PHB Content Research Articles

Fructose based hyper production of poly-3-hydroxybutyrate from Halomonas sp. YLGW01 and impact of carbon sources on bacteria morphologies

Polyhydroxyalkanoates (PHA), such as poly (3-hydroxybutyrate) (PHB), have emerged as potential alternatives to petroleum-based plastics and can be produced through the appropriate selection of marine bacteria that are already adapted to high salt and low temperature conditions without the requirement of antibiotic treatment. The present study, thus, aimed to screen and characterize thirteen PHA-producing microbial strains isolated from the Gwangalli beach in Busan, South Korea. Among them, Halomonas sp. YLGW01 produced the highest amount of PHB (94.6 ± 1.8% (w/w)) using fructose. Interestingly Halomonas sp. YLGW01 showed increase in cell size (8.39 ± 3.63 μm) with fructose as carbon source as compared to glucose (2.34 ± 0.44 μm). Fructose syrup was investigated as carbon source under unsterilized conditions and 95.26 ± 1.78% of PHB was produced. Overall, this strain showed the highest PHB contents in halotolerant bacteria.

Effect of Cultivation Conditions on Poly(3-hydroxybutyrate) Synthesis by Nodule Bacteria Rhizobium phaseoli

The synthesis of poly(3-hydroxybutyric acid) (PHB) in nodule bacteria Rhizobium phaseoli grown in submerged culture was studied. The Law method for the determination of PHB was modified, which made it possible to isolate the polymer and to carry out its quantitative assessment directly in the biomass. Only S forms of R. phaseoli cells could synthesize PHB. Its accumulation was more active on carbon-rich medium (by ~50%), and the polymer granules were more clearly visible. The PHB synthesis increased with a decrease in aeration, and the maximum content was reached after 35−40 h of growth. The effect of the oxidative stress caused by t-butyl hydroperoxide and benzyl viologen on the growth of PHB-synthesizing bacteria was studied. Both compounds negatively affected bacterial growth. The growth was reactivated only in PHB-synthesizing S forms. We can conclude that PHB is a protector for nodule bacteria under the conditions of oxidative stress. The maximal PHB content in R. phaseoli cells reached 54% of the dry cell weight, which exceeded the values previously described for rhizobia. This value is close to those reported for the bacterial strains producing the polymer and used in biotechnology. We can conclude that rhizobia grown in the submerged culture can be potential producers of PHB and other polyhydroxyalkanoates.

Facile Preparation of Porous Microfiber from Poly-3-(R)-Hydroxybutyrate and Its Application.

In this study, we described the development of a simplified wet spinning method of the production of a novel type of porous continuous fiber based on poly-3-(R)-hydroxybutyrate (PHB). The principle of this method is precipitation of PHB dissolved in chloroform solution into the ethanol precipitation bath. The influence of various PHB concentrations and feed rates on specific surface area (measured by nitrogen absorption method) was studied. Materials were also characterized by SEM. Surface areas of fibers achieved by wet spinning were in the range of tens of m2.g−1, and the biggest surface area value was 55 m2.g–1. The average diameter of fibers was in the range of 20–120 μm and was dependent on both PHB concentration and feed rate. Optimum conditions for reaching stable fibers of high surface area were 3–5 % w.t. of PHB and feed rate 0.5–3 ml.h−1. Fibers were functionalized by adsorption of some natural plant extracts. The incorporation of active substances into fibers was confirmed by infrared spectroscopy. High antioxidant and antimicrobial effect of PHB-fibers with cloves extract was found, as well as excellent long-term stability and optimal dynamics of the release of active compounds. The newly produced material would be applicable in pharmacy, cosmetics, and wound healing.

Biokinetics of fed-batch production of poly (3-hydroxybutyrate) using microbial co-culture.

A novel fed-batch strategy based on carbon/nitrogen (C/N) ratio in a microbial co-culture production medium broth was carried out in a biocalorimeter for improved production of poly (3-hydroxybutyrate) (PHB). Shake flask study suggested that the C/N ratio of 10 increased the yield of PHB by 2.8 times. Online parameters monitored during the C/N ratio of 10 in biocalorimeter (BioRC1e) indicated that the heat profile was maintained in the fed-batch mode resulting in a PHB yield of 30.3 ± 1.5g/L. The oxy-calorific heat yield coefficient during the fed-batch strategy was found to be 394.24 ± 18.71kJ/O2 due to the oxidative metabolism of glucose. The reported heat-based model adapted for PHB concentration prediction in the present fed-batch mode. The heat-based model has a Nash-Sutcliffe efficiency of 0.9758 for PHB prediction. PHB obtained by fed-batch-mode was characterized using gas chromatography-mass spectrometry (GC-MS) for the monomer-acid analysis, Thermogravimetric analysis (TGA) for thermal stability of PHB, and Fourier transform infrared spectroscopy (FT-IR) for confirmation of functional groups. Here, we establish a favorable C/N ratio for achieving optimal PHB yield and a predictive heat-based model to monitor its production.

Microbial production of poly (3-hydroxybutyrate) (PHB) from rubber seed oil using Cupriavidus necator H16

Rubber seed oil is inedible plant oil that is a potential replacement for the traditional organic building blocks used in PHB biosynthesis. Herein we describe the biosynthesis of PHB by Cupriavidus necator H16 grown in media with rubber seed oil and urea serving as carbon and nitrogen sources, respectively. The method was optimized over a temperature range of 30-50 °C for batch culture studies. A maximized PHB concentration of 15.4 g/L, and dry well weight of 19.2 g/L was obtained using 20 g/L of rubber seed oil at 30°C. Experimental results were used to generate a logistic curve, and the Luedeking-Piret model was implemented to predict the specific growth rate of C. necator at 0.1898 1/h. The estimated rate constants were in good agreement with experimental values. This study demonstrated that rubber seed oil is a viable alternative to conventional carbon sources for the production of PHB using C.necator H16.

Polyhydroxybutyrate and glycogen production in photobioreactors inoculated with wastewater borne cyanobacteria monocultures

The aim of this study was to investigate the PHB and glycogen accumulation dynamics in two photobioreactors inoculated with different monocultures of wastewater-borne cyanobacteria, using a three-stage feeding strategy (growth phase, feast-famine phase and feast phase). Two cyanobacterial monocultures containing members of Synechocystis sp. or Synechococcus sp. were collected from treated wastewater and inoculated in lab-scale photobioreactors to evaluate the PHB and glycogen accumulation. A third photobioreactor with a complex microbial community grown in real wastewater was also set up. During each experimental phase different concentrations of inorganic carbon were applied to the cultures, these shifts allowed to discern the accumulation mechanism of carbon storage polymers (PHB and glycogen) in cyanobacteria. Conversion of one into the other was directly related to the carbon content. The highest PHB and glycogen contents (5.04%dcw and 69%dcw, respectively) were achieved for Synechocystis sp.

Utilization of waste fish oil and glycerol as carbon sources for polyhydroxyalkanoate production by Salinivibrio sp. M318

A moderately halophilic bacterium isolated from fermenting shrimp paste, Salinivibrio sp. M318 was found capable of using fish sauce and mixtures of waste fish oil and glycerol as nitrogen and carbon sources, respectively, for poly(3-hydroxybutyrate) (PHB) production. A cell dry weight (CDW) of up to 10 g/L and PHB content of 51.7 wt% were obtained after 48 h of cultivation in flask experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] was synthesized when 1,4-butanediol, γ-butyrolactone, or sodium 4-hydroxybutyrate was added as precursors to the culture medium. The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was achieved by supplying precursors such as sodium valerate, sodium propionate, and sodium heptanoate. Salinivibrio sp. M318 was able to accumulate the above mentioned PHAs during the growth phase. High CDW of 69.1 g/L and PHB content of 51.5 wt% were obtained by strain Salinivibrio sp. M318 after 78 h of cultivation in fed-batch culture. The results demonstrate Salinivibrio sp. M318 to be a promising wild-type bacterium for the production of PHA from aquaculture residues.

Understanding the biocalorimetric and respirometric behaviour of co-culture (R. eutropha, P. putida and A. vinelandii) in poly (3-hydroxybutyrate) batch production

Limiting nitrogen and excess carbon sources induce accumulation of the bioplastic, Poly-(3-hydroxybutyrate) (PHB) as inclusion bodies in microbes. Here, we designed a co-culture (R. eutropha, P.putida, A.vinelandii) that produced higher PHB yield than in monocultures or pairwise combination. PHB concentration of 8.3 ± 0.42 g/L, which is 63% of total Cell Dry Mass (CDM), was achieved using co-culture in batch mode of operation in biocalorimeter. Maximum total biomass based specific growth rate (μX: 0.49 h−1) was achieved in co-culture and bioenergetics profiles were compared for individual and co-culture. In this co-culture, P. putida and R. eutropha greatly contributed to PHB accumulation, while A. vinelandii induced faster growth. Aeration and agitation rates were optimized in the biocalorimeter, 1 liter per minute (lpm) and 150 rotations per minute (rpm) respectively for improved PHB yield. Oxygen uptake rate and carbon dioxide evolution rates correlated well with heat profiles, suggesting aerobic metabolism in consortia. Cellular accumulation of PHB granules in the co-culture was visualized using fluorescent microscopy.

Polyhydroxyalkanoates production from methane emissions in Sphagnum mosses: Assessing the effect of temperature and phosphorus limitation

The isolation of highly efficient methanotrophic communities is crucial for the optimization of methane bioconversion into products with a high market value such as polyhydroxyalkanoates (PHA). The research here presented aimed at enriching a methanotrophic consortium from two different inocula (Sphagnum peat moss (Sp) and Sphagnum and activated sludge (M)) able to accumulate PHA while efficiently oxidizing CH4. Moreover, the effect of the temperature and phosphorus limitation on the biodegradation rate of CH4 and the PHA accumulation potential was investigated. Higher CH4 degradation rates were obtained under P availability at increasing temperature (25, 30 and 37 °C). The biomass enriched from the mixed inoculum always exhibited a superior biodegradation performance regardless of the temperature (a maximum value of 84.3 ± 8.4 mg CH4 h−1 g biomass−1 was recorded at 37 °C). The results of the PHB production showed that phosphorus limitation is required to promote PHB accumulation, the highest PHB content being observed with the Sphagnum inoculum at 25 °C (13.6 ± 5.6%).The differential specialization of the microbial communities depending on the enrichment temperature supported the key role of this parameter on the results obtained. In all cases after the completion of the enrichment process and of the P limitation tests, Methylocystis, a type II methanotroph known for its ability to accumulate PHA, was the genus that became dominant (reaching percentages from 16 to 46% depending on the enrichment temperature). Thus, the results here obtained demonstrated for the first time the relevance of the temperature used for the enrichment of the methanotrophic bacteria to boost PHA production yields under P limiting condition, highlighting the importance of optimizing culture conditions to improve the cost-efficiency of bioprocesses based on using methane as the primary feedstock for the PHA industrial market.

Fed-Batch Cultivations of Rhodospirillum rubrum Under Multiple Nutrient-Limited Growth Conditions on Syngas as a Novel Option to Produce Poly(3-Hydroxybutyrate) (PHB).

Syngas from gasified organic waste materials is a promising feedstock for the biotechnological synthesis of the bioplastic poly([R]-3-hydroxybutyrate) (PHB) with Rhodospirillum rubrum. In a first approach, growth studies were carried out with this strain in gas-tight serum vials. When syngas (40% CO, 40% H2, 10% CO2, and 10% N2 v/v) was diluted with N2 to 60%, a 4-fold higher biomass production was detected compared to samples grown on 100% syngas, thus indicating a growth inhibitory effect. The best performing syngas-mixture was then used for C-, C,N-, and C,P-limited fed-batch fermentations in a bioreactor with continuous syngas and acetate supply. It was found that C,P-limited PHB productivity was 5 times higher than for only C-limited growth and reached a maximal PHB content of 30% w/w. Surprisingly, growth and PHB production stopped when N, as a second nutrient, became growth-limiting. Finally, it was concluded that a minimal supply of 0.2 g CO g−1 biomass h−1 has to be guaranteed in order to cover the cellular maintenance energy.

Compound‐specific 13C stable isotope probing confirms synthesis of polyhydroxybutyrate by soil bacteria

Many bacteria synthesize carbon (C) and energy storage compounds, including water-insoluble polyester lipids composed mainly or entirely of poly(3-hydroxybutyrate) (PHB). Despite the potential significance of C and energy storage for microbial life and C cycling, few measurements of PHB in soil have been reported. A new protocol was implemented, based on an earlier sediment extraction and derivatization procedure, with quantification by gas chromatography/mass spectrometry (GC/MS) and 13 C-isotopic analysis by GC/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The PHB content was 4.3 μg C g-1 in an agricultural soil and 1.2 μg C g-1 in a forest topsoil. This was an order of magnitude more PHB than obtained by the existing extraction method, suggesting that native PHB in soil has been previously underestimated. Addition of glucose increased the PHB content by 135% and 1,215% over 5 days, with the largest increase in the relatively nutrient-poor forest soil. In the agricultural soil, 68% of the increase was derived from added 13 C-labeled glucose, confirming synthesis of PHB from glucose for the first time in soil. The presence and responsiveness of PHB in both these contrasting soils show that PHB could provide a useful indicator of bacterial nutritional status and unbalanced growth. Microbial storage could be important to C and nutrient cycling and be a widespread strategy in the life of soil bacteria. The presented method offers new insight into the significance of this compound in soil.

Production of poly(3-hydroyxybutylate) by a novel alginolytic bacterium Hydrogenophaga sp. strain UMI-18 using alginate as a sole carbon source

A novel alginolytic bacterium Hydrogenophaga sp. strain UMI-18 that produces poly(3-hydroyxybutylate) (PHB) in the alginate-mineral salt (AMS) medium containing 1% (w/v) sodium alginate as a sole carbon source was isolated from a decayed brown seaweed litter. The yield of PHB produced by strain UMI-18 was 1.1±0.15g/L of AMS and the PHB content in dried cell pellet was 58±4% (w/w). Glucose, fructose, galactose, mannose, mannitol, sucrose and lactose were also available for the production of PHB by strain UMI-18. The yield of PHB in 1% (w/v) carbohydrate media reached 2.03-2.24g/L for glucose and fructose, 0.75-1.64g/L for alginate, galactose, mannitol and sucrose, and ∼0.15g/L for lactose. The PHB produced by strain UMI-18 showed a glass-transition temperature (Tg) at 4°C, a melting temperature at 175°C, and an average molecular mass of 860kDa. Draft genome analysis of the strain UMI-18 revealed that an alginate-assimilating gene cluster is located in contig 8 comprising 453,520bp and the PHB-synthesis gene cluster is in contig 15 comprising 653,793bp.

Effect of carbon sources for PHB production in bubble column bioreactor: Emphasis on improvement of methane uptake

In this study, poly(3-hydroxybutyrate) (PHB) production was investigated for the first time using high cell density cultivation of Methylocystis hirsuta from natural gas. Various carbon sources including acetate, ethanol and glucose were used in a loop bioreactor. Then, effects of magnesium and phosphorus contents on PHB production were investigated. Results revealed that the bacterial strain had the capability to grow on various substrates. Furthermore, the cell dry mass (CDM) reached to 4.5 g/L with a PHB content of 85% (w/w) using a ratio of 1:1 of methanol:ethanol. Deficiencies of magnesium and phosphorus were shown to play a key role to achieve high cell densities in bioreactors. In addition, PHB was produced in a bubble column bioreactor at optimum conditions, resulting in the production of 8 g/L of CDM with a PHB content of 73.4% (w/w).

Scale-up challenges and requirement of technology-transfer for cyanobacterial poly (3-hydroxybutyrate) production in industrial scale

ABSTRACTEven though extensive research has been directed toward optimization of the strain, media, and the process, yet there is limited knowledge on the performance and viability of large-scale PHB production lines exploiting cyanobacteria. In this study, the scale-up challenges associated with photosynthetic PHB production are listed. The high PHB producing cyanobacterial mutant, MT_a24, a randomly mutated strain of Synechocystis sp. PCC 6714, has been tested in pilot-scale trials for photosynthetic PHB production under non-sterile conditions. The MT_a24 obtained PHB content of 0.356 g L−1 and 1.7 g L−1 of glycogen from CO2 after 10 days of cultivation using a self-limiting media and non-optimized cultivation parameters. The results obtained here suggest that in order to achieve high PHB productivity values of the lab and to overcome the existing scalability issues reassessment of the optimized parameters needs to be performed during the pilot-scale trials.

Poly(3-hydroxybutyrate) Production from Natural Gas by a Methanotroph Native Bacterium in a Bubble Column Bioreactor

Reducing the total cost of poly(3-hydroxybutyrate) (PHB) production as an attractive substitute for conventional petrochemical plastics still remains an unsolved problem. The aim of this research was the screening of PHB-producing microorganisms and selection of the best suitable medium for microbial growth and PHB production from methane. A new isolated methanotroph for PHB production from natural gas was studied in different media. After selection of the suitable medium, the effect of five process variables (content of nitrogen source, disodium hydrogen phosphate, methane to air ratio, seed age, and pH) on PHB production was investigated in a bubble column bioreactor. Also, hydrodynamic and mass transfer factors (flow regime, mixing time, gas hold up, and kLa) were considered. At optimum operating conditions and engineering parameters in a bubble column, PHB content in the dried biomass reached 25 % w/w. The results showed that pH is the most important variable in the selected conditions.

Poly(3-hydroxybutyrate) accumulation by Azotobacter vinelandii under different oxygen transfer strategies.

Azotobacter vinelandii OP is a bacterium that produces poly(3-hydroxybutyrate) (PHB). PHB production in a stirred bioreactor, at different oxygen transfer strategies, was evaluated. By applying different oxygen contents in the inlet gas, the oxygen transfer rate (OTR) was changed under a constant agitation rate. Batch cultures were performed without dissolved oxygen tension (DOT) control (using 9% and 21% oxygen in the inlet gas) and under DOT control (4%) using gas blending. The cultures that developed without DOT control were limited by oxygen. As result of varying the oxygen content in the inlet gas, a lower OTR (4.6mmolL-1h-1) and specific oxygen uptake rate (11.6mmolg-1h-1) were obtained using 9% oxygen in the inlet gas. The use of 9% oxygen in the inlet gas was the most suitable for improving the intracellular PHB content (56 ± 6w w-1). For the first time, PHB accumulation in A. vinelandii OP cultures, developed with different OTRs, was compared under homogeneous mixing conditions, demonstrating that bacterial respiration affects PHB synthesis. These results can be used to design new oxygen transfer strategies to produce PHB under productive conditions.

Preparation and characterization of thermoplastic starch composite reinforced by plasma-treated poly (hydroxybutyrate) PHB

The present work aims to study the effect of the addition of poly (hydroxybutyrate) PHB as reinforcement in thermoplastic cornstarch (TPS) produced by the thermal compression molding method. Initially, the physical and chemical properties of TPS with different amounts of glycerol as the plasticizer (25, 30 and 35 wt%), are evaluated. Then, the composites, including 35% glycerol and different PHB contents (10, 20 and 30 wt%), are prepared. Additionally, the surface of the PHB granules is modified by plasma treatment using atmospheric air and sulfur hexafluoride (SF6) gas. In order to evaluate the influence of the PHB contents before and after the plasma treatment, the composites are characterized by FTIR, TGA, DSC, XRD, SEM, and mechanical tests. SEM showed a homogeneous distribution of PHB granules in the TPS matrix. On the other hand, the micrographs of the composites, using the high concentration of plasma-treated PHB, showed the agglomerated particles in the starch matrix, which represented stress concentrators, and showed weak interfacial adhesion leading to the poor mechanical properties. The thermogravimetric analysis of the composites with PHB treated by plasma showed a higher thermal stability compared to the composites of TPS and untreated PHB.

Increased carbohydrate production from carbon dioxide in randomly mutated cells of cyanobacterial strain Synechocystis sp. PCC 6714: Bioprocess understanding and evaluation of productivities

Recently, several mutants of Synechocystis sp. PCC 6714 were obtained showing superior PHB content and productivities. Here, the most promising mutant named MT_a24 is compared in detail with the wild-type in controlled photobioreactors. In order to provide an easily scalable and alternative approach to the normally done two-step process -comprising of growth phase and limitation phase- a one-step cultivation was optimized. The multivariate experimental design approach was used for the optimization of the one-step, self-limiting media. During one-step cultivation of MT_a24 with optimized media 30 ± 4% (DCW) corresponding to 1.16 g L−1 PHB was obtained. Using pulse experiments it was demonstrated that phosphate is the key driver of glycogen synthesis in Synechocystis sp. PCC 6714 and it can be used to boost glycogen productivity. The maximum glycogen content acquired was 2.6 g L−1 (76.2% DCW) for mutant MT_a24 using phosphate feeding and carbon dioxide as carbon source.

Biodegradable PHB-Rubber Copolymer Toughened PLA Green Composites with Ultrahigh Extensibility

The sustainable biopolymer, poly(lactide) (PLA), has been intensely researched over the past decades because of its excellent biodegradability, renewability, and sustainability. The boundless potential of this sustainable biopolymer could resolve the adverse negative impact caused by the petroleum-based polymers. However, the inherent drawback of PLA such as brittleness, low heat distortion temperature, and slow recrystallization rate narrowed the broad applications in biomedical, automotive, and structural fields. In this study, we successfully synthesized a PHB-based filler (PHB-di-rub) displaying synergetic functions of (1) effective nucleation and (2) extreme toughening of the PLA matrix at only 5% (1.5 wt % PHB content). Remarkably, the storage modulus improves by 15%; tensile elongation extends by 57-fold (300% strain) and toughness by 38-fold while maintaining its original strength and stiffness. Likewise, 10% of PHB-di-rub (3 wt % PHB content) has an even higher improvement with a storage modulus ...

Assessment of poly(3-hydroxybutyrate) synthesis from a novel obligate alkaliphilic Bacillus marmarensis and generation of its composite scaffold via electrospinning

In this study, poly(3-hydroxybutyrate) (PHB) production from a newly isolated obligate alkaliphilic Bacillus marmarensis DSM 21297 was investigated to evaluate the ability of obligate alkaliphilic strain to produce a biopolymer. Additionally, electrospun nanofibers from B. marmarensis PHB (Bm-PHB) were generated using Bm-PHB/polycaprolactone (PCL) blend to evaluate the applicability of Bm-PHB. According to the experimental results, the metabolic activity of B. marmarensis decreased the pH of the medium by generating H+ ions to initiate Bm-PHB production, which was achieved at pH below 9.0. Regarding medium components, the addition of MgSO4.7H2O and KH2PO4 to the medium containing 1% glucose enhanced the amount of Bm-PHB synthesis, and an approximately 60% increase in PHB concentration was obtained in the presence of mineral salts.Based on FTIR analysis, the chemical structures of Bm-PHB and commercial PHB were found to be highly similar. Additionally, the Tg and Tm values of Bm-PHB were determined to be 17.77 °C and 165.17 °C, respectively. Moreover, Bm-PHB/PCL composite scaffold was generated by electrospinning method that produced nanofibers between 150 and 400 nm in diameter, with an average of 250 nm. To our knowledge, this is the first report to produce PHB from an obligate alkaliphilic Bacillus strain and PHB scaffold.