Polyhydroxybutyrate Accumulation Research Articles

Effects of methane to oxygen ratio on cell growth and polyhydroxybutyrate synthesis in high cell density cultivation of Methylocystis sp. MJC1.

Polyhydroxybutyrate (PHB) production through CH4 conversion by methanotrophs offers a solution for greenhouse gas emissions and plastic waste concerns. In this study, we aimed to achieve high cell density cultivation of Methylocystis sp. MJC1 for efficient PHB production. Cultivating MJC1 using CH4 and air (3:7, v/v) yielded a final cell density of 52.9 g/L with a 53.7% (28.4 g/L) PHB content after 210 h, showcasing PHB mass production potential. However, long-term cultivation led to a low volumetric productivity of 0.200g/L/h. To address this, we conducted cultivation at various O2/CH4 ratios using O2 instead of air, which significantly improved the PHB productivity. Under high O2 conditions (O2/CH4 ratio of 1.5), biomass productivity increased 1.51-fold compared to that under low O2 conditions in the same time frame; however, PHB accumulation was delayed. Using an equal ratio of CH4 and O2 induced active cell growth and selective PHB production, achieving the highest PHB productivity (0.365 g/L/h) with a final cell density of 55.9 g/L and PHB content of 61.7% (34.5 g/L) in 162 h. This study highlighted the significance of the O2/CH4 ratio in CH4 conversion and PHB production by M. sp. MJC1.

Polyhydroxyalkanoate Production from Eucalyptus Bark's Enzymatic Hydrolysate.

In recent years, polyhydroxyalkanoates (PHAs) have gained notoriety because of their desirable properties that include proven biodegradability, biocompatibility, and thermal stability, which make them suitable alternatives to fossil-based polymers. However, the widespread use of PHAs is still challenging because of their production costs, which are greatly associated with the cultivation medium used for bacterial cultivation. In Portugal, one-quarter of the forest area is covered by Eucalyptus globulus wood, making its residues a cheap, abundant, and sustainable potential carbon source for biotechnological uses. In this work, eucalyptus bark was used as the sole feedstock for PHA production in a circular bioeconomic approach. Eucalyptus bark hydrolysate was obtained after enzymatic saccharification using Cellic® CTec3, resulting in a sugar-rich solution containing glucose and xylose. Although with differing performances, several bacteria were able to grow and produce PHA with distinct compositions, using the enzymatic hydrolysate as the sole carbon source. Pseudomonas citronellolis NRRL B-2504 achieved a high cellular growth rate in bioreactor assays (24.4 ± 0.15 g/L) but presented a low accumulation of a medium-chain-length PHA (mcl-PHA) comprising the monomers hydroxydecanoate (HD, 65%), hydroxydodecanoate (HDd, 25%), and hydroxytetradecanoate (HTd, 14%). Burkholderia thailandensis E264, on the other hand, reached a lower cellular growth rate (8.87 ± 0.34 g/L) but showed a higher biopolymer accumulation, with a polyhydroxybutyrate (PHB) content in the cells of 12.3 wt.%. The new isolate, Pseudomonas sp., revealed that under nitrogen availability, it was able to reach a higher accumulation of the homopolymer PHB (31 wt.%). These results, although preliminary, demonstrate the suitability of eucalyptus bark's enzymatic hydrolysate as a feedstock for PHA production, thus offering an exciting avenue for achieving sustainable and environmentally responsible plastic products from an undervalued forestry waste.

Production of poly-3-hydroxybutyrate (PHB) nanoparticles using grape residues as the sole carbon source

The production of poly-3-hydroxybutyrate (PHB) on an industrial scale remains a major challenge due to its higher production cost compared to petroleum-based plastics. As a result, it is necessary to develop efficient fermentative processes using low-cost substrates and identify high-value-added applications where biodegradability and biocompatibility properties are of fundamental importance. In this study, grape residues, mainly grape skins, were used as the sole carbon source in Azotobacter vinelandii OP cultures for PHB production and subsequent nanoparticle synthesis based on the extracted polymer. The grape residue pretreatment showed a high rate of conversion into reducing sugars (fructose and glucose), achieving up to 43.3 % w w−1 without the use of acid or external heat. The cultures were grown in shake flasks, obtaining a biomass concentration of 2.9 g L−1 and a PHB accumulation of up to 37.7 % w w−1. PHB was characterized using techniques such as Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The formation of emulsified PHB nanoparticles showed high stability, with a particle size between 210 and 240 nm and a zeta potential between −12 and − 15 mV over 72 h. Owing to these properties, the produced PHB nanoparticles hold significant potential for applications in drug delivery.

The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate

BackgroundLack of nutrients, in particular nitrogen and phosphorus, has been known in the field to sense glutamate production via 2-oxoglutarate and subsequently accelerate carbon storage, including glycogen and polyhydroxybutyrate (PHB), in cyanobacteria, but a few studies have focused on arginine catabolism. In this study, we first time demonstrated that gene manipulation on proC and adc1, related to proline and polyamine syntheses in arginine catabolism, had a significant impact on enhanced PHB production during late growth phase and nutrient-modified conditions. We constructed Synechocystis sp. PCC 6803 with an overexpressing proC gene, encoding Δ1pyrroline-5-carboxylate reductase in proline production, and adc1 disruption resulted in lower polyamine synthesis.ResultsThree engineered Synechocystis sp. PCC 6803 strains, including a ProC-overexpressing strain (OXP), adc1 mutant, and an OXP strain lacking the adc1 gene (OXP/Δadc1), certainly increased the PHB accumulation under nitrogen and phosphorus deficiency. The possible advantages of single proC overexpression include improved PHB and glycogen storage in late phase of growth and long-term stress situations. However, on day 7 of treatment, the synergistic impact created by OXP/Δadc1 increased PHB synthesis by approximately 48.9% of dry cell weight, resulting in a shorter response to nutrient stress than the OXP strain. Notably, changes in proline and glutamate contents in engineered strains, in particular OXP and OXP/Δadc1, not only partially balanced the intracellular C/N metabolism but also helped cells acclimate under nitrogen (N) and phosphorus (P) stress with higher chlorophyll a content in comparison with wild-type control.ConclusionsIn Synechocystis sp. PCC 6803, overexpression of proC resulted in a striking signal to PHB and glycogen accumulation after prolonged nutrient deprivation. When combined with the adc1 disruption, there was a notable increase in PHB production, particularly in situations where there was a strong C supply and a lack of N and P.

Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

Polyhydroxybutyrate (PHB) Biosynthesis by an Engineered Yarrowia lipolytica Strain Using Co-Substrate Strategy

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h−1. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica.

Simultaneous production of polyhydroxybutyrate and biogas from paper mill sludge through sodium citrate-mediated disperser-induced phase separated pretreatment

Paper mill waste activated sludge is an excess sludge produced from the paper industry wastewater treatment plant. Because of the bulk nature, its disposal concerns both economic and environmental perspectives. However, it widely consists of organic components and is considered a major attraction for its valorization to produce bioenergy and valued products. During the bioenergy and valued product retrieval, the enzymatic hydrolysis got hampered due to the presence of exopolymeric substances and thus require proper pretreatment prior to the product recovery. The application of two different phases of pretreatment by removing the flocs in the first step and then followed by the cell disintegration by mechanical means helps in enhancing the organics efficiently in soluble phase. In this study, the upshot of sodium citrate mediated disperser disintegration on sludge from a paper mill was explored enhance enzymatic hydrolysis. Firstly, the floc deaggregation results at 0.05 g sodium citrate per g suspended solid with the maximum floc deaggregation degree of 89 %. Further, the consequence of disperser disintegration on the floc deaggregated sludge sample shows solid reduction and solubilization of 12.5 % and 17.5 %, respectively, higher than the disperser alone disintegration (9.57 % and 13.4 %). The anaerobic biodegradability assay shows the maximum biogas production of 174.3 mL/g of chemical oxygen demand while utilizing pretreated settled sludge as substrate. There was no accumulation of polyhydroxybutyrate using pretreated sludge supernatant. Thus, the phase-separated pretreatment by sodium citrate-induced disperser pretreatment is a suitable approach for enhancing hydrolysis during bioenergy recovery.

Population Dynamics in the Biogenesis of Single-/Multi-Layered Membrane Vesicles Revealed by Encapsulated GFP-Monitoring Analysis

Various generations of membrane vesicles (MV) have been observed in Escherichia coli in terms of triggering events and populations of single-layered (s)/multi-layered (m) forms. Previously, we proposed a novel mechanism for MV generation triggered by the intracellular accumulation of biopolyester polyhydroxybutyrate (PHB). This was designated as the Polymer Intracellular Accumulation-triggered system for Membrane Vesicle Production (PIA-MVP). Herein, we attempted to determine the conditions for the change in the population between s-MV and m-MV using glucose concentration-dependent PIA-MVP. PIA-MVP was established using the good correlation between the glucose concentration-dependent PHB accumulation and MV generation. Thus, we assumed the presence of a critical glucose concentration could determine the population ratio of s-MV to m-MV, indicating that s-MV generation is a dominant component in the extracellular environment. Cytoplasmic green fluorescent protein (GFP) was used to evaluate the glucose concentration, enabling the selective generation of s-MV. The glucose concentration was determined to be 15 g/L to satisfy this purpose under the culture conditions. In conclusion, we established a biological system allowing us to selectively generate both single- and multi-layered MVs based on PIA-VIP encapsulation of GFP, providing a versatile toolkit to gain insights into the MV generation mechanism and achieve progress in various engineering applications.

Calcium peroxide induced mechanical disintegration in acidic environment for efficient biogas and biopolymer generation from paper mill sludge

The proposed research envisioned to improve the accessibility of organics and hydrolysis of paper mill waste activated sludge for polyhydroxybutyrate production and anaerobic digestion. In order to fill this research gap an exploration has been made by adding lower dosage of calcium peroxide at acidic pH for the removal of exopolymeric substances thereby improving the disintegration efficacy. The results indicate that the calcium peroxide dosage of 0.06 g/g SS at pH 5 was optimum for floc fragmentation. The influence of floc fragmented sludge on disintegration by disperser shows the solubilization and solid decrement of 18.8% and 12.4% at the specific energy input of 4729.24 kJ/kg total solid. The impact of pretreated supernatant on polyhydroxybutyrate accumulation shows the maximum polyhydroxybutyrate content of 85.03 % while utilizing 60% (v/v) of pretreated sludge supernatant as a carbon source at 42 h. Subsequently, the settled sludge was directed towards an anaerobic biodegradability assay and found about 167 mL/g of chemical oxygen demand of biogas generation, which is higher than the crude sample (52 mL/g of chemical oxygen demand). The higher solubilization in pretreated sample reveals the easier accessibility of substrate by the microorganisms, thereby enhances bioenergy and valued product generation.

Eliminating genes for a two-component system increases PHB productivity in Cupriavidus basilensis 4G11 under PHB suppressing, nonstress conditions.

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two-component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, nonstress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Due to uncertainty in the two-component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single-stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single-stage continuous systems. This work expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.

Polyhydroxybutyrate (PHB) accumulation by a mangrove isolated cyanobacteria Limnothrix planktonica using fruit waste

Cyanobacterial polyhydroxybutyrate (PHB) is preferred over bacteria for low-cost production due to its photoautotrophic nature and lower carbon requirement. Considering its impact on the environment and circular economy, the valorization of fruit waste is the need of the hour. In the present study, fruit peels of banana, orange, pea, jackfruit, watermelon and waste flowers were tried as carbon sources for mangrove-isolated cyanobacteria Limnothrix planktonica to accumulate PHB. Alterations in the ASN-III culture medium and the introduction of untreated and pre-treated (acid/alkali-treated) peels as carbon sources are tried to enhance PHB. Banana peel showed the maximum PHB accumulation potential of 25.73 mg/L on the 12th day of incubation, followed by jackfruit (22.46 mg/L) and watermelon peels (20.72 mg/L); whereas, commercial carbon sources showed lower PHB accumulation up to 19.26 mg/L and 18.21 mg/L with fructose and glucose respectively. PHB accumulation was boosted to 5-fold higher (39.39 mg/L) in NP deficiency medium along with banana peel supplement, as compared to photoautotrophic conditions (8.49 mg/L) after the 9th day of incubation. Additionally, the PHB obtained by using the fruit wastes has a higher molecular weight than the PHB accumulated during photoautotrophic conditions. Optimization of parameters using fruit wastes and characterization of PHB would lead to its potential use.

Integrating proteomic data with metabolic modeling provides insight into key pathways of Bordetella pertussis biofilms.

Pertussis, commonly known as whooping cough is a severe respiratory disease caused by the bacterium, Bordetella pertussis. Despite widespread vaccination, pertussis resurgence has been observed globally. The development of the current acellular vaccine (ACV) has been based on planktonic studies. However, recent studies have shown that B. pertussis readily forms biofilms. A better understanding of B. pertussis biofilms is important for developing novel vaccines that can target all aspects of B. pertussis infection. This study compared the proteomic expression of biofilm and planktonic B. pertussis cells to identify key changes between the conditions. Major differences were identified in virulence factors including an upregulation of toxins (adenylate cyclase toxin and dermonecrotic toxin) and downregulation of pertactin and type III secretion system proteins in biofilm cells. To further dissect metabolic pathways that are altered during the biofilm lifestyle, the proteomic data was then incorporated into a genome scale metabolic model using the Integrative Metabolic Analysis Tool (iMAT). The generated models predicted that planktonic cells utilised the glyoxylate shunt while biofilm cells completed the full tricarboxylic acid cycle. Differences in processing aspartate, arginine and alanine were identified as well as unique export of valine out of biofilm cells which may have a role in inter-bacterial communication and regulation. Finally, increased polyhydroxybutyrate accumulation and superoxide dismutase activity in biofilm cells may contribute to increased persistence during infection. Taken together, this study modeled major proteomic and metabolic changes that occur in biofilm cells which helps lay the groundwork for further understanding B. pertussis pathogenesis.

A new type of phasin characterized by the presence of a helix-hairpin-helix domain is required for normal polyhydroxybutyrate accumulation and granule organization in Caulobacter crescentus.

Bacteria frequently store excess carbon in hydrophobic granules of polyhydroxybutyrate (PHB) that in some growth conditions can occupy most of the cytoplasmic space. Different types of proteins associate to the surface of the granules, mainly enzymes involved in the synthesis and utilization of the reserve polymer and a diverse group of proteins known as phasins. Phasins have different functions, among which are regulating the size and number of the granules, modulating the activity of the granule-associated enzymes and helping in the distribution of the granules inside the cell. Caulobacter crescentus is an oligotrophic bacterium that shows several morphological and regulatory traits that allow it to grow in very nutrient-diluted environments. Under these conditions, storage compounds should be particularly relevant for survival. In this work, we show an initial proteomic characterization of the PHB granules and describe a new type of phasin (PhaH) characterized by the presence of an N-terminal hydrophobic helix followed by a helix-hairpin-helix (HhH) domain. The hydrophobic helix is required for maximal PHB accumulation and maintenance during the stationary phase while the HhH domain is involved in determining the size of the PHB granules and their distribution in the cell.

Polyhydroxybutyrate Metabolism in Azospirillum brasilense and Its Applications, a Review.

Gram-negative Azospirillum brasilense accumulates approximately 80% of polyhydroxybutyrate (PHB) as dry cell weight. For this reason, this bacterium has been characterized as one of the main microorganisms that produce PHB. PHB is synthesized inside bacteria by the polymerization of 3-hydroxybutyrate monomers. In this review, we are focusing on the analysis of the PHB production by A. brasilense in order to understand the metabolism during PHB accumulation. First, the carbon and nitrogen sources used to improve PHB accumulation are discussed. A. brasilense accumulates more PHB when it is grown on a minimal medium containing a high C/N ratio, mainly from malate and ammonia chloride, respectively. The metabolic pathways to accumulate and mobilize PHB in A. brasilense are mentioned and compared with those of other microorganisms. Next, we summarize the available information to understand the role of the genes involved in the regulation of PHB metabolism as well as the role of PHB in the physiology of Azospirillum. Finally, we made a comparison between the properties of PHB and polypropylene, and we discussed some applications of PHB in biomedical and commercial areas.

Valorization of Waste from Argan Seeds for Polyhydroxybutyrate Production Using Bacterial Strains Isolated from Argan Soils.

The aim of this work was to study the valorization of argan seed pulp, a waste material obtained from argan oil extraction, for the biosynthesis of polyhydroxybutyrate (PHB). A new species that showed the metabolic capacity for the conversion of argan waste into the bio-based polymer was isolated from an argan crop located in Teroudant, a southwestern region of Morocco, where the arid soil is exploited for goat grazing. The PHB accumulation efficiency of this new species was compared to the previously identified species 1B belonging to the genus Sphingomonas, and results were reported as dry cell weight residual biomass and PHB final yield measured. Temperature, incubation time, pH, NaCl concentration, nitrogen sources, residue concentrations, and culture medium volumes were analyzed with the aim of obtaining a maximum accumulation of PHB. UV-visible spectrophotometry and FTIR analysis confirmed that PHB was present in the material extracted from the bacterial culture. The results of this wide investigation indicated that the new isolated species 2D1 had a higher efficiency in PHB production compared to the previously identified strain 1B, which was isolated from a contaminated argan soil in Teroudant. PHB final yield of the two bacterial species, i.e., the new isolated and 1B, cultivated under optimal culture conditions, in 500 mL MSM enriched with 3% argan waste, were 21.40% (5.91 ± 0.16 g/L) and 8.16% (1.92 ± 0.23 g/L), respectively. For the new isolated strain, the result of the UV-visible spectrum indicates the absorbance at 248 nm, while the FTIR spectrum showed peaks at 1726 cm-1 and 1270 cm-1: these characteristic peaks indicated the presence of PHB in the extract. The data from the species 1B UV-visible and FTIR spectra were previously reported and were used in this study for a correlation analysis. Furthermore, additional peaks, uncharacteristic of standard PHB, suggest the presence of impurities (e.g., cell debris, solvent residues, biomass residues) that persisted after extraction. Therefore, a further enhancement of the sample purification during extraction is recommended for more accuracy in the chemical characterization. If 470,000 tons of argan fruit waste can be produced annually, and 3% of waste is consumed in 500 mL culture by 2D1 to produce 5.91 g/L (21.40%) of the bio-based polymer PHB, it can be estimated that the amount of putative PHB that can be extracted annually from the total argan fruit waste is about 2300 tons.

Ability of converting sugarcane bagasse hydrolysate into polyhydroxybutyrate (PHB) by bacteria isolated from stressed environmental soils

We isolated 109 potential polyhydroxybutyrate (PHB)-producing bacteria from various soil sources. Using Sudan Black B staining, lipid granules were observed in 65 isolates. UV spectrophotometry-based quantification revealed that 14 isolates effectively produced PHB, and all isolates carried the phaC gene encoding polyhydroxyalkanoate synthase required for PHB polymerization. After cultivation at 30 °C for 72 h in E2 medium containing 3.5% (w/v) glucose as the carbon source and 0.354% (w/v) NH4Y as the nitrogen source, the highest PHB content (%-CDW) and yield (g-PHB/g-glucose) with values of 20.4% and 0.28 and 19.7% and 0.07 were obtained for Priestia megaterium KKR5 and Burkholderia cepacia ASL22, respectively. TEM analysis revealed PHB granules with diameters of 0.2–0.7 μm inside KKR5 and ASL22 cells. We achieved the bioconversion of sugarcane bagasse hydrolysate (BGH) into PHB by these strains via separate hydrolysis and fermentation. H3PO4 steam-pretreated BGH generated reducing sugars containing glucose and xylose without toxic chemicals. Using the reducing sugars of BGH as the carbon source, ASL22 exhibited a higher PHB yield (0.22 g-PHB/g-reducing sugar) than KKR5 (0.12 g-PHB/g-reducing sugar). PHB from KKR5 and ASL22 had FTIR and NMR spectra that were similar to those of standard PHB. A GC/MS analysis confirmed the molecular structure of PHB as methyl hydroxybutyrate with a mass value of 43–103. By DSC analysis, the melting temperatures of PHB from KKR5 and ASL22 and standard PHB were 167.8, 168.5, and 176.6 °C, respectively. Thus, PHB-producing bacteria isolated from stressed soils successfully utilize a low-cost BGH substrate for PHB accumulation.

Utilization of pulp and paper industrial wastewater for production of polyhydroxybutyrate by Bacillus sonorensis NAM5

Given the environmental pollution caused by petroleum-based plastics, finding alternative substitutes for sustainability has become critical. Polyhydroxybutyrate (PHB), a storage food material that is accumulated by several bacteria, is biodegradable, safe, environment friendly and comparable to conventional plastics. However, scale-up is an issue due to high production cost. Substrate replacement using renewable, plentiful, sustainable and low-cost carbon sources derived from industrial waste facilitates waste reduction, while also enabling the synthesis of value-added products. In this context, inexpensive pulp and paper industrial waste as carbon source was exploited for production of PHB by using previously isolated (Source: hot springs of Manikarn, Himachal Pradesh, India) thermophilic bacteria Bacillus sonorensis NAM5 under optimized conditions in a fermenter. Production was done in a fermenter under optimized conditions (72 h of incubation at 50 °C temperature and 7 pH) to enhance the accumulation of PHB. The bacterial strain was able to produce 5.28 ± 0.11 g L−1 after 72 h of growth without any carbon and nitrogen source supplementation to the industrial effluent. The culture accumulated 66% PHB of cell dry weight (CDW). The produced polymer was characterized through FTIR, NMR and TGA. Additionally, bacteria-treated industrial wastewater was used for phytotoxicity assay on agriculturally important crops such as wheat, maize and mung, which exhibited considerable difference in growth parameters.

A mechanistic mathematical model for photo fermentative hydrogen and polyhydroxybutyrate production.

An original mathematical model describing the photo fermentation process is proposed. The model represents the first attempt to describe the photo fermentative hydrogen production and polyhydroxybutyrate accumulation, simultaneously. The mathematical model is derived from mass balance principles and consists of a system of ordinary differential equations describing the biomass growth, the nitrogen and the substrate degradation, the hydrogen and other catabolites production, and the polyhydroxybutyrate accumulation in photo fermentation systems. Moreover, the model takes into account important inhibiting phenomena, such as the self-shading and the substrate inhibition, which can occur during the evolution of the process. The calibration was performed using a real experimental data set and it was supported by the results of a sensitivity analysis study. The results showed that the most sensitive parameters for both hydrogen and PHB production were the hydrogen yield on substrate, the catabolites yield on substrate, and the biomass yield. Successively, a different experimental data set was used to validate the model. Performance indicators showed that the model could efficiently be used to simulate the photo fermentative hydrogen and polyhydroxybutyrate production by Rhodopseudomonas palustris. For instance, the index of agreement of 0.95 was observed for the validated hydrogen production trend. Moreover, the model well predicted the maximum PHB accumulation in bacterial cells. Indeed, the predicted and observed accumulated PHB were 4.5 and 4.8%, respectively. Further numerical simulations demonstrated the model consistency in describing process inhibiting phenomena. Numerical simulations showed that the acetate and nitrogen inhibition phenomena take place when concentrations are higher than 12.44 g L-1 and lower than 4.76 mg L-1, respectively. Finally, the potential long term hydrogen production from accumulated polyhydroxybutyrate in bacterial cells was studied via a fast-slow analysis technique.

Use of exogenous substrate in Chlorella cultivation: Strategy for biomass and polyhydroxybutyrate production

The aim of this study was to investigate the influence of exogenous carbon supplementation and nitrogen source reduction on Chlorella fusca LEB 111 growth, biomass composition, and polyhydroxybutyrate accumulation. First, assays were performed with 50 % and 25 % reduced nitrogen source concentrations (NaNO3). In the second stage, the influence of culture supplementation with 10, 20, and 30 mg L−1 D-xylose, associated with 50 and 25 % reductions in NaNO3, was evaluated. The experiments conducted with a 25 % reduction in NaNO3 and supplementation with 10 mg L−1 D-xylose resulted in a positive effect on the biomass productivity of C. fusca LEB 111, with production as high as 354.4 mg L−1 d−1. The maximum concentration of PHB extracted from C. fusca LEB 111 was 3.7 % (w w−1) and was obtained when the microalgae were cultivated with a 25 % of reduction in NaNO3 and supplementation of D-xylose at 20 mg L−1. Therefore, this study brings new perspectives regarding reducing the use of nutritional sources and using exogenous carbon sources in using microalgae to produce molecules of high biotechnological potential.