at sub-species level. Based on our results, we propose that both S. baekryungensis DSM16222 and Sphingopyxis sp. LPB0140 strains should not be included under genus Sphingopyxis. Core-analysis revealed, 1422 genes were shared which included essential pathways and genes for conferring adaptation against stress environment. Polyhydroxybutyrate degradation, anaerobic respiration, type IV secretion were notable abundant pathways and exopolysaccharide, hyaluronic acid production and toxin-antitoxin system were differentially present families. Interestingly, genome of S. witflariensis DSM14551, Sphingopyxis sp. MG and Sphingopyxis sp. FD7 provided a hint of probable pathogenic abilities. Protein-Protein Interactome depicted that membrane proteins and stress response has close integration with core-proteins while aromatic compounds degradation and virulence ability formed a separate network. Thus, these should be considered as strain specific attributes.

Microbial functional diversity plays an important role in the degradation of polyhydroxybutyrate (PHB) in soil.

Towards bioremediation of recalcitrant materials like synthetic polymer, soil has been recognized as a traditional site for disposal and subsequent degradation as some microorganisms in soil can degrade the polymer in a non-toxic, cost-effective, and environment friendly way. Microbial functional diversity is a constituent of biodiversity that includes wide range of metabolic activities that can influence numerous aspects of ecosystem functioning like ecosystem stability, nutrient availability, ecosystem dynamics, etc. Thus, in the current study, we assumed that microbial functional diversity could play an important role in polymer degradation in soil. To verify this hypothesis, we isolated soil from five different sites of landfill and examined several microbiological parameters wherein we observed a significant variation in heterotrophic microbial count as well as microbial activities among the soil microcosms tested. Multivariate analysis (principle component analysis) based on the carbon sources utilization pattern revealed that soil microcosms showed different metabolic patterns suggesting the variable distribution of microorganisms among the soil microcosms tested. Since microbial functional diversity depends on both microbial richness and evenness, Shannon diversity index was determined to measure microbial richness and Gini coefficient was determined to measure microbial evenness. The tested soil microcosms exhibited variation in both microbial richness and evenness suggesting the considerable difference in microbial functional diversity among the tested microcosms. We then measured polyhydroxybutyrate (PHB) degradation in soil microcosms after desired period of incubation of PHB in soil wherein we found that soil microcosms having higher functional diversity showed enhanced PHB degradation and soil microcosms having lower functional diversity showed reduced PHB degradation. We also noticed that all the tested soil microcosms showed similar pattern in both microbial functional diversity and PHB degradation suggesting a strong positive correlation (r = 0.95) between microbial functional diversity and PHB degradation. Thus, the results demonstrate that microbial functional diversity plays an important role in PHB degradation in soil by exhibiting versatile microbial metabolic potentials that lead to the enhanced degradation of PHB.

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16.

In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutrophaIMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing.

Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen–thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.

Effect of influent C/N ratio on nitrogen removal using PHB as electron donor in a post‐denitritation SBR

AbstractBackgroundThe post‐denitritation sequencing batch reactor (SBR) is widely‐used and can achieve high levels of nitrogen removal. In this study the effect of influent COD/TN (total nitrogen) ratio (i.e. C/N ratio) on nitrogen removal performance was investigated.ResultsThe experimental results showed that polyhydroxybutyrate (PHB) was the internal carbon source for denitritation, so PHB degradation rate following first‐order kinetics was the rate‐limiting step both for simultaneous nitritation–denitritation (SND) in the substrate famine period of the oxic stage and endogenous denitritation in the anoxic stage. Higher influent C/N ratio resulted in more PHB fractions in microorganisms, which facilitated a higher efficiency of SND and a faster endogenous denitritation rate (DNR). Consequently, mean TN removal ratio in oxic stage dropped from 32.81% to 8.61%, and average endogenous DNR in the anoxic stage fell from 1.50 to 0.27 mgN h‐1 gVSS‐1, when influent C/N ratio changed from 6.82 to 1.89. Furthermore, PHB fraction in the biomass did not drop drastically when influent C/N ratio dropped for a short‐term period, which facilitated better resistance to shock loads.ConclusionHigh influent C/N ration benefits nitrogen removal in this process, and an influent C/N ratio of 4.00 was suitable for advanced nitrogen removal. © 2013 Society of Chemical Industry

Microstructure and properties of polyhydroxybutyrate-calcium phosphate cement composites

AbstractThe biopolymer (polyhydroxybutyrate) microparticles-calcium phosphate composites were prepared by mechanical mixing of the basic composite components with the addition of hardening liquid after ethanol-composite mixture suspension moulding. The composite microstructures were more compact than the pure cement samples as confirmed by the lower values of specific areas and mesopore volumes. Both the specific areas and mesopore volumes decreased with soaking time in a simulated body fluid. The low polyhydroxybutyrate degradation in composites was found after soaking in simulated body fluid, which was terminated after one week. The formation of a dense apatite layer bonded directly to the surface of polyhydroxybutyrate microparticles was observed. The highest diametral tensile strength (13 MPa) and compressive strength (95 MPa) values of up to 50 % higher than in pure cement were measured in samples with 10 % of polyhydroxybutyrate. The addition of polyhydroxybutyrate microparticles had no effect on the setting time.

Improvement of thermal properties of polyhydroxybutyrate by grafted chemicals

AbstractThe crystallization and thermal degradation behaviors of polyhydroxybutyrate (PHB) grafted with maleic acid (MAc) and exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride (ETA) are analyzed with differential scanning calorimetry (DSC), dynamic thermogravimetric analysis (TGA), and gel permeation chromatography (GPC). It is obtained that the PHB grafted with MAc or ETA has a faster crystallization rate, larger crystallinity, and better thermal stability than the as-received PHB. The thermal stability of specimens are in the order of the as-received PHB < PHB grafted with ETA < PHB grafted with MAc. The grafting ratio of MAc is larger than that of ETA. The thermal stability of PHB can be greatly improved by grafting a small amount of MAc. The activation energy of the PHB degradation reaction are little affected by the grafted chemicals, nonetheless, the reaction order and frequency factor are significantly affected by the grafted MAc.

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1-18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.

Dynamics of Activity of the Key Enzymes of Polyhydroxyalkanoate Metabolism in Ralstonia eutropha B5786

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation. The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.

Sinorhizobium meliloti strain 1021 bioS and bdhA gene transcriptions are both affected by biotin available in defined medium.

Sinorhizobium meliloti 1021 responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Immunogold labeling and electron microscopy revealed that the BioS protein is located within the S. meliloti cytoplasm. Under biotin-limiting conditions the S. meliloti cell lumen was filled with polyhydroxybutyrate (PHB) granules suggesting that either PHB synthesis or degradation are influenced by biotin. To test this hypothesis a 3-hydroxybutyrate-dehydrogenase-lacZ (bdhA-lacZ) fusion was mobilized into S. meliloti. beta-galactosidase tests revealed an overall 3.6-5.2-fold higher bdhA transcription in the presence of added biotin. Comparison of the bdhA and the bioS promoter regions identified several common motifs.

Sinorhizobium meliloti strain 1021 bioS and bdhA gene transcriptions are both affected by biotin available in defined medium

Sinorhizobium meliloti 1021 responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Immunogold labeling and electron microscopy revealed that the BioS protein is located within the S. meliloti cytoplasm. Under biotin-limiting conditions the S. meliloti cell lumen was filled with polyhydroxybutyrate (PHB) granules suggesting that either PHB synthesis or degradation are influenced by biotin. To test this hypothesis a 3-hydroxybutyrate-dehydrogenase- lacZ ( bdhA-lacZ) fusion was mobilized into S. meliloti. β-galactosidase tests revealed an overall 3.6–5.2-fold higher bdhA transcription in the presence of added biotin. Comparison of the bdhA and the bioS promoter regions identified several common motifs.