PHBV Production Research Articles

Long-term effects of cycle time and volume exchange ratio on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from food waste digestate by Haloferax mediterranei cultivated in sequencing batch reactors for 450 days

Food wastedigestatewas fed into a sequencing batch reactor (SBR) forHaloferax mediterranei(HM) to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This SBR was operated uninterruptedly for 450 days to test its stability, during which the cycle time and volume exchange ratiowere varied to understand their impacts on the PHBV fermentation performance under ranged organic loading rates (OLR). Results showed that 1) PHBV productivity was proportional to OLR of food wastedigestate; 2) substrate and product inhibitions were two limiting factors constraining substrate utilization and PHBV yields; 3) PHBV titer was dependent on the hydraulic retention time of the SBR while a volume exchange ratio lower than 0.5 is unfavorable due to the product inhibitor accumulation. This study for the first time demonstrated that the long-term stability of food waste-fed PHBV production byHMand revealed that inhibition effects could be barriers in SBR limiting the full-scale application of the technology.

Characterization of the conversion system of natural rubber to poly(3-Hydroxyalkanoate) in Piscinibacter gummiphilus strain NS21T

Poly(3-hydroxyalkanoate) (PHA), a bacteria-synthesized biodegradable polyester, is a useful alternative to fossil resources, and current systems for its production rely predominantly on edible resources, raising concerns about microbial competition for nutrients. Therefore, we investigated mechanisms underlying PHA production from non-edible resources by Piscinibacter gummiphilus strain NS21T. Strain NS21T can utilize natural rubber as a carbon source on solid media and potentially produces PHA. Gas chromatography and nuclear magnetic resonance analyses of NS21T cell extracts revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate) from natural rubber and glucose, respectively. Transcriptional analysis suggested that phaC is involved in PHA production. An increased PHBV accumulation rate under nitrogen-limiting conditions indicates the potential of this strain to be used as a PHBV production enhancement strategy. Furthermore, the disruption of PHA depolymerase genes resulted in enhanced PHA production, indicating the involvement of these genes in PHA degradation. These findings highlight the potential of NS21T for PHBV production from natural rubber, a non-edible resource.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei Using Candy Industry Waste as Raw Materials.

The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical properties of biotechnological and biomedical interest and can be used as an alternative to plastics made from chemical synthesis (which are not environmentally friendly). The versatile metabolism of H. mediterranei makes the use of waste as a carbon source for cellular growth and PHA synthesis possible. In this work, cellular growth and the production and characterization of PHBV using two different types of confectionery waste were analyzed and compared with cellular growth and PHBV synthesis in a standard culture media with glucose of analytical grade as a carbon source. The PHBV granules produced were analyzed by TEM and the biopolymer was isolated and characterized by GC-MS, FTIR NMR, and DSC. The results reveal that H. mediterranei can use these two residues (R1 and R2) for pure PHBV production, achieving 0.256 and 0.983 g PHBV/L, respectively, which are among the highest yields so far described using for the first-time waste from the candy industry. Thus, a circular economy-based process has been designed to optimize the upscaling of PHBV production by using haloarchaea as cell factories and valorizing confectionery waste.

A novel approach for poly(hydroxybutyrate-co-hydroxyvalerate) production from vegetable wastes and pig manure: Potential in economy, environment and sustainability

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising alternative to conventional petroleum-based plastics, owing to its considerable biodegradability and physicochemical properties. However, the high raw material costs limit its large-scale production. This study established a novel integrated process by coupling the anaerobic fermentation of vegetable wastes (VW) and pig manure (PM) with PHBV biosynthesis. The results demonstrated appreciable PHBV yields of 0.326 and 0.261 gPHBV/gVFA for VW and PM, respectively. Considering the magnitude of VM and PM in China, this novel approach could result in CO2 emission reduction by up to 12.70 million tons per year, as indicated by environmental impact assessment. Additionally, a preliminary economic analysis suggested economic benefits of up to 9.81 billion USD per year. These findings demonstrate that this integrated process can play a significant role in enabling large-scale production of PHBV while simultaneously resolving environmental pollution related problems associated with conventional plastics.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Biorefinery of volatile fatty acids for the synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using Paracoccus sp. TOH: Fermentation performance and metabolic pathway

The residual microbes and uncertain volatile fatty acids (VFAs) components in anaerobic fermentation hydrolysate cause microbial contamination and multi-type polyhydroxyalkanoates (PHA) generation in pure microbial synthesis systems. Meanwhile, the regulation of biosynthesis of poly(3-hydroxybuturate-co-3-hydroxyvalerate) (PHBV), one type of excellent PHA, by re-utilization of waste-organics as carbon substrates is still elusive. In this study, four pretreatment methods for sludge hydrolysate containing 4746 mg-COD·L−1 of VFAs were established to assess the accumulation of PHBV by using Paracoccus sp. TOH as the chassis. Results showed that the autoclaving system exhibited an optimal PHBV concentration of 202.9 mg L−1 with the highest relative abundance of Paracoccus sp. TOH (92.4%). Valeric acid was identified as the primary substrate for 3-hydroxyvalerate accumulation, and the highest PHBV production of 3.76 g L−1 was achieved using 3 g L−1 valeric acid and 5 g L−1 glycerol as co-substrates. The increased carbon metabolism flows through the sequential action of acyl-coenzyme A dehydrogenase and enoyl-coenzyme A hydratase in the fatty acid β-oxidation pathway facilitated PHBV accumulation. This study provides a blueprint for the microbial synthesis of PHBV with oriented acid production from organic wastes.

How temperature shapes the biosynthesis of polyhydroxyalkanoates in mixed microbial cultures.

Three sequential batch reactors were operated for the enrichment in microbial communities able to store polyhydroxyalkanoates (PHAs) using activated sludge as inoculum. They ran simultaneously under the same operational conditions (organic loading rate, hydraulic and solids retention time, cycle length, C/N ratio) just with the solely difference of the working temperature: psychrophilic (15°C), mesophilic (30°C), and thermophilic (48°C). The microbial communities enriched showed different behaviors in terms of consumption and production rates. In terms of PHA accumulation, the psychrophilic community was able to accumulate an average amount of 17.7 ± 5.7wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the mesophilic 40.3 ± 7.0wt% PHBV, and the thermophilic 14.8 ± 0.3wt% PHBV in dry weight over total solids. The average PHBV production yields for each selected community were 0.41 ± 0.12 CmmolPHBV /CmmolVFA at 15°C, 0.64 ± 0.05 CmmolPHBV /CmmolVFA at 30°C, and 0.39 ± 0.14 CmmolPHBV /CmmolVFA at 48°C. The overall performance of the mesophilic reactor was better than the other two, and the copolymers obtained at this temperature contained a higher PHV fraction. The physico-chemical properties of the obtained biopolymers at each temperature were also measured, and major differences were found in the molecular weight, following an increasing trend with temperature. PRACTITIONER POINTS: PHBV molecular weight is influenced by the operational temperature increasing with it. Increasing temperatures promote the production of HB over HV. The best accumulation performance was found at 30°C for the tested operational conditions.

Enhanced poly(3-hydroxybutyrateco-3-hydroxyvalerate) production from high-concentration propionate by a novel halophile Halomonas sp. YJ01: Detoxification of the 2-methylcitrate cycle

As a carbon substrate, propionate can be used to synthesize poly(3-hydroxybutyrateco-3-hydroxyvalerate) [PHBV] biopolymer, but high concentrations can inhibit PHBV production. Therefore, novel PHBV producers that can utilize high propionate concentrations are needed. Here, a novel halophile, Halomonas sp. YJ01 was applied to PHBV production via a propionate-dependent pathway, and optimal culture growth conditions were determined. The maximum poly(3-hydroxybutyrate) [PHB] content and yield in the presence of glucose were 89.5 wt% and 5.7 g/L, respectively. This strain utilizes propionate and volatile fatty acids (VFAs) for PHBV accumulation. Multiple genes related to polyhydroxyalkanoate (PHA) synthesis were identified using whole-genome annotation. The PHBV yield and 3HV fraction obtained by strain YJ01 utilizing 15 g/L propionate were 0.86 g/L and 29 mol%, respectively, but in cultures with glucose-propionate, it decreased its copolymer dry weight. This indicates that propionyl-CoA was converted to pyruvate through the 2-methylcitrate cycle (2MCC), which reduced propionate detoxification for the strain.

Simultaneous production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from recovered volatile fatty acid with treatment of leachate by Pilot-Scale Mechanical Vapor Recompression

Serious global problems faced due to many petroleum-based materials in the last century, which is called the plastic age, constitute the main motivation of this research. Considering wastewater treatment from this perspective, both the recovery of organic acids from wastewater and their conversion into bioplastics are extremely important in terms of reducing petroleum dependency. In this study, while the treatment of landfill leachate was provided with biological process integrated into Mechanical Vapor Recompression (MVR), simultaneously PHBV production was carried out with 84.9% recovered VFA as carbon source. The effects of C/N/P ratio and feeding regime on PHBV storage were investigated by Cupriavidus necator. PHBV storage of 96% (g PHBV/g DCW) was maximized by 2-stage feeding and nitrogen restriction. The ratio of 3HV to 3HB of PHBV was 45%. In addition, extracted PHBV was compared with standard PHA in terms of thermal and chemical properties with FTIR, XRD, TGA and DSC analyses.

Continuous bioreactor production of polyhydroxyalkanoates in Haloferax mediterranei.

In this work, the viability of continuous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production with controlled composition in Haloferax mediterranei when fed volatile fatty acids is demonstrated. Continuous fermentations showed to greatly outperform batch fermentations with continuous feeding. Operating the bioreactor continuously allowed for PHBV productivity normalised by cell density to increase from 0.29 to 0.38mgL-1 h-1, in previous continuously fed-fed batch fermentations, to 0.87 and 1.43mgL-1 h-1 in a continuous mode of operation for 0.1 and 0.25M carbon concentrations in the media respectively. Continuous bioreactor experiments were carried out for 100h, maintaining control over the copolymer composition at around 30mol% 3-hydroxyvalerate 3HV. This work presents the first continuous production of PHBV in Haloferax mediterranei which continuously delivers polymer at a higher productivity, compared to fed-batch modes of operation. Operating bioreactors continuously whilst maintaining control over copolymer composition brings new processing opportunities for increasing biopolymer production capacity, a crucial step towards the wider industrialisation of polyhydroxyalkanoates (PHAs).

Valorization of lignin-derived compounds into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by engineered Halomonas sp. Y3

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biopolyester with great potential, but its high production cost via the propionate-dependent pathway has hindered its development. Herein, we engineer Halomonas sp. Y3 to achieve efficient conversion of various LDCs into PHBV without propionate supplement. Initially, we successfully achieve PHBV production without propionate supplement by overexpressing threonine synthesis. The resulting biopolyester exhibits a 3 HV proportion of up to 7.89 mol%, comparable to commercial PHBV (8 mol%) available from Sigma Aldrich (403105). To further enhance PHBV production, we rationally design the reconstruction of aromatic compound catabolism. The engineered strain Y3_18 efficiently assimilates all LDCs containing syringyl (S), guaiacyl (G), and p-hydroxyphenyl-type (H) units. From 1 g/L of S-, G-, and H-type LDCs, Y3_18 produces PHBV at levels of 449 mg/L, 488 mg/L, and 716 mg/L, respectively, with yields of 44.9 % (g/g), 48.8 % (g/g), and 71.6 % (g/g). Moreover, to improve PHBV yield from lignin, we integrate laccase-secretion and PHBV production modules. This integration leads to the accumulation of 425.84 mg/L of PHBV with a yield of 21.29 % (g/g) and a 3 HV proportion of 6.38 mol%. By harnessing the capabilities of Halomonas sp. Y3, we demonstrate an efficient and sustainable approach for PHBV production from a variety of LDCs.

Inexpensive Production of Poly (3‑hydroxybutyrate‑co‑ 3‑hydroxyvalerate) from Bacillus megaterium PP-10 Using Pineapple Peel Waste

Pineapple peel waste has recently been interested in being utilized as a low-cost carbon source in PHA biosynthesis to reduce the production cost of PHA. The production of copolymer Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV] by the new Bacillus megaterium PP-10 was investigated. The bacteria were grown in a mixture of pineapple peel hydrolysate (PPH) and 3-hydroxyvalerate (3HV) precursor such as sodium propionate or sodium valerate at ratios of 1:1. Remarkably, the microbial growth and PHBV production in a mixture of PPH and sodium valerate exhibited higher biomass and higher PHA amount than that of sodium propionate, accounted about 2.40 ± 0.07 g/L of DCW and 0.71 ± 0.03 g/L of PHA concentration (PHA content of 29.6%DCW). Moreover, to control the 3HV molar fraction in PHBV, various sodium valerate concentration from 2 to 18 g/L was supplemented with PPH, and the result showed that the 3HV fraction increased linear trend with an increase in valerate concentration and was in the range between 6-35 mol%HV. In contrast, a maximum PHA concentration of 1.65 ± 0.04 g/L content (about 49%DCW) was obtained when B. megaterium PP-10 was cultivated in 18 g/L of total reducing sugar in PPH with 2 g/L of sodium valerate at 12 h of cultivation. Finally, the produced PHBV containing 20 mol%HV was further determined by some thermal properties and found that it possessed the melting and glass transition temperatures of 148°C and -10°C, respectively. Therefore, PHBV synthesized by B. megaterium PP-10 with various 3HV fractions was an excellent choice for biopolymer production.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated 3-hydroxyvalerate fraction by overexpressing acetolactate synthase in Cupriavidus necator H16

The elastomeric properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable copolymer, strongly depend on the molar composition of 3-hydroxyvalerate (3HV). This paper reports an improved artificial pathway for enhancing the 3HV component during PHBV biosynthesis from a structurally unrelated carbon source by Cupriavidus necator H16. To increase the intracellular accumulation of propionyl-CoA, a key precursor of the 3HV monomer, we developed a recombinant strain by genetically manipulating the branched-chain amino acid (e.g., valine, isoleucine) pathways. Overexpression of the heterologous feedback-resistant acetolactate synthase (alsS), (R)-citramalate synthase (leuA), homologous 3-ketothiolase (bktB), and the deletion of 2-methylcitrate synthase (prpC) resulted in biosynthesis of 42.5 % (g PHBV/g dry cell weight) PHBV with 64.9 mol% 3HV monomer from fructose as the sole carbon source. This recombinant strain also accumulated the highest PHBV content of 54.5 % dry cell weight (DCW) with 24 mol% 3HV monomer from CO2 ever reported. The lithoautotrophic cell growth and PHBV production by the recombinant C. necator were promoted by oxygen stress. The thermal properties of PHBV showed a decreasing trend of the glass transition and melting temperatures with increasing 3HV fraction. The average molecular weights of PHBV with modulated 3HV fractions were between 20 and 26 × 104 g/mol.

Aerobic-anaerobic transition boosts poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Rhodospirillum rubrum: the key role of carbon dioxide

BackgroundMicrobially produced bioplastics are specially promising materials since they can be naturally synthesized and degraded, making its end-of-life management more amenable to the environment. A prominent example of these new materials are polyhydroxyalkanoates. These polyesters serve manly as carbon and energy storage and increase the resistance to stress. Their synthesis can also work as an electron sink for the regeneration of oxidized cofactors. In terms of biotechnological applications, the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), or PHBV, has interesting biotechnological properties due to its lower stiffness and fragility compared to the homopolymer poly(3-hydroxybutyrate) (P3HB). In this work, we explored the potentiality of Rhodospirillum rubrum as a producer of this co-polymer, exploiting its metabolic versatility when grown in different aeration conditions and photoheterotrophically.ResultsWhen shaken flasks experiments were carried out with limited aeration using fructose as carbon source, PHBV production was triggered reaching 29 ± 2% CDW of polymer accumulation with a 75 ± 1%mol of 3-hydroxyvalerate (3HV) (condition C2). Propionate and acetate were secreted in this condition. The synthesis of PHBV was exclusively carried out by the PHA synthase PhaC2. Interestingly, transcription of cbbM coding RuBisCO, the key enzyme of the Calvin-Benson-Bassham cycle, was similar in aerobic and microaerobic/anaerobic cultures. The maximal PHBV yield (81% CDW with 86%mol 3HV) was achieved when cells were transferred from aerobic to anaerobic conditions and controlling the CO2 concentration by adding bicarbonate to the culture. In these conditions, the cells behaved like resting cells, since polymer accumulation prevailed over residual biomass formation. In the absence of bicarbonate, cells could not adapt to an anaerobic environment in the studied lapse.ConclusionsWe found that two-phase growth (aerobic-anaerobic) significantly improved the previous report of PHBV production in purple nonsulfur bacteria, maximizing the polymer accumulation at the expense of other components of the biomass. The presence of CO2 is key in this process demonstrating the involvement of the Calvin-Benson-Bassham in the adaptation to changes in oxygen availability. These results stand R. rubrum as a promising producer of high-3HV-content PHBV co-polymer from fructose, a PHBV unrelated carbon source.

Thermal pre-processing before extraction of polyhydroxyalkanoates for molecular weight quality control

Past studies have repeatedly demonstrated the technical feasibility to produce polyhydroxyalkanoate (PHA) using bacterial biomass of mixed microbial cultures (MMCs). Commercial quality grades of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV, can be produced with control of average monomer composition. However, demonstration of PHBV production and recovery with quality control of molecular weight (MW) distribution has been lacking in the research literature. Towards this goal, a workflow has been developed for characterizing molecular weight control by thermal treatment pre-processing of dried PHA-rich biomass before solvent extraction. Dimethyl carbonate (DMC) was a suitable solvent in this workflow in the routine evaluation of extractable PHA. From assessments of DMC extraction using differential scanning calorimetry, 125 °C was selected for nominally 100 percent extraction yield independent of polymer 3-hydroxyvalerate (3HV) content (2 to 41 wt.% 3HV) and molecular weight (100 to 1400 kDa). Intrinsic viscosity measurements of PHBV in DMC at 60 °C was used for molecular weight monitoring. Mark-Houwink constants, α (0.738 ± 0.010) and LogK (-2.016 ± 0.025), were estimated for a PHBV co-polymer blend having 36 wt.% 3HV. A model of random scission supported that weight average molecular weight (Mw) was a more robust metric, compared to number average molecular weight (Mn), for assessing the polymer scission rates. During isothermal heat treatment for a given biomass batch, interpreted scission rate was reproducible and commonly, but not always, constant in time. Scission rates between biomass batches were also variable. Measured properties of the polymer in the biomass (thermal stability, biomass PHA content, PHBV grade, initial moisture content) could not be correlated to this observed batch-to-batch variation of scission rate. Molecular weight loss before extraction did not influence the melting temperatures of the co-polymer blends of PHBV evaluated over a wide sub-eutectic range of average 3HV content. Molecular weight changes for these PHBV co-polymer blends were considered to have likely influenced the nature of blend 3HV distribution, and consequently, crystallization behaviour. Molecular weight loss effects on crystallization behaviour at constant PHBV average 3HV wt.% content could then have contributed to the observed variability for glass transition temperatures and melting enthalpies. However, a reproducible correlation between this variability and MW change was not observed.

Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.

Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxvalerate) from volatile fatty acids by Cupriavidus necator.

A promising strategy to alleviate the plastic pollution from traditional petroleum-based plastics is the application of biodegradable plastics, in which polyhydroxyalkanoates (PHAs) have received increasing interest owing to their considerable biodegradability. In the PHAs family, poly(3-hydroxybutyrate-co-3-hydroxvalerate) (PHBV) has better mechanical properties, which possesses broader application prospects. With this purpose, the present study adopted Cupriavidus necator to synthesize PHBV utilizing volatile fatty acids (VFAs) as sole carbon sources. Results showed that the concentration and composition of VFAs significantly influenced the production of PHAs. Especially, even carbon VFAs (acetate and butyrate) synthesized only poly(3-hydroxybutyrate) (PHB), while the addition of odd carbon VFAs (propionate and valerate) resulted in PHBV production. The 3-hydroxyvalerate (3HV) contents in PHBV were directly determined by the specific VFAs compositions, in which valerate was the preferred substrate for 3HV accumulation. After optimization by response surface methodology, the highest PHBV accumulation achieved 79.47% in dry cells, and the conversion efficiency of VFAs to PHBV reached 40%, with the PHBV production of 1.20 ± 0.05 g/L. This study revealed the metabolic rule of VFAs converting into PHAs by C. necator and figured out the optimal VFAs condition for PHBV accumulation, which provides a valuable reference for developing downstream strategies of PHBV production in industrial applications in future.

Exploitation of wasted bread as substrate for polyhydroxyalkanoates production through the use of Haloferax mediterranei and seawater.

The use of the halophile microorganism Haloferax mediterranei, able to synthesize poly(hydroxybutyrate-hydroxyvalerate) (PHBV), is considered as a promising tool for the industrial production of bioplastic through bioprocessing. A consistent supplementation of the growth substrate in carbohydrates and minerals is overall necessary to allow its PHBV production. In this work, wasted bread was used as substrate for bioplastic production by microbial fermentation. Instead of the consistent and expensive minerals supplement required for Hfx. mediterranei DSM1411 growth, microfiltered seawater was added to the wasted bread-derived substrate. The suitable ratio of wasted bread homogenate and seawater, corresponding to 40:60, was selected. The addition of proteases and amylase to the bread homogenate promoted the microbial growth but it did not correspond to the increase of bioplastic production by the microorganism, that reach, under the experimental conditions, 1.53 g/L. An extraction procedure of the PHBV from cells, based on repeated washing with water, followed or not by a purification through ethanol precipitation, was applied instead of the conventional extraction with chloroform. Yield of PHBV obtained using the different extraction methods were 21.6 ± 3.6 (standard extraction/purification procedure with CHCl3:H2O mixture), 24.8 ± 3.0 (water-based extraction), and 19.8 ± 3.3 mg PHAs/g of wasted bread (water-based extraction followed by ethanol purification). Slightly higher hydroxyvalerate content (12.95 vs 10.78%, w/w) was found in PHBV obtained through the water-based extraction compared to the conventional one, moreover, the former was characterized by purity of 100% (w/w). Results demonstrated the suitability of wasted bread, supplemented with seawater, to be used as substrate for bioplastic production through fermentation. Results moreover demonstrated that a solvent-free extraction, exclusively based on osmotic shock, could be used to recover the bioplastic from cells.

Evaluation of different nutrient limitation strategies for the efficient production of poly(hydroxybutyrate‐co‐hydroxyvalerate) from waste frying oil and propionic acid in high cell density fermentations of Cupriavidus necator H16

Because of its application potential and biodegradability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate;PHBV), a member of the polyhydroxyalkanoates (PHA) biopolymer family, is one of the most extensively studied PHA. High PHBV productivity with a significant amount of hydroxyvalerate (HV) content is very appealing for commercial scale production. The goal of this study was to investigate the efficiency of various defined limitation strategies, namely nitrogen, phosphorus, and oxygen-limitation, for high yield PHBV production by Cupriavidus necator H16 with increased HV unit using waste frying vegetable oil (WFO) and propionic acid (PA) in a high cell density culture (5 L bioreactor). With optimized WFO and PA feeding, highest PHBV harvest (121.7 ± 2.59 g/L; HV 13.9 ± 0.44% (w/w)) and volumetric productivity (2.03 ± 0.04 gPHBV/L·h) were obtained in oxygen-limited operation, while highest HV content (19.8 ± 0.28 wt%) and yield coefficient (0.43 ± 0.017 gHV/gPA) were observed during phosphorus-limited cultivation. Although nitrogen limitation is widely applied in the production of PHA, nitrogen-limited cultivation had the lowest cell dry matter, PHBV production, volumetric productivity, oil-to-HB and PA-to-HV yield coefficients for the given conditions. The results of the present study demonstrate the highest PHBV yield together with the highest HV content using WFO as main carbon source and PA as the HV precursor ever reported in the literature.

Study on the production of high 3HV content PHBV via an open fermentation with waste silkworm excrement as the carbon source by the haloarchaeon Haloferax mediterranei.

Silkworm excrement is hard to be degraded or bio-utilized by environmental microorganisms due to its high content of heavy metals and antimicrobial biomacromolecules in mulberry leaves. In traditional Chinese silk industry, the silkworm excrement results in environmental problems. In this study, the silkworm excrement after chlorophyll ethanol-extraction was researched. An open fermentation strategy was developed using the silkworm excrement as the sole or partial carbon source by haloarchaea to accumulate polyhydroxyalkanoates. As a haloarchaeon with strong carbon source utilization ability, Haloferax mediterranei was found to accumulate a certain amount of poly(3-hydroxybutyrate-co-3-hydroxyvalerate; PHBV) using waste silkworm excrement. The results showed that the addition of silkworm excrement into glucose based fermentation medium can significantly improve the production of PHBV. Using a mixture carbon source including the extract of silkworm excrement and glucose (with a 1:1 carbon content ratio), the yield of PHBV was 1.73 ± 0.12 g/l, which showed a 26% increase than that of fermentation without the silkworm excrement addition. When the NaCl content of medium was set to approximately 15%, fermentation without sterilization was performed using silkworm excrement as the carbon source. Moreover, the addition of the silkworm excrement extract could increase the 3-hydroxyvalerate (3 HV) content of PHBV regardless of the sterilization or non-sterilization fermentation conditions. When using silkworm excrement as the sole carbon source, the 3 HV content was as high as 16.37 ± 0.54 mol %. The real-time quantitative PCR results showed that the addition of the silkworm excrement could specifically enhance the expression of genes involved in the aspartate/2-ketobutyric acid pathway related to 3 HV synthesis in H. mediterranei, and further analysis of the amino acid of the silkworm excrement suggested that the high content of threonine in the silkworm excrement might be the reason for the increase of 3 HV content. Taken together, the success of non-sterile fermentation in hypersaline condition using haloarchaea implied a novel way to reuse the silkworm excrement, which not only reduces the production costs of PHBV, but also is conducive to environmental protection.