Polyhydroxybutyrate Content Research Articles

METHODS OF STERILIZATION OF MEDICAL POLYMER COMPOSITES BASED ON POLYHYDROXYBUTYRATE WITH ELASTOMERIC ADDITIVE

Introduction. Polyhydroxybutyrate is a biodegradable and completely biocompatible component, and in combination with various modifying additives can be suitable for the manufacture of medical products used in surgical practice as bone implants or their parts. There implants have a number of advantages over traditional metal products, but for their integration into the body they require thorough sterilization cleaning, which in the case of poly-mer compositions has a number of limitations associated with the possible destruction of the material structure during various cleaning stages. Purpose of the study. Find optimal methods for sterilization and disinfection of materials based on polyhydroxybutyrate (PHB) and an elastomeric additive – butadiene-nitrile rubber (NBR-28). Material and methods. Two-component PHB-NBR compositions with PHB content from 30 to 90% were studied. Four methods of sterilization and disinfection were used: autoclaving, air sterilization, disinfection with chlorine solution and ethanol solution. Determined mechanical characteristics are strength and elongation of the material at break. Sterility control – by the method of washings with subsequent observation of the growth of bacteria and fungi in Sabouraud's medium and thioglycollate medium. Results. Sterilized and disinfected samples showed no microbial growth in both culture media. No change in mechanical characteristics was detected for samples subjected to solution cleaning methods. High temperature cleaning reduced the mechanical properties of samples by 20–80% depending on the sterilization mode. Conclusions. The data obtained show that for sterilization and disinfection of PCM based on the biodegradable polymer PHB, solution methods are suitable without restrictions and the autoclave sterilization method is suitable with minor restrictions, while air sterilization leads to the destruction of PCM. The work was financially supported by the Ministry of Science and Higher Education of Russian Federation (Research theme state registration number 122041300207-2).

Nonsterilized Fermentation of Crude Glycerol for Polyhydroxybutyrate Production by Metabolically Engineered Vibrio natriegens.

Polyhydroxybutyrate (PHB) is an attractive biodegradable polymer that can be produced through the microbial fermentation of organic wastes or wastewater. However, its mass production has been restricted by the poor utilization of organic wastes due to the presence of inhibitory substances, slow microbial growth, and high energy input required for feedstock sterilization. Here, Vibrio natriegens, a fast-growing bacterium with a broad substrate spectrum and high tolerance to salt and toxic substances, was genetically engineered to enable efficient PHB production from nonsterilized fermentation of organic wastes. The key genes encoding the PHB biosynthesis pathway of V. natriegens were identified through base editing and overexpressed. The metabolically engineered strain showed 166-fold higher PHB content (34.95 wt %) than the wide type when using glycerol as a substrate. Enhanced PHB production was also achieved when other sugars were used as feedstock. Importantly, it outperformed the engineered Escherichia coli MG1655 in PHB productivity (0.053 g/L/h) and tolerance to toxic substances in crude glycerol, without obvious activity decline under nonsterilized fermentation conditions. Our work demonstrates the great potential of engineered V. natriegens for low-cost PHB bioproduction and lays a foundation for exploiting this strain as a next-generation model chassis microorganism in synthetic biology.

Tryptophan-based in vivo coloring of polyhydroxybutyrate through co-production with indigo and effect on the properties of polymer

With the continuous growth of the demand for eco-friendly alternatives to petroleum-based plastics, polyhydroxybutyrate (PHB) has emerged as a promising alternative because of its biodegradability. However, certain characteristics, such as its color, rigidity, and brittleness, have limited its range of applications. In this study, we investigated the in vivo coloring of PHB with indigo by combining the biosynthesis operons of PHB and indigo, which involved the use of tryptophan as the substrate for the biosynthesis of indigo. The heterologous and homologous tryptophanase genes converted tryptophan to indole, which was further oxidized to indoxyl by flavin monooxygenase (FMO), followed by spontaneous dimerization to indigo in the presence of oxygen. Different indigo-PHB films were obtained by controlling the concentration of tryptophan. Additionally, we investigated the mechanical properties of the indigo-PHB films using UTM analysis and found that their mechanical properties varied according to the amount of indigo incorporated into PHB. Notably, elongation at break in indigo-PHB film, which was produced using 10 mM of tryptophan resulting in a 1.1 mg indigo/g PHB content, significantly improved to 70 % compared to the 7.2 % observed in normal PHB films. Furthermore, indigo-PHB films were subjected to FE-SEM, GPC, and DSC analyses to identify differences, and it was observed that there was a decrease in Tc and Tg values, more obvious in Tg value, in indigo PHB film with 1.1 mg indigo/g PHB content. Considering many approaches to improving PHB properties, our study showed the possibility of different changes in color and improved flexibility via the co-production of indigo and PHB.

Production of polyhydroxybutyrate from waste cooking oil using magnetically recoverable microbial-based nanocomposites as reusable inocula

Polyhydroxybutyrate (PHB) is produced during bacterial metabolism and can be used for the production of biodegradable plastics. The utilization of wastes as carbon sources and inoculum reuse are potential strategies to reduce production costs. In this study, a method for PHB production from waste cooking oil using a reusable inoculum was developed. Two microbial-based nanocomposites (fabricated bead and bacterial nanocomposites) were used as reusable inocula, and 6% waste cooking oil was used as a carbon source. The addition of 0.1% iron oxide (Fe3O4) increased PHB production and oil removal efficiency. Supplementation with 0.1% pumice enhanced the compressive strength and Young’s modulus of the fabricated bead nanocomposite containing a photosynthetic bacterium, alginate, Fe3O4, and pumice. The bead nanocomposite was reused for nine cycles with single harvesting of PHB. To improve the recycling time, free cells released from the bead nanocomposite were immobilized to generate a bacterial nanocomposite containing bacteria and Fe3O4. Bacterial nanocomposites showed the highest oil removal rates (38%–51%) and PHB contents in multiple harvests (19%–30%). The bacterial nanocomposite was recycled in 11 batches without deterioration and simplified using magnetic harvesting, which eliminated the incubation time and medium required for inoculum preparation. These results suggest that bead nanocomposites can be used to treat cooking oil until they disintegrate and release free cells that are immobilized with Fe3O4 to generate bacterial nanocomposites for unlimited recycling. This study introduces technology for PHB production from waste cooking oil.

CoP-Fe2O3/g-C3N4 photocathode enhances the microbial electrosynthesis of polyhydroxybutyrate production via CO2 reduction

The hydrogen energy and CO2 reduction in-situ recombination has drawn increasing attention. Microbial electrosynthesis (MES) integrated with photocatalytic materials is a novel technology for CO2 utilization. Here, MES with the photocathode CoP-Fe2O3/g-C3N4 was constructed using Ralstonia eutropha as biocatalyst to generate polyhydroxybutyrate (PHB). The integration of CoP and Fe2O3/g-C3N4 facilitated electron–hole pair separation and electron transfer, which enhanced hydrogen evolution reaction and provided additional reducing power for CO2 conversion. Simultaneously, the concentration of reactive oxygen species was also considerably reduced, increasing the production of PHB. In particular, 87.54 mg/L of PHB was obtained in CoP-Fe2O3/g-C3N4 at − 1.05 V, which is about three times higher than that in carbon felt. At − 0.9 V, PHB concentration further increased to 142.20 mg/L. This study provides a new approach for converting CO2 into multicarbon compounds in situ electrolysis of water under visible light.

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.

Polyhydroxybutyrate (PHB) production from sugar cane molasses and tap water without sterilization using novel strain, Priestia sp. YH4

The production cost of biodegradable polymer like polyhydroxybutyrate (PHB) is still higher than that of petroleum-based plastics. A potential solution for reducing its production cost is using a cheap carbon source and avoiding a process of sterilization. In this study, a novel PHB-producing microbial strain, Priestia sp. YH4 was screened from the marine environment using sugarcane molasses as the carbon source without sterilization. Culture conditions, such as carbon, NaCl, temperature, pH, inoculum size, and cultivation time, were optimized for obtaining the highest PHB production by YH4 resulting in 5.94 g/L of dry cell weight (DCW) and 61.7 % of PHB content in the 5 mL culture. In addition, it showed similar PHB production between the cultures with or without sterilization in Marine Broth media. When cultured using only tap water, sugarcane molasses, and NaCl in a 5 L fermenter, 24.8 g/L DCW was produced at 41 h yielding 13.9 g/L PHB. Finally, DSC (Differential Scanning Calorimetry) and GPC (Gel Permeation Chromatography) were used to analyze thermal properties and molecular weights resulting in Tm = 167.2 °C, Tc = 67.3 °C, Mw = 2.85 × 105, Mn = 1.05 × 105, and PDI = 2.7, respectively. Therefore, we showed the feasibility of more economical process for PHB production by finding novel strain, utilizing molasses with minimal media components and avoiding sterilization.

Continuous polyhydroxybutyrate production from biogas in an innovative two-stage bioreactor configuration.

Biogas biorefineries have opened up new horizons beyond heat and electricity production in the anaerobic digestion sector. Added-value products such as polyhydroxyalkanoates (PHAs), which are environmentally benign and potential candidates to replace conventional plastics, can be generated from biogas. This work investigated the potential of an innovative two-stage growth-accumulation system for the continuous production of biogas-based polyhydroxybutyrate (PHB) using Methylocystis hirsuta CSC1 as cell factory. The system comprised two turbulent bioreactors in series to enhance methane and oxygen mass transfer: a continuous stirred tank reactor (CSTR) and a bubble column bioreactor (BCB) with internal gas recirculation. The CSTR was devoted to methanotrophic growth under nitrogen balanced growth conditions and the BCB targeted PHB production under nitrogen limiting conditions. Two different operational approaches under different nitrogen loading rates and dilution rates were investigated. A balanced nitrogen loading rate along with a dilution rate (D) of 0.3 day-1 resulted in the most stable operating conditions and a PHB productivity of ~53 g PHB m-3 day-1 . However, higher PHB productivities (~127 g PHB m-3 day-1 ) were achieved using nitrogen excess at a D = 0.2 day-1 . Overall, the high PHB contents (up to 48% w/w) obtained in the CSTR under theoretically nutrient balanced conditions and the poor process stability challenged the hypothetical advantages conferred by multistage vs single-stage process configurations for long-term PHB production.

Semi-continuous production of polyhydroxybutyrate (PHB) in the Chlorophyta Desmodesmus communis

Polyhydroxyalkanoates (PHAs) are promising alternatives that accumulate as energy and carbon storage material in various microorganisms, including bacteria and microalgae, being biodegradable and suitable for a wide variety of applications. Among these compounds, the most prevalent and well-characterized biopolymer is polyhydroxybutyrate (PHB), which belongs to the short-chain PHAs.The present study was designed to evaluate algae-based PHB production in two Chlorophyta (Desmodesmus communis and Chlorella vulgaris) under a two-phase nutritional mode of cultivation, namely a phototrophic growth phase (PGP) and a mixotrophic stress phase (MSP) with N,P-depleted media and organic carbon supply (i.e., glucose or sodium acetate, NaOAc). The highest PHB productivity (0.11 g PHB/g biomass/d; 0.015 g PHB/L/d), corresponding to 32.1 % w/w of intracellular PHB, was observed for D. communis after 3 days of cultivation under mixotrophic conditions in batch cultures (e.g., low light, phosphorus-free medium, 1 g/L of NaOAc). A scaled-up cultivation (10 L) was set up to evaluate for the first time PHB yields and biomass composition in a semi-continuous system. A PHB content of 34 % w/w was achieved on day 8, corresponding to a maximum PHB productivity of 0.10 g PHB/g biomass/d (or 0.011 g PHB/L/d), which increased up to 54 % w/w on day 15. The biomass was composed of about 30 % w/w proteins, 6 % w/w polysaccharides, and 11 % w/w lipids, which can be valorised from a biorefinery perspective. The scaled-up D. communis cultivation in 10 L PBRs confirmed the potential utilization of this algal species for PHB production with productivity up to 2-times higher than those reported for several cyanobacterial species and similar to the maximum value obtained with batch cultures in previous works performed with Scenedesmaceae.

Utilizing microalgal hydrolysate from dairy wastewater-grown Chlorella sorokiniana SU-1 as sustainable feedstock for polyhydroxybutyrate and β-carotene production by engineered Rhodotorula glutinis #100-29

The objective of this study was to explore the potential of utilizing Chlorella sorokiniana SU-1 biomass grown on dairy wastewater-amended medium as sustainable feedstock for the biosynthesis of β-carotene and polyhydroxybutyrate (PHB) by Rhodotorula glutinis #100-29. To break down the rigid cell wall, 100 g/L of microalgal biomass was treated with 3% sulfuric acid, followed by detoxification using 5% activated carbon to remove the hydroxymethylfurfural inhibitor. The detoxified microalgal hydrolysate (DMH) was used for flask-scale fermentation, which yielded a maximum biomass production of 9.22 g/L, with PHB and β-carotene concentration of 897 mg/L and 93.62 mg/L, respectively. Upon scaling up to a 5-L fermenter, the biomass concentration increased to 11.2 g/L, while the PHB and β-carotene concentrations rose to 1830 mg/L and 134.2 mg/L. These outcomes indicate that DMH holds promise as sustainable feedstock for the production of PHB and β-carotene by yeast.

Electron uptake from solid electrodes promotes the more efficient conversion of CO2 to polyhydroxybutyrate by using Rhodobacter sphaeroides

Microbial electrosynthesis (MES) is a promising strategy for the conversion of CO2 to useful chemicals. Nevertheless, the characteristics of electrode-associated cells in MES and their metabolic pathway regulation in CO2 fixation have not been elucidated. This study examined the electrode-driven polyhydroxybutyrate (PHB) production from CO2 in Rhodobacter sphaeroides. The electron uptake and regulation of the metabolic pathways differed in electrode-associated and suspended R. sphaeroides. The electrode-associated cells produced PHB at concentrations up to 23.50 ± 2.8% of the dry cell weight (DCW), whereas the suspended cells grew faster but with a lower cellular PHB content. Gene expression analyses showed that phaA expression was upregulated in electrode-associated R. sphaeroides, whereas phaB expression was downregulated in suspended cells. The electrode-associated cells expressed unconventional CO2 fixation enzymes, such as isocitrate dehydrogenase and formate dehydrogenase, with more PHB synthesis. These results show that CO2 can be upcycled to polymeric substances and provide novel insights into the genetic regulation of electrode-associated cells in MES.

Increased Biomass and Polyhydroxybutyrate Production by Synechocystis sp. PCC 6803 Overexpressing RuBisCO Genes.

The overexpression of the RuBisCO (rbc) gene has recently become an achievable strategy for increasing cyanobacterial biomass and overcoming the biocompound production restriction. We successfully constructed two rbc-overexpressing Synechocystis sp. PCC 6803 strains (OX), including a strain overexpressing a large subunit of RuBisCO (OXrbcL) and another strain overexpressing all large, chaperone, and small subunits of RuBisCO (OXrbcLXS), resulting in higher and faster growth than wild type under sodium bicarbonate supplementation. This increased biomass of OX strains significantly contributed to the higher polyhydroxybutyrate (PHB) production induced by nutrient-deprived conditions, in particular nitrogen (N) and phosphorus (P). As a result of higher PHB contents in OX strains occurring at days 7 and 9 of nutrient deprivation, this enhancement was apparently made possible by cells preferentially maintaining their internal lipids while accumulating less glycogen. The OXrbcLXS strain, with the highest level of PHB at about 39 %w/dry cell weight (DCW) during 7 days of BG11-NP treatment, contained a lower glycogen level (31.9 %w/DCW) than wild type control (40 %w/DCW). In contrast, the wild type control strain exposed to N- and NP-stresses tended to retain lipid levels and store more glycogen than PHB. In this model, we, for the first time, implemented a RuBisCO-overexpressing cyanobacterial factory for overproducing PHB, destined for biofuel and biomaterial biotechnology.

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.

Characterization and Process Optimization for Enhanced Production of Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Bacillus flexus Isolated from Municipal Solid Waste Landfill Site.

In recent years, there has been a growing interest in bio-based degradable plastics as an alternative to synthetic plastic. Polyhyroxybutyrate (PHB) is a macromolecule produced by bacteria as a part of their metabolism. Bacteria accumulate them as reserve materials when growing under different stress conditions. PHBs can be selected as alternatives for the production of biodegradable plastics because of their fast degradation properties when exposed to natural environmental conditions. Hence, the present study was undertaken in order to isolate the potential PHB-producing bacteria isolated from the municipal solid waste landfill site soil samples collected from the Ha'il region of Saudi Arabia to assess the production of PHB using agro-residues as a carbon source and to evaluate the growth of PHB production. In order to screen the isolates for producing PHB, a dye-based procedure was initially employed. Based on the 16S rRNA analysis of the isolates, Bacillus flexus (B. flexus) accumulated the highest amount of PHB of all the isolates. By using a UV-Vis spectrophotometer and Fourier-transform infrared spectrophotometer (FT-IR), in which a sharp absorption band at 1721.93 cm-1 (C=O stretching of ester), 1273.23 cm-1 (-CH group), multiple bands between 1000 and 1300 cm-1 (stretching of the C-O bond), 2939.53 cm-1 (-CH3 stretching), 2880.39 cm-1 (-CH2 stretching) and 3510.02 cm-1 (terminal -OH group), the extracted polymer was characterized and confirmed its structure as PHB. The highest PHB production by B. flexus was obtained after 48 h of incubation (3.9 g/L) at pH 7.0 (3.7 g/L), 35 °C (3.5 g/L) with glucose (4.1 g/L) and peptone (3.4 g/L) as carbon and nitrogen sources, respectively. As a result of the use of various cheap agricultural wastes, such as rice bran, barley bran, wheat bran, orange peel and banana peel as carbon sources, the strain was found to be capable of accumulating PHB. Using response surface methodology (RSM) for optimization of PHB synthesis using a Box-Behnken design (BBD) proved to be highly effective in increasing the polymer yield of the synthesis. With the optimum conditions obtained from RSM, PHB content can be increased by approximately 1.3-fold when compared to an unoptimized medium, resulting in a significant reduction in production costs. Thus, isolate B. flexus is a highly promising candidate for the production of industrial-size quantities of PHB from agricultural wastes and is capable of removing the environmental concerns associated with synthetic plastics from the industrial production process. Moreover, the successful production of bioplastics using a microbial culture provides a promising avenue for the large-scale production of biodegradable and renewable plastics with potential applications in various industries, including packaging, agriculture and medicine.

Polyhydroxybutyrate production from crude glycerol using a highly robust bacterial strain Halomonas sp. YLGW01

Petrochemical-based plastics are hardly biodegradable and a major cause of environmental pollution, and polyhydroxybutyrate (PHB) is attracting attention as an alternative due to its similar properties. However, the cost of PHB production is high and is considered the greatest challenge for its industrialization. Here, crude glycerol was used as a carbon source for more efficient PHB production. Among the 18 strains investigated, Halomonas taeanenisis YLGW01 was selected for PHB production due to its salt tolerance and high glycerol consumption rate. Furthermore, this strain can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3 HV)) with 17 % 3 HV mol fraction when a precursor is added. PHB production was maximized through medium optimization and activated carbon treatment of crude glycerol, resulting in 10.5 g/L of PHB with 60 % PHB content in fed-batch fermentation. Physical properties of the produced PHB were analyzed, i.e., weight average molecular weight (6.8 × 105), number average molecular weight (4.4 × 105), and the polydispersity index (1.53). In the universal testing machine analysis, the extracted intracellular PHB showed a decrease in Young's modulus, an increase in Elongation at break, greater flexibility than authentic film, and decreased brittleness. This study confirmed that YLGW01 is a promising strain for industrial PHB production using crude glycerol.

One-Step Oxidation of Orange Peel Waste to Carbon Feedstock for Bacterial Production of Polyhydroxybutyrate.

Orange peels are an abundant food waste stream that can be converted into useful products, such as polyhydroxyalkanoates (PHAs). Limonene, however, is a key barrier to building a successful biopolymer synthesis from orange peels as it inhibits microbial growth. We designed a one-pot oxidation system that releases the sugars from orange peels while eliminating limonene through superoxide (O2• -) generated from potassium superoxide (KO2). The optimum conditions were found to be treatment with 0.05 M KO2 for 1 h, where 55% of the sugars present in orange peels were released and recovered. The orange peel sugars were then used, directly, as a carbon source for polyhydroxybutyrate (PHB) production by engineered Escherichia coli. Cell growth was improved in the presence of the orange peel liquor with 3 w/v% exhibiting 90-100% cell viability. The bacterial production of PHB using orange peel liquor led to 1.7-3.0 g/L cell dry weight and 136-393 mg (8-13 w/w%) ultra-high molecular weight PHB content (Mw of ~1900 kDa) during a 24 to 96 h fermentation period. The comprehensive thermal characterization of the isolated PHBs revealed polymeric properties similar to PHBs resulting from pure glucose or fructose. Our one-pot oxidation process for liberating sugars and eliminating inhibitory compounds is an efficient and easy method to release sugars from orange peels and eliminate limonene, or residual limonene post limonene extraction, and shows great promise for extracting sugars from other complex biomass materials.

Characterization of polyhydroxybutyrate (PHB) synthesized by newly isolated rare actinomycetes Aquabacterium sp. A7-Y

Polyhydroxyalkanoates (PHAs) as biodegradable plastics have attracted increasing attention due to its biodegradable, biocompatible and renewable advantages. Exploitation some unique microbes for PHAs production is one of the most competitive approaches to meet complex industrial demand, and further develop next-generation industrial biotechnology. In this study, a rare actinomycetes strain A7-Y was isolated and identified from soil as the first PHAs producer of Aquabacterium genus. Produced PHAs by strain A7-Y was identified as poly(3-hydroxybutyrate) (PHB) based on its structure characteristics, which is also similar with commercial PHB. After optimization of fermentation conditions, strain A7-Y can produce 10.2 g/L of PHB in 5 L fed-batch fermenter, corresponding with 54 % PHB content of dry cell weight, which is superior to the reported actinomycetes species. Furthermore, the phaCAB operon in stain A7-Y was excavated to be responsible for the efficient PHB production and verified in recombinant Escherichia coli. Our results indicate that strain A7-Y and its biosynthetic gene cluster are potential candidates for developing a microbial formulation for the PHB production.

Production of polyhydroxyalkanoates by the thermophile Cupriavidus cauae PHS1

Thermophilic production of polyhydroxyalkanoate is considered a very promising way to overcome the problems that may arise when using mesophilic strains. This study reports the first thermophilic polyhydroxybutyrate-producing Cupriavidus species, which are known as the best polyhydroxybutyrate-producing microorganisms. Cupriavidus cauae PHS1 harbors a phbCABR cluster with high similarity to the corresponding proteins of C. necator H16 (80, 93, 96, and 97 %). This strain can produce polyhydroxybutyrate from a range of substrates, including acetate (5 g/L) and phenol (1 g/L), yielding 7.6 % and 18.9 % polyhydroxybutyrate, respectively. Moreover, the strain produced polyhydroxybutyrate at temperatures ranging from 25 to 50 °C, with the highest polyhydroxybutyrate content (47 °C) observed at 45 °C from gluconate. Additionally, the strain could incorporate 3-hydroxyvalerate (12.5 mol. %) into the polyhydroxybutyrate polymer using levulinic acid as a precursor. Thus, Cupriavidus cauae PHS1 may be a promising polyhydroxybutyrate producer as alternative for mesophilic polyhydroxybutyrate-producing Cupriavidus species.

Response Surface Methodology and Artificial Neural Network Optimization and Modeling of the Saccharification and Fermentation Conditions of the Polyhydroxybutyrate from Corn Stover

In this study, a response surface methodology (RSM) and artificial neural network (ANN) were used for the modeling and optimization of the enzymatic hydrolysis and fermentation conditions of the polyhydroxybutyrate (PHB) production from pretreated corn stover. Using three factors in each process, a face-centered central composite Design (FCD) was employed to optimize the reducing sugar yield (RSY), biomass, and PHB production of the saccharification and fermentation, respectively. The optimal conditions (RSY of 0.539 g/g) for enzymatic saccharification were 9.776% w/v solids loading, 83.656 FPU/g enzyme loading, and 18 h saccharification time. It was found that solids loading had the greatest impact on RSY. On the other hand, the optimum fermentation medium conditions were 19.727 g/g C/N ratio, 18.421 g/g C/P ratio, and 9.623 mL of trace elements solution (TES) per liter of fermentation medium, with a maximum PHB concentration of 7.083 g/L. During the fermentation of corn stover hydrolysate medium, the C/N ratio positively affected the growth and the PHB productivity of the microorganism. Moreover, the reliability of the RSM and ANN methods in modeling and predicting the RSY, biomass, and PHB concentrations were compared. The ANN showed higher accuracy compared to the RSM models. This study proved that the microorganism could synthesize PHB under optimal fermentation conditions using the reducing sugars from the enzymatic saccharification of pretreated corn stover.

Production of phycobiliproteins, bioplastics and lipids by the cyanobacteria Synechocystis sp. treating secondary effluent in a biorefinery approach.

Cyanobacteria have been identified as promising organisms to reuse nutrients from waste effluents and produce valuable compounds such as lipids, polyhydroxyalkanoates (PHAs), and pigments. However, almost all studies on cyanobacterial biorefineries have been performed under lab scale and short cultivation periods. The present study evaluates the cultivation of the cyanobacterium Synechocystis sp. in a pilot scale 30 L semi-continuous photobioreactor fed with secondary effluent for a period of 120 days to produce phycobiliproteins, polyhydroxybutyrate (PHB) and lipids. To this end, the harvested biomass from the semi-continuous photobioreactor was transferred into 5 L vertical column batch photobioreactors to perform PHB and lipid accumulation under nutrient starvation. Three hydraulic retention times (HRT) (6, 8 and 10 days) were tested in the semi-continuous photobioreactor to evaluate its influence on biomass growth and microbial community. A maximum biomass concentration of 1.413 g L-1 and maximum productivity of 173 mg L-1 d-1 was reached under HRT of 8 days. Microscopy analysis revealed a shift from Synechocystis sp. to Leptolyngbya sp. and green algae when HRT of 6 days was used. Continuous, stable production of phycobiliproteins in the semi-continuous photobioreactor was obtained, reaching a maximum content of 7.4%dcw in the biomass. In the batch photobioreactors a PHB content of 4.8%dcw was reached under 7 days of nitrogen and phosphorus starvation, while a lipids content of 44.7%dcw was achieved under 30 days of nitrogen starvation. PHB and lipids production was strongly dependent on the amount of nutrients withdrawn from the grow phase. In the case of lipids, their production was stimulated when there was only phosphorus depletion. While Nitrogen and phosphorus limitation was needed to enhance the PHB production. In conclusion, this study demonstrates the feasibility of cultivating cyanobacteria in treated wastewater to produce bio-based valuable compounds within a circular bioeconomy approach.