PHA Accumulation Research Articles

The metabolic pathways of carbon assimilation and polyhydroxyalkanoate production by Rhodospirillum rubrum in response to different atmospheric fermentation.

The purple nonsulfur bacteria, Rhodospirillum rubrum, is recognized as a potential strain for PHAs bioindustrial processes since they can assimilate a broad range of carbon sources, such as syngas, to allow reduction of the production costs. In this study, we comparatively analyzed the biomass and PHA formation behaviors of R. rubrum under 100% CO and 50% CO gas atmosphere and found that pure CO promoted the PHA synthesis (PHA content up to 23.3% of the CDW). Hydrogen addition facilitated the uptake and utilization rates of CO and elevated 3-HV monomers content (molar proportion of 3-HV up to 9.2% in the presence of 50% H2). To elucidate the genetic events culminating in the CO assimilation process, we performed whole transcriptome analysis of R. rubrum grown under 100% CO or 50% CO using RNA sequencing. Transcriptomic analysis indicated different CO2 assimilation strategy was triggered by the presence of H2, where the CBB played a minor role. An increase in BCAA biosynthesis related gene abundance was observed under 50% CO condition. Furthermore, we detected the α-ketoglutarate (αKG) synthase, converting fumarate to αKG linked to the αKG-derived amino acids synthesis, and series of threonine-dependent isoleucine synthesis enzymes were significantly induced. Collectively, our results suggested that those amino acid synthesis pathways represented a key way for carbon assimilation and redox potential maintenance by R. rubrum growth under syngas condition, which could partly replace the PHA production and affect its monomer composition in copolymers. Finally, a fed-batch fermentation of the R. rubrum in a 3-l bioreactor was carried out and proved H2 addition indeed increased the PHA accumulation rate, yielding 20% ww-1 PHA production within six days.

Unlocking growth potential in Halomonas bluephagenesis for enhanced PHA production with sulfate ions.

The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.

Characterization of polyhydroxybutyrate production from Halomonas titanicaeKHS3 and manufacturing of electrosprayed nanoparticles

AbstractPolyhydroxyalkanoates (PHAs) are biopolymers accumulated by a diversity of bacterial strains as carbon and energy reserve when grown under unbalanced nutritional conditions. Among the wide spectrum of applications for these biopolymers, the generation of nano and microparticles has drawn huge attention. PHA nanoparticles showed a high surface‐to‐volume ratio that makes them interesting for pharmaceutical uses. Bacteria from the Halomonas genus became interesting tools for biopolymer production in the last years, due to its metabolic plasticity that allows them to grow in a wide spectrum of compounds and salt concentrations. Halomonas titanicae KHS3 was previously isolated based on their ability to grow using aromatic hydrocarbons as the sole carbon source. In this work, the novelty lies in the evaluation of PHA accumulation by H. titanicae KHS3. Accumulation was successfully observed and thoroughly characterized on various carbon sources. Irrespective of the carbon source employed for growth, our experimental conditions consistently yielded PHB as the sole material identified. The truly intriguing aspect of this study is that PHB solutions in glacial acetic acid demonstrated exceptional suitability for electrospraying processing. This groundbreaking development led to the creation of nanoparticles with unique characteristics that hold immense promise for a wide range of applications.

Utilization of chicken feathers for PHA accumulation by bacteria isolated from slaughterhouse soil.

Utilization of chicken feathers for PHA accumulation by bacteria isolated from slaughterhouse soil.

Production of medium-chain-length PHA in octanoate-fed enrichments dominated by Sphaerotilus sp.

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by using microbial enrichments is a promising but largely unexplored approach to obtain elastomeric biomaterials from secondary resources. In this study, several enrichment strategies were tested to select a community with a high mcl-PHA storage capacity when feeding octanoate. On the basis of analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen-limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation experiments resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with an mcl fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with an mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction with a higher Mw and a hydroxyhexanoate/hydroxyoctanoate-rich fraction with a lower Mw . The ratio between the two fractions could be adjusted by changing the environmental conditions, such as oxygen availability and pH. Almost all enrichments were dominated by Sphaerotilus sp. This is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains.

Process conditions affect properties and outcomes of polyhydroxyalkanoate accumulation in municipal activated sludge

The developments of mixed culture polyhydroxyalkanoate production has been directed to maximize the biomass PHA content with limited attention to polymer quality. Direct comparison of PHA accumulation literature is challenging, and even regularly contradicting in reported results, due to underlying differences that are not well expressed. A study was undertaken to systematically compare the commonly reported process conditions for PHA accumulation by full-scale municipal activated sludge. A biomass acclimation step combined with a pulse-wise feeding strategy resulted in maximum average PHA contents and product yields. pH control and active nitrification did not result in observable effects on the PHA productivity. Under these conditions a high molecular weight polymer (1536 ± 221 kDa) can be produced. Polymer extraction recoveries were influenced by the PHA molecular weight. A standard protocol for an activated sludge PHA accumulation test including downstream processing and standardized extraction has been developed and is available as supplementary material.

Quantification of polyhydroxyalkanoate accumulated in waste activated sludge

Polyhydroxyalkanoate accumulation experiments at pilot scale were performed with fullscale municipal waste activated sludge. Development of biomass PHA content was quantified by thermogravimetric analysis. Over 48 h the biomass reached up to 0.49 ± 0.03 gPHA/gVSS (n=4). Samples were processed in parallel to characterise the distribution of PHA in the biomass. Selective staining methods and image analysis were performed by Confocal Laser Scanning Microscopy. The image analysis indicated that nominally 55% of this waste activated sludge was engaged in PHA storage activity. Thus even if the biomass PHA content reached 0.49gPHA/gVSS, the accumulating fraction of the biomass was estimated to have attained about 0.64gPHA/gVSS. The combination of quantitative microscopy and polymer mass assessment enabled to distinguish the effect of level of enrichment in PHA storing bacteria and the average PHA storage capacity of the accumulating bacteria. The distribution of microbial 16S rRNA levels did not follow a measurable trend during PHA accumulation.

Engineering Burkholderia sacchari to enhance poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] production from xylose and hexanoate

Burkholderia sacchari LFM101 LMG19450T is a Brazilian bacterium isolated from sugarcane crops soil and a promising biotechnological platform for bioprocesses. It is an efficient producer of poly(3-hydroxybutyrate) from carbohydrates including xylose. In the present work, the expression of B. sacchari xylose consumption genes (xylA, xylB and tktA) was combined with the expression of Aeromonas sp. phaC (PHA synthase), aiming to increase both the growth rates in xylose and the 3-hydroxyhexanoate (3HHx) molar fractions in the produced PHA. Genes were cloned into pBBR1MCS-2 vectors and then expressed in the B. sacchari PHA- mutant LFM344. Maximum specific growth rates on xylose and PHA accumulation capacity of all recombinants were evaluated. In bioreactor experiments, up to 55.5 % CDW was accumulated as copolymer, hexanoate conversion to 3HHx raised from 2 % to 54 % of the maximum theoretical value, compared to wild type. 3HHx mol% ranged from 8 to 35, and molecular weights were between 111 and 220 kg/mol. Thermal analysis measurement showed a decrease in Tg and Tm values with higher 3HHx fraction, indicating improved thermomechanical characteristics. Recombinants construction and bioreactor strategies allowed the production of P(3HB-co-3HHx) with controlled monomeric composition from xylose and hexanoate, allowing its application in diverse fields, including the medical area.

Engineered Nanogel Particles Enhance the Photoautotrophic Biosynthesis of Polyhydroxyalkanoate in Marine Photosynthetic Bacteria

Improving polyhydroxyalkanoate (PHA, a biodegradable plastic) production under photoautotrophic cultivation is challenging for sustainable bioproduction. In this study, we demonstrated the use of engineered nanogel particles to enhance PHA accumulation in the marine photosynthetic bacterium Rhodovulum sulfidophilum under photoautotrophic culture. We screened the effect of 13 engineered nanogel particles on the cell growth and PHA accumulation of R. sulfidophilum. The addition of anionic nanogel particles significantly enhanced PHA accumulation in R. sulfidophilum up to 157-fold compared to that without nanogel particles. By performing 13C tracer experiments and gas chromatography–mass spectrometry analysis, we confirmed that HCO3– was assimilated throughout the central carbon metabolism and that the accumulated PHA was indeed incorporated from HCO3–. Our results indicate successful PHA production with the supplementation of engineered nanogel particles under photoautotrophic cultivation in R. sulfidophilum. Furthermore, the strategy of using engineered nanoparticles demonstrated in this study may be applicable to other microbial cell factories to produce other commodity metabolites.

Microbial conversion of lignin rich biomass hydrolysates to medium chain length polyhydroxyalkanoates (mcl-PHA) using Pseudomonas putida KT2440

As world moves toward increasing number of products being produced from renewable lignocellulosic agricultural and forest residues, the major classes of products that will shift to greener routes on priority are energy, fuels, and materials in that order. In materials segment, polyhydroxyalkanoates are an emerging class of biopolyesters with several potential industrial uses. The present work investigates medium chain length polyhydroxyalkanoates (mcl-PHA) producing capabilities of Pseudomonas putida KT2440 from a mixture of compounds produced from lignocellulosic biomass deconstruction. The hydrolysates obtained from nitric acid pretreatment of lignin rich cotton stalk (CS) and palm empty fruit bunch (EFB) were used as substrates for production of mcl-PHA. Presence of 3-hydroxydecanoate and 3-hydroxyocytanoate observed on GC-MS confirmed PHA accumulation in the cells. PHA accumulation was estimated between 20% and 35% of cell dry weight when grown on both model substrates as well as biomass hydrolysates. PHA titers obtained on hydrolysates of CS and EFB were 0.24 g/L and 0.21 g/L, respectively.

Synthetic Control of Metabolic States in Pseudomonas putida by Tuning Polyhydroxyalkanoate Cycle.

ABSTRACTPolyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications.

Production of Polyhydroxyalkanoate by Bacillus thuringiensis Isolated from Agricultural Soils of Cascas-Peru

HIGHLIGHTS Bacillus thuringiensis SP7-1 from agricultural soils producing polyhydroxyalkanoate. The strains SP7-1 with a PHA accumulation of 0.54 g/L and 19 % in dry biomass. PHA was characterized with FTIR, DSC and TGA with remarkable chemical properties. A thermic degradation in a range of 270-303°C and a Melting Temperature of 166.88°C.

Effect of carbon source type on phosphorus removal efficiency of MBBR and characteristics of fluorescent substances in water

In this paper, Moving Bed Bioreactor (MBBR) reactor was used as the research object to explore the effects of carbon source type on phosphorus removal performance of MBBR system and the changes of microbial metabolic characteristics in typical cycles. The results showed that the absorption and release of phosphorus by sodium propionate and starch were obvious. The accumulation and degradation of PHA and glycogen were observed in the three reactors, and PHA was prior to glycogen decomposition. When sodium acetate was used as carbon source, glycogen decreased during the stationary phase, and the other two did not change significantly. The intensity of fluorescent substances varies greatly with the type of carbon source. The fluorescent substances in the effluent are mainly tyrosine-like and protein-like substances, fulvic acid-like substances and humic acid-like substances. Therefore, the suitable carbon source type should be selected according to the microbial characteristics in the water treatment process to improve the treatment efficiency.

Bioconversion of Organic Pollutants in Fish-Canning Wastewater into Volatile Fatty Acids and Polyhydroxyalkanoate.

The wastewater from the cookers of a tuna-canning plant was used as feedstock for the process. It was acidified in a continuous stirred tank reactor (CSTR) of 1.5 L to produce a mixture of volatile fatty acids (VFAs). The effluent contained 28.3 ± 8.7 g CODS/L and 25.0 ± 4.6 g CODVFA/L, 4.4 ± 1.6 g NH4+/L, and 10.9 ± 4.0 g Na+/L, which corresponds to about 28 g NaCl/L approximately. This was used to feed a PHA production system. The enriched MMC presented a capacity to accumulate PHAs from the fermented tuna wastewater. The maximum PHA content of the biomass in the fed-batch (8.35 wt% PHA) seemed very low, possibly due to the variable salinity (from 2.2 up to 12.3 g NaCl/L) and the presence of ammonium (which promoted the biomass growth). The batch assay showed a PHA accumulation of 5.70 wt% PHA, but this is a much better result if the productivity of the reactor is taken into account. The fed-batch reactor had a productivity of 10.3 mg PHA/(L h), while the batch value was about five times higher (55.4 mg PHA/(L h)). At the sight of the results, it can be seen that the acidification of fish-canning wastewater is possible even at high saline concentrations (27.7 g NaCl/L). On the other hand, the enrichment and accumulation results show us promising news and which direction has to be followed: PHAs can be obtained from challenging substrates, and the feeding mode during the accumulation stage has an important role to play when it comes to inhibition.

Development of a biorefinery platform for urban waste valorisation into biogas and added-value products

This study focuses on the application of the circular economy approach, with generation of energy and production of added-value products from organic waste, while minimizing environmental impacts. Within this purpose, an urban biorefinery technology chain has been designed at pilot scale for the production of biogas and biopolymers (polyhydroxyalkanoates, PHA). The pilot system (100-400 L) comprised different units: a) biowaste acidogenic fermentation; b) solid/liquid separation unit (a coaxial centrifuge and a tubular ultrafiltration membrane); c) a Sequencing Batch Reactor (SBR) for the production of aerobic PHA-storing biomass; d) aerobic fed-batch PHA accumulation reactor and e) anaerobic co-digestion (ACoD). The thermal pre-treatment of the biowaste before mesophilic fermentation increased the carbon conversion into volatile fatty acids (VFA), being the final VFA/CODSOL (soluble COD) higher than 0.80. The VFA-rich stream was utilized in a high-rate SBR for the enrichment of PHA-accumulating biomass, at short solid retention time (SRT of 1 d), 12 h of cycle length and 4.0 gCOD/L d as organic loading rate (OLR). The aerobic biomass was characterized by a high accumulating capacity (with PHA content around 60% on cell dry weight). The global PHA yield of 0.1 kg PHA/kg VS (volatile solids) was estimated as the best scenario. The excess sludge and the solid-rich biorefinery overflows were utilized for biogas production in a dedicated anaerobic digestion section to sustain a closed loop approach and to prevent secondary wastes production.

Metabolic potential of the moderate halophile Yangia sp. ND199 for co-production of polyhydroxyalkanoates and exopolysaccharides.

Yangia sp. ND199 is a moderately halophilic bacterium isolated from mangrove samples in Northern Vietnam, which was earlier reported to grow on several sugars and glycerol to accumulate poly(hydroxyalkanoates) (PHA). In this study, the potential of the bacterium for co‐production of exopolysaccharides (EPS) and PHA was investigated. Genome sequence analysis of the closely related Yangia sp. CCB‐M3 isolated from mangroves in Malaysia revealed genes encoding enzymes participating in different EPS biosynthetic pathways. The effects of various cultivation parameters on the production of EPS and PHA were studied. The highest level of EPS (288 mg/L) was achieved using sucrose and yeast extract with 5% NaCl and 120 mM phosphate salts but with modest PHA accumulation (32% of cell dry weight, 1.3 g/L). Growth on fructose yielded the highest PHA concentration (85% of CDW, 3.3 g/L) at 90 mM phosphate and higher molecular weight EPS at 251 mg/L yield at 120 mM phosphate concentration. Analysis of EPS showed a predominance of glucose, followed by fructose and galactose, and minor amounts of acidic sugars.

Efficient bioconversion of sugarcane bagasse into polyhydroxybutyrate (PHB) by Lysinibacillus sp. and its characterization

In this study, Lysinibacillus sp. RGS was evaluated to synthesize polyhydroxybutyrate (PHB) from a broad range of pure carbon sources and residual sugars of chemically pretreated sugarcane bagasse (SCB) hydrolysates. Effects of supplementation of nutrients and various experimental variables to enhance PHB accumulation were investigated. Results of optimized parameters were identified as 48 h, 37 °C, pH 7; inoculums concentration (2.5% v/v) and shaking condition (100 rpm). Growth kinetics and bioprocess parameters of Lysinibacillus sp. using SCB hydrolysates with corn steep liquor (2%) accounted for the maximum cell growth (8.65 g/L) and PHA accumulation (61.5%) with PHB titer of (5.31 g/L) under optimal conditions. The produced biopolymer was studied by Fourier Transform Infrared (FTIR) spectroscopy and the results revealed the obtained to be PHB. Thus Lysinibaciluus sp. exhibits high potential in industrial scale manufacture of PHB using SCB as an inexpensive substrate.

Isolation and identification of polyhydroxyalkanoates producing bacteria from biopolymers waste in soil

The production of polyhydroxyalkanoates PHAs from biopolymer degrading bacteria was examined in situ by screening isolates using Sudan B Black staining process as potential PHAs detecting, and Nile Blue staining as a proof method detection. Five bacterial strains isolated from biopolymer waste buried in a garden soil were able to produce high rate of PHA. AK1P and AK2P strains demonstrated high productivity of biopolymer by converting 5% (w/v) lactose as the only carbon source to PHA during fermentation. AY2P strain converted 5% (w/v) of glucose with less PHA accumulation. The favorite temperature for those strains to produce a high rate of PHA was at 30° C.

Development of ultrasound aided chemical pretreatment methods to enrich saccharification of wheat waste biomass for polyhydroxybutyrate production and its characterization

Development of an effective pretreatment method is a vital stage to enhance biomass-based biopolymers production. The present study aims on the pretreatment of wheat waste biomass (WS) with alkaline and in combination with ultrasound (US) treatment to enhance enzymatic saccharification of WS into monomeric sugars. Effects of chemical dosage, substrate loading, US power and US pretreatment time on the pretreatment efficiency were systematically investigated. Under optimized conditions; US + NaOH pretreatment attained maximum 70% delignification of WS, with 84.5% hydrolysis yield, 90% glucose yield and 65% xylose yield after enzymatic hydrolysis (30 FPU/g of dry WS) which is substantially higher than the individual pretreatment. Analytical studies (XRD, FTIR, and SEM) revealed that the combined US + NaOH pretreatment can effectively destroyed the ultrastructure of biomass and improved the accessibility to hydrolyzing enzymes. The resulted enzymatic hydrolysates were utilized as a possible biomass feedstock for polyhydroxybutyrate (PHB) production using Ralstonia eutropha. Effects of supplementation of nutrients, stressing agents and initial concentration of US + NaOH pretreated WS hydrolysates (20 to 40 g/L) on PHB production were evaluated. The maximum 74% PHA accumulation, PHB titer of 7.85 g/L and yield of PHB about 0.441 g/g of reducing sugar production was obtained. Analytical characterization and thermal characteristics of extracted biopolymer showed the poperies of standard PHB. The foregoing results suggested the potential of WS as an alternate renewable resource for sustainable biopolymer production and enables WS waste biomass disposal concerns.

Engineering the Osmotic State of Pseudomonas putida KT2440 for Efficient Cell Disruption and Downstream Processing of Poly(3-Hydroxyalkanoates).

In the last decade, the development of novel programmable cell lytic systems based on different inducible genetic constructs like the holin–endolysin and lysozyme appears as a promising alternative to circumvent the use of costly enzymes and mechanical disrupters for downstream processing of intracellular microbial products. Despite the advances, upon activation of these systems the cellular disruption of the biocatalyst occurs in an extended period, thus delaying the recovery of poly(3-hydroxyalkanoate) (PHA). Herein the osmotic state of Pseudomonas putida KT2440 was engineered by inactivating the inner-membrane residing rescue valve MscL, which is responsible mainly for circumventing low-osmolarity challenges. Then the major outer membrane porin OprF and the specific porin OprE were overproduced during PHA producing conditions on decanoate-grown cells. The engineered P. putida strains carrying each porin showed no impairment on growth rate and final biomass and PHA yield after 48 h cultivation. Expression of both porins in tandem in the mutant strain KTΔmscL-oprFE led to a slight reduction of the biomass synthesis (∼10%) but higher PHA accumulation (%wt) relative to the cell dry mass. Each strain was then challenged to an osmotic upshift for 1 h and subsequently to a rapid passage to a hypotonic condition where the membrane stability of the KTΔmscL-oprFE suffered damage, resulting in a rapid reduction of cell viability. Cell disruption accounted for >95% of the cell population within 3 h as reported by colony forming units (CFU), FACS analyses, and transmission electron microscopy. PHA recovery yielded 94.2% of the biosynthesized biopolymer displaying no significant alterations on the final monomer composition. This study can serve as an efficient genetic platform for the recovery of any microbial intracellular compound allowing less unit operation steps for cellular disruption.