PHB Production Research Articles

Techno-economic assessment of a sustainable and cost-effective bioprocess for large scale production of polyhydroxybutyrate

The rapid depletion of crude-oil resource which sustains a conventional petroleum refinery together with its environmental impact has led to the search for more sustainable alternatives. In this context, biorefinery serves to fulfil the aim by utilizing waste resources. Hence, this study focused on techno-economic assessment of PHB production at large scale from waste carob pods in a closed-loop biorefinery setup. Firstly, the use of pure sugars in SC1 was shifted to use of carob pods as feedstock in SC2, upgradation of stirred tank bioreactor with novel annular gap bioreactor in SC3 and replacing the conventional centrifugation process with the upcoming ceramic membrane separation process in SC4. An Aspen plus™ flowsheet was developed by including the aforementioned novel strategies for PHB production. The effectiveness of PHB production under various scenarios was evaluated based on its pay-out period and turnover accumulated at the end of 7th year of a PHB plant operation. Instead of pure sugars as the feedstock (SC1), carob pod extract (SC2) reduced the pay-out period from 12.6 to 6.8 years. Likewise, switching onto ABR from the conventional STBR further decreased the pay-out period to 4.8 years. Finally, the use of ceramic membranes (SC4) instead of centrifugation resulted in a similar pay-out period of 4.8 years with increased turnover of about 1.4 billion USD. Thus, the use of carob pods along with an improved PHB titre in ABR and incorporation of affordable ceramic membrane technology for PHB rich biomass separation resulted in a highly cost-effective PHB production strategy.

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaCA–04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA–04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA–04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA–04 protein with high YP/S and PHB productivity. Soluble phaCA–04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA–04 from pColdTF-phaCABA–04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCABA–04 enhanced soluble protein formation to approximately 3.09−4.1 times higher than that from pColdI-phaCABA–04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCABA–04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Developing Rhodobacter sphaeroides for cathodic biopolymer production

In this work, Rhodobacter sphaeroides was identified as a potential cathodic production strain for photoautotrophic production processes. First, a stable cultivation in a bioelectrochemical system (BES) was established under conditions in which hydrogen produced by a poised cathode served as an electron donor. It was shown that both the introduction of a plasmid vector and exposure to the corresponding antibiotic selection pressure caused a strong improvement in both cathodic biofilm formation and electrochemical properties. A quantitative proteomic analysis identified key players in the molecular adaptation to biofilm growth on the cathodic surface. Furthermore, biofilm formation kinetics were quantified by optical coherence tomography measurements, which showed a strong tendency for biofilm formation together with a robust biofilm architecture. A media switch to N2-limited conditions resulted in increased cathodic poly(3-hydroxybutyrate) (PHB) accumulation, suggesting R. sphaeroides as a potential strain for photoautotrophic PHB production in future industrial applications.

Increased bioproduction of glycogen, lipids, and poly(3-hydroxybutyrate) under partial supply of nitrogen and phosphorus by photoautotrophic cyanobacterium Synechocystis sp. PCC 6803

Under an adequate supply of nitrogen (N) and phosphorus (P), the cyanobacterium Synechocystis sp. PCC 6803 produced a high biomass yield but with a low storage level of glycogen and poly(3-hydroxybutyrate) (PHB). In contrast, deprivation of N or P stimulated accumulation of glycogen and PHB, but reduced biomass yield. In addition, deprivation of N or P had no apparent effect on total lipid accumulation. Here, we examined how partial supply of N or P has effects on the biomass yield and production of glycogen, PHB, and lipids. The N or P supply in the culture media was orderly decreased from 100% (set according to the standard BG11 medium) to 50, 35, 20, 10, and 0%. A 100% supply of both N and P produced biomass of up to 1.4 g L−1, while a 35–50% N supply slightly reduced the biomass to 1.2–1.3 g L−1, and no apparent change in the biomass was detected under a 20–50% P supply. A maximum glycogen accumulation of 51.8% (w/w dry weight; DW) was obtained under 20% P supply, while the maximum PHB storage of 6.3% (w/w DW) was obtained with 10% P supply. Lipid accumulation peaked at 12.7% (w/w DW) under 0% P supply. The maximal co-production of these three bioproducts was obtained under 20% P supply at 765 mg L−1 which is the sum of 72 mg L−1 PHB (corresponding to 5.1% w/w DW PHB), 618 mg L−1 glycogen (43.8% w/w DW glycogen), and 75 mg L−1 lipid (5.3% w/w DW lipid) from the total biomass production of 1.4 g L−1. The glycogen production (618 mg L−1) was higher than those previously reported for Synechocystis. The results indicated that a proper adjustment of initial N and P supplies in the culture is crucial for maximal production of these three bioproducts by Synechocystis.

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04 and pColdTF-phaCABA–04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Valorization of agro-wastes for the biosynthesis and characterization of polyhydroxybutyrate by Bacillus sp. isolated from rice bran dumping yard.

Investigations have been made to determine the usage of inexpensive agro-waste products as an alternative carbon source for the production of degradable bacterial polyester. Among 33 bacterial isolates, a gram-positive bacterium PPECLRB-16 isolated from rice bran dumping yard was found to accumulate a relatively higher quantity of PHB and identified as Bacillus sp. through 16S rRNA gene sequence analysis. The higher PHB producing bacterial isolate was grown with different inexpensive agro-wastes to determine the suitable carbon source for its growth and PHB production. The one-factor-at-a-time approach comparatively enhanced PHB yield (5.64g/L) when grown for 48h with 1.5% (w/v) of defatted oil cake at a pH of 7.0. The bacterially accumulated PHB was isolated from the cells, purified, and characterized using solid-state 13C NMR, FT-IR, Powder XRD, TGA, GPC, Tensile and HR-SEM analyses. The hydrophobicity and printing accessibility of recovered PHB were demonstrated using contact angle measurement by coating on different surfaces. The results obtained in the present investigation have thrown light on the potential usage of agro-waste by-products, mainly oil cake, as an appropriate carbon source for the commercial production of PHB by Bacillus sp. in a cost-effective way.

Production of Poly(3-Hydroxybutyrate) by Haloarcula, Halorubrum, and Natrinema Haloarchaeal Genera Using Starch as a Carbon Source

Microbial production of bioplastics, derived from poly(3-hydroxybutyrate) (PHB), have provided a promising alternative towards plastic pollution. Compared to other extremophiles, halophilic archaea are considered as cell factories for PHB production by using renewable, inexpensive carbon sources, thus decreasing the fermentation cost. This study is aimed at screening 33 halophilic archaea isolated from three enrichment cultures from Tunisian hypersaline lake, Chott El Jerid, using starch as the sole carbon source by Nile Red/Sudan Black staining and further confirmed by PCR amplification of phaC and phaE polymerase genes. 14 isolates have been recognized as positive candidates for PHA production and detected during both seasons. The identification of these strains through 16S rRNA gene analyses showed their affiliation to Halorubrum, Natrinema, and Haloarcula genera. Among them, three PHB-producing strains, CEJ34-14, CEJ5-14, and CEJ48-10, related to Halorubrum chaoviator, Natrinema pallidum, and Haloarcula tradensis were found to be the best ones reaching values of 9.25, 7.11, and 1.42% of cell dry weight (CDW), respectively. Our findings highlighted that Halorubrum, Natrinema, and Haloarcula genera were promising candidates for PHB production using soluble starch as a carbon source under high salinity (250 g L−1 NaCl).

Techno-economic evaluation and life-cycle assessment of poly(3-hydroxybutyrate) production within a biorefinery concept using sunflower-based biodiesel industry by-products

This study presents techno-economic evaluation of a biorefinery concept using biodiesel industry by-products (sunflower meal and crude glycerol) to produce poly(3-hydroxybutyrate) (PHB), crude phenolic extracts (CPE) and protein isolate (PI). The PHB production cost at two annual production capacities ($12.5/kg for 2,500 t PHB/year and $7.8/kg for 25,000 t PHB/year) was not cost-competitive to current PHB production processes when the revenues derived from co-products were not considered. Sensitivity analysis projected the economic viability of a biorefinery concept that could achieve a minimum selling price of $1.1/kg PHB similar to polypropylene. The annual PHB production capacity and the identification of marketable end-uses with respective market prices for the co-products CPE and PI were crucial in attaining process profitability. Greenhouse gas emissions (ca. 0.64 kg CO2-eq/kg PHB) and abiotic depletion potential (61.7 MJ/kg PHB) were lower than polypropylene. Biorefining of sunflower meal and crude glycerol could lead to sustainable PHB production.

Exploration of Cupriavidus necator ATCC 25207 for the Production of Poly(3-hydroxybutyrate) Using Acid Treated Beet Molasses

Poly (3-hydroxybutyrate) (PHB) is a good alternative to petroleum-based plastics due to its biodegradable and biocompatible properties. In the present work, Cupriavidus necator ATCC 25207 was used for the first time to produce PHB using fructose, glucose, and acid or enzyme hydrolyzed molasses, a cheap carbon source. The bacterium produced highest PHB with 4.99 g/L, 3.94 g/L, and 15.28 g/L using fructose, glucose, and acid hydrolyzed molasses, respectively. Sulfuric acid and invertase treated molasses yielded 1288% and 529% more PHB than untreated molasses. In addition, acid treated molasses resulted in 151% more PHB but 34.04% less cell growth than enzyme hydrolyzed molasses. This novel finding could have applications in fermentations using molasses in growth or production media. The utilization of glucose by the bacterium was increased by 67.03% through spontaneous mutagenesis. The characterizations of the produced polymers were carried out using FTIR, NMR, XRD, DSC, TGA, and GPC. The molecular weights of PHB samples from acid hydrolyzed molasses and fructose are 3.84 $$\times $$ 105 and 1.43 $$\times $$ 106 g/mol, respectively. This study shows that C. necator ATCC 25207 has a potential to be used in PHB production using acid hydrolyzed molasses.

Model-based Optimization of Biopolymer Production from Glycerol

The present study focuses on sustainable production of biodegradable polymers by <br /> Cupriavidus necator DSMZ 545 using glycerol as substrate. The batch growth and biopolymer production kinetics were established in a 7-L bioreactor, which resulted in a <br /> total biomass of 8.88 g L–1 and poly(3-hydroxybutyrate) (PHB) accumulation of 6.76 g L–1. The batch kinetic and independently acquired substrate inhibition data were then used to develop a mathematical model for PHB production process. This was eventually used to design different nutrient feeding strategies under constant feed rate, decreasing feed rate, and pseudo steady state of substrate (glycerol) to optimize the PHB production during fed-batch cultivation. Among all the fed-batch cultivation strategies, the highest PHB accumulation and productivity of 13.12 g L–1 and 0.27 g L–1 h–1, respectively, was <br /> achieved in fed-batch bioreactor cultivation where a pseudo steady state with respect to <br /> glycerol was maintained.

Improved fermentation strategies in a bioreactor for enhancing poly(3-hydroxybutyrate) (PHB) production by wild type Cupriavidus necator from fructose

Poly(3-hydroxybutyrate) (PHB) belongs to the family of polyhydroxyalkanoates, biopolymers used for agricultural, industrial, or even medical applications. However, scaling up the production is still an issue due to the myriad of parameters involved in the fermentation processes. The present work seeks, firstly, to scale up poly(3-hydroxybutyrate) (PHB) production by wild type C. necator ATCC 17697 from shaken flasks to a stirred-tank bioreactor with the optimized media and fructose as carbon source. The second purpose is to improve the production of PHB by applying both the batch and fed-batch fermentation strategies in comparison with previous works of wild type C. necator with fructose. Furthermore, thinking of biomedical applications, physicochemical, and cytotoxicity analyses of the produced biopolymer, are presented.Fed-batch fermentation with an exponential feeding strategy enabled us to achieve the highest values of PHB concentration and productivity, 25.7 g/l and 0.43 g/(l h), respectively. The PHB productivity was 3.3 and 7.2 times higher than the one in batch strategy and shaken flask cultures, respectively. DSC, FTIR, 1H, and 13C NMR analysis led to determine that the biopolymer produced by C. necator ATCC 17697 has a molecular structure and characteristics in agreement with the commercial PHB. Additionally, the biopolymer does not induce cytotoxic effects on the NIH/3T3 cell culture.Due to the improved fermentation strategies, PHB concentration resulted in 40 % higher of the already reported one for wild type C. necator using other fed-batch modes and fructose as a carbon source. Thus the produced PHB could be attractive for biomedical applications, which generate a rising interest in polyhydroxyalkanoates during recent years.

Screening, optimization and characterization of poly hydroxy butyrate from fresh water microalgal isolates

ABSTRACT Microalgae, especially Chlorella sp., have been used for centuries as food and currently their biotechnological potential is notable for the presence of several compounds relevant to the market, like PHB. Microalgae are appealing because of the increasing demand for biopolymers like PHB. Therefore, the present study is aimed to determine the efficacy of biomass production and yield of PHB producing microalgae isolated and identified by phylogenetic analysis. The parameter was optimized as pH 7, temperature 30°C, under sunlight with 0.2% of sodium bicarbonate for higher biomass and PHB production. The extracted PHB were characterized through SEM, EDAX, XRD and FT-IR. In GC-MS analysis the major peak represents Benzyl butyl phthalate confirming the polymer of PHB. DSC-TGA, was performed and the melting peak of PHB was found as 342.1°C conferring to thermogravimetric analysis, the range of temperature for rapid thermal degradation of PHB was at 418°C to 420°C with the degradation peaking at 418.2°C the total weight loss within this temperature range was 98.5%. Hence, maximum 80% of PHB was produced from Chlorella sp. in addition to sequestering CO2.

Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC

BackgroundPHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon–neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW).ResultsWe have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic.ConclusionThe amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

Revealing of sugar utilization systems in Halomonas sp. YLGW01 and application for poly(3-hydroxybutyrate) production with low-cost medium and easy recovery

Poly(3-hydroxybutyrate) (PHB) is a common polyhydroxyalkanoate (PHA) with potential as an alternative for petroleum-based plastics. Previously, we reported a new strain, Halomonas sp. YLGW01, which hyperproduces PHB with 94% yield using fructose. In this study, we examined the PHB production machinery of Halomonas sp. YLGW01 in more detail by deep-genome sequencing, which revealed a 3,453,067-bp genome with 65.1% guanine-cytosine content and 3054 genes. We found two acetyl-CoA acetyltransferases (Acetoacetyl-CoA thiolase, PhaA), one acetoacetyl-CoA reductase (PhaB), two PHB synthases (PhaC1, PhaC2), PHB depolymerase (PhaZ), and Enoyl-CoA hydratase (PhaJ) in the genome, along with two fructose kinases and fructose transporter systems, including the phosphotransferase system (PTS) and ATP-binding transport genes. We then examined the PHB production by Halomonas sp. YLGW01 using high-fructose corn syrup (HFCS) containing fructose, glucose, and sucrose in sea water medium, resulting in 7.95 ± 0.11 g/L PHB (content, 67.39 ± 0.34%). PHB was recovered from Halomonas sp. YLGW01 using different detergents; the use of Tween 20 and SDS yielded micro-sized granules with high purity. Overall, these results reveal the distribution of PHB synthetic genes and the sugar utilization system in Halomonas sp. YLGW01 and suggest a possible method for PHB recovery.

From Organic Wastes to Bioplastics: Feasibility of Nonsterile Poly(3-hydroxybutyrate) Production by Zobellella denitrificans ZD1.

Poly(3-hydroxybutyrate) (PHB)—a renewable and biodegradable polymer—is a promising alternative to nonbiodegradable synthetic plastics that are derived from petrochemicals. The methods currently employed for PHB production are costly, in part, due to the expensive cultivation feedstocks and the need to sterilize the culture medium, which is energy-intensive. This study investigates the feasibility of nonsterile PHB production from several saline organic wastes using a salt-tolerant strain, Zobellella denitrificans ZD1 (referred to as strain ZD1). Factors such as the pH, salinity, carbon/nitrogen (C/N) ratio, nitrogen source, and electron acceptor that might affect the growth of strain ZD1 and its PHB production were determined. Our results showed successful nonsterile PHB production by growing the strain ZD1 on nonsterile synthetic crude glycerol, high-strength saline wastewater, and real municipal wastewater-activated sludge under saline conditions. The PHB production was significantly enhanced when the levels of salts and nitrate-nitrogen in the culture medium were increased. This study suggested a promising low-cost nonsterile PHB production strategy from organic wastes using strain ZD1.

Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.

Poly(3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible thermoplastic, and synthesized from the central metabolite acetyl-CoA. The acetyl-CoA synthesis from glucose presents low atomic economy due to the release of CO2 in pyruvate decarboxylation. As ethanol and acetate can be converted into acetyl-CoA directly, they were used as carbon source for PHB production in this study. The reductase mutant AdhE A267T/E568K was introduced into Escherichia coli to enable growth on ethanol, and acetate utilization was improved by overexpression of acetyl-CoA synthetase ACS. Comparison of the PHB production using glucose, ethanol or acetate as sole carbon source showed that the production and yield from ethanol was much higher than those from glucose and acetate, and metabolome analysis revealed the differences in metabolism of glucose, ethanol and acetate. Furthermore, other acetyl-CoA derived chemicals including 3-hydroxypropionate and phloroglucinol were produced from those three feedstocks, and similar results were achieved, suggesting that ethanol could be a suitable carbon source for the production of acetyl-CoA derivatives.

Increasing PHB production with an industrially scalable hardwood hydrolysate as a carbon source

In this study, we report an industrially feasible conversion of wood hydrolysate from a pilot-scale forest biorefinery. The wood hydrolysate was compared to a synthetic hydrolysate with a similar content of carbon sources (glucose, xylose, and acetate). To maximize the PHB concentration, high-cell density cultivations were conducted in bioreactors for 52 h using the bacterium Paraburkholderia sacchari IPT 101 LMG 19450. The conversion of wood hydrolysate yielded a maximum PHB concentration of 34.5 g/L, which is among the highest reported for lignocellulose hydrolysates. In comparison, the use of a synthetic hydrolysate resulted in substantially lower PHB concentration (22.0 g/L). This could be attributed to a higher maximum specific growth rate of 0.36 vs. 0.33 per h for cells grown in wood hydrolysate vs. synthetic hydrolysate. For the wood hydrolysate, the final PHB content per cell mass reached 58 % g/g and a maximum PHB productivity of 0.72 g/(Lh), while the synthetic hydrolysate reached 55 % g/g and 0.46 g/(Lh) respectively. Given the increase in bacterial growth and PHB productivity when using the wood hydrolysate, the chosen lignocellulose conversion process is beneficial for subsequent biotechnological conversion to key bioproducts.

Dynamic Metabolic Analysis of Cupriavidus necator DSM545 Producing Poly(3-hydroxybutyric acid) from Glycerol

Cupriavidus necator DSM 545 can utilise glycerol to synthesise poly(3-hydroxybutyric acid) under unbalanced growth conditions, i.e., nitrogen limitation. To improve poly(3-hydroxybutyric acid) (PHB) batch production by C. necator through model-guided bioprocessing or genetic engineering, insights into the dynamic effect of the fermentation conditions on cell metabolism are crucial. In this work, we have used dynamic flux balance analysis (DFBA), a constrained-based stoichiometric modelling approach, to study the metabolic change associated with PHB synthesis during batch cultivation. The model employs the ‘minimisation of all fluxes’ as cellular objectives and measured extracellular fluxes as additional constraints. The mass balance constraints are further adjusted based on thermodynamic considerations. The resultant flux distribution profiles characterise the evolution of metabolic states due to adaptation to dynamic extracellular conditions and provide further insights towards improvements that can be implemented to enhance PHB productivity.

Productivity and scale‐up of poly(3‐hydroxybutyrate) production under different oxygen transfer conditions in cultures of Azotobacter vinelandii

AbstractBackgroundPoly(3‐hydroxybutyrate) (PHB) is a polymer produced by Azotobacter vinelandii. The production of PHB in a bioreactor is affected by oxygen transfer conditions. The aim of this study was to evaluate PHB synthesis in extended batch of A. vinelandii, to obtain an operation curve (productivity versus oxygen transfer rate) and to scale up the bioprocess.ResultsPHB production by A. vinelandii under dinitrogen fixation was evaluated using an extended batch modality under different oxygen transfer rates (OTRs). Extended batch cultures were performed using different agitation rates (400, 600, 800 and 1000 rpm), which determined different OTRs in the cultures. Under the conditions evaluated, it was possible to establish an operation curve of PHB productivity at different OTRs, allowing for the establishment of the maximum productivity of PHB. The maximum PHB productivity obtained was 0.40 ± 0.05 g L−1 h−1, which was reached under an OTR of 22.1 ± 1.5 mmol L−1 h−1. Using the OTR as a criterion to scale up PHB production from 3 to 30 L, it was possible to obtain a similar PHB concentration (7.9 ± 0.0 g L−1) and volumetric productivity (0.43 ± 0.04 g L−1 h−1) on a 30 L scale.ConclusionsThe results lead to the conclusion that in extended cultures and under dinitrogen fixation in A. vinelandii cultures, the OTR value determines the maximum PHB productivity. This study provides evidence that the OTR is an adequate parameter as a criterion for the successful scaling up of PHB synthesis for the first time. © 2020 Society of Chemical Industry

Acid soaking followed by steam flash-explosion pretreatment to enhance saccharification of rice husk for poly(3-hydroxybutyrate) production

In order to exploit an abundant and cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, the rice husk was pretreated with dilute sulfuric acid and steam flash-explosion to enhance the enzymatic saccharification. The yield of reducing sugars of pretreated rice husk hydrolysate came up to 266.5 mg/g rice husk, much higher than that of untreated rice husk hydrolysate (72.67 mg/g rice husk). This result indicated that the pretreatment can significantly enhance the yield of reducing sugars. Then the hydrolysate was used as a sole carbon source for PHB production by using Cupriavidus necator. Response surface method was used to optimize the fermentation conditions. Under optimum fermentation conditions (carbon source, 31.81 gL−1; nitrogen source, 1.8 gL−1; pH-value, 7.0; temperature, 27.1 °C), the PHB yield was 5.0 gL−1, which was in good agreement with the predicted value.