Polyhydroxybutyrate Yield Research Articles

Production of Polyhydroxybutyrate (PHB) by Novel Bacillus megaterium SWU01 Isolated from Activated Sludge

Polyhydroxybutyrate (PHB) is a natural polymer accumulated in granules as intracellular energy storage in stress condition. The aim of this research was to isolate potential PHB producing bacteria from canned tuna wastewater activated sludge and cow dung. Five positive bacterial isolates were confi rmed the accumulation of PHB using lipophilic staining and TEM. To identify the bacterial species, the phylogenetic analyses inferred from 16S rDNA gene sequence demonstrated that the bacterial strains were closely related to Bacillus genera. The comparison of PHB production from bacterial isolates showed that SWU01 strain isolated from the activated sludge exhibited the highest yield of PHB production. The cultivation of SWU01 stain in optimization condition (modifi ed M9 medium supplemented with 2% sodium acetate and 0.5% yeast extract at pH 7 for 48 h) exhibited the yield of PHB up to 0.95 g/L or 7.5 compared with M9 medium supplemented with 2% glucose. Transmission electron micrograph showed high among of PHB in the granule of the bacterial cell. From all results, the bacterial strain SWU01, effi cient PHB producers, exhibited the potential for their utilization in commercial PHB production.

Random genome reduction coupled with polyhydroxybutyrate biosynthesis to facilitate its accumulation in Escherichia coli.

Genome reduction has been emerged as a powerful tool to construct ideal chassis for synthetic biology. Random genome reduction couple genomic deletion with growth and has the potential to construct optimum genome for a given environment. Recently, we developed a transposon-mediated random deletion (TMRD) method that allows the random and continuous reduction of Escherichia coli genome. Here, to prove its ability in constructing optimal cell factories, we coupled polyhydroxybutyrate (PHB) accumulation with random genome reduction and proceeded to reduce the E. coli genome. Five mutants showed high biomass and PHB yields were selected from 18 candidates after ten rounds of genome reduction. And eight or nine genomic fragments (totally 230.1–270.0 Kb) were deleted in their genomes, encompassing 4.95%–5.82% of the parental MG1655 genome. Most mutants displayed better growth, glucose utilization, protein expression, and significant increase of electroporation efficiency compared with MG1655. The PHB content and concentration enhanced up to 13.3%–37.2% and 60.2%–102.9% when batch fermentation was performed in M9-glucose medium using the five mutants. Particularly, in mutant H16, lacking 5.28% of its genome, the increase of biomass and PHB concentration were more than 50% and 100% compared with MG1655, respectively. This work expands the strategy for creating streamlined chassis to improve the production of high value-added products.

Phosphate-inducible poly-hydroxy butyrate production dynamics in CO2 supplemented upscaled cultivation of engineered Phaeodactylum tricornutum

Diatoms such as Phaeodactylum tricornutum are emerging as sustainable alternatives to traditional eukaryotic microbial cell factories. In order to facilitate a viable process for production of heterologous metabolites, a rational genetic design specifically tailored to metabolic requirements as well as optimised culture conditions are required. In this study we investigated the effect of constitutive and inducible expression of the heterologous poly-3-hydroxybutyrate (PHB) pathway in P. tricornutum using non-integrative episomes in 3 different configurations. Constitutive expression led to downregulation of at least one individual gene out of three (phaA, phaB and phaC) and was outperformed by inducible expression. To further asses and optimise the dynamics of PHB accumulation driven by the inducible alkaline phosphatase 1 promoter, we upscaled the production to lab-scale bioreactors and tested the effect of supplemented CO2 on biomass and PHB accumulation. While ambient CO2 cultivation resulted in a maximum PHB yield of 2.3% cell dry weight (CDW) on day 11, under elevated CO2 concentrations PHB yield peaked at 1.7% CDW on day 8, coincident with PHB titres at 27.9 mg L−1 that were approximately threefold higher than ambient CO2. With other more valuable bio-products in mind, these results highlight the importance of the genetic design as well as substrate availability to supply additional reduction equivalents to boost biomass accumulation and relieve potential enzymatic bottlenecks for improved product accumulation.

A Two-stage strategy for polyhydroxybutyrate (PHB) production by continuous Biohydrogen fermenter and sequencing batch reactor from food industry wastewater

The present investigation aimed to enhance the production of polyhydroxybutyrate (PHB). The volatile fatty acid (VFA) was obtained from food industry wastewater using a high-yield VFA extraction system. The experimentation was conducted in two stages: continuous production of VFA and batch production of PHB. During the VFA production, the optimal VFA yield was determined at different hydraulic retention times (HRT) and high chemical oxidation demand (COD). To find the highest PHB production yield, the level of glycerin, ammonia nitrogen, and substrate inoculation in the batch PHB production stage was recorded. At 35 °C and 150 rpm, the wastewater substrate was mixed with urea to achieve an ammonia nitrogen concentration of 2 g/L. The mixed microorganisms were employed to produce PHB. Results revealed that the volatile solids had a PHB yield of 40% (g PHB/g VSS), while the volatile solids (VSS) had 6,900 mg/L. The effluent from the acidification tank can effectively generate PHB to produce VFAs from waste sewage and food industry wastewater. Using various kinds of wastewaters to generate value-added products is an excellent approach to bioenergy generation.

Isolation of local strain of Bacillus sp. SM-11, producing PHB using different waste raw as substrate

Polyhydroxyalkalonates (PHAs) have numerous industrial and medical applications, is being used in various medical applications such as scaffold, suture, heart valve and drug delivery etc. Poly 3-hydroxy butyrate (PHB) is the most common and important family member of PHAs. The bacterial strains which are able to produce higher quantities of PHA using low-cost substrates are always in demand. In the present study, a local strain of Bacillus sp. SM11 isolated from soil was screened for ability to produce biopolymer i.e., PHB (polyhydroxybutyrate). Various factors which have been found to have an impact on PHB production by the selected capable bacterial isolates were optimized viz., organic waste source (soya extract, whey, molasses, corn extract, and distillery waste liquor), nitrogen source (peptone, beef extract, yeast extract, ammonium chloride, and ammonium sulphate), pH, and trace elements. An optimized PHB yield of 3.53g/L was obtained using whey as a source of carbon, added with 1% of yeast extract as a nitrogen source at pH 6.0 in presence of calcium as a trace element. The outcome of the present study indicates that isolate is among one of the high PHB producing microorganism, using whey as a carbon source.

Statistical optimization of medium components and growth conditions to enhance Polyhydroxybutyrate yield by Bacillus endophyticus MTCC 13038

Polyhydroxybutyrate (PHB) is a biodegradable polymer accumulated as carbon and energy reserve granules by several bacteria and is ideal substitute for petroleum-based polymers. The present study aimed to isolate PHB producing bacteria and optimize medium components and growth conditions to enhance PHB yield. Potent PHB accumulating bacterial strain was isolated from oil spilled soil and identified as Bacillus endophyticus based on its 16S rRNA analysis. Sudan Black B staining method was used to detect PHB granules and affirmed by using Fourier Transform Infrared Spectroscopy. Differential scanning calorimetry analysis was conducted for thermal analysis of extracted PHB polymer. Bacillus endophyticus showed highest PHB accumulation utilizing glucose as the carbon and potassium nitrate as the nitrogen source which were further optimized along with physical parameters like pH and temperature using Box and Behnken method. The highest biomass (7.49 g/L) and PHB (3.67 g/L) were observed at pH 7, temp. 37ºC, glucose 25 g/L and potassium nitrate 0.1 g/L. Bacillus endophyticus was found to accumulate up to 49% PHB under optimized conditions. These results indicate the potential application of Bacillus endophyticus at the industrial level for PHB production.

Production and characterization of polyhydroxybutyrate (PHB) BY Burkholderia cepacia BPT1213 using waste glycerol as carbon source

This study focused on the utilization of waste glycerol, a by-product of the biodiesel industry, used as a carbon source to grow Burkholderia cepacia BPT1213 for the production of PHB. B. cepacia BPT1213 was cultivated in minimal salt media (MSM) supplemented with waste glycerol at concentrations ranging from 2% to 9% (v/v). A gradual decrease was depicted by the cell dry weight (CDW) and PHB content with increasing waste glycerol content. B. cepacia BPT1213 is able to synthesize and accumulate up to 2.41 g/L and 66.4% dry biomass and PHB yield respectively when supplemented with 2% (v/v) waste glycerol. This resulted in a total PHB concentration of 1.6 g/L. The characterization of extracted PHB was carried out by Nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR) for structural composition evaluation. Both analyses showed expected signals for PHB as reported in other studies which depicted the methyl, methylene and methine groups for NMR analyses, and certain functional groups which were the hydroxyl group, carbonyl stretch and ester group for FTIR. This study demonstrated the effects of waste glycerol concentration on the end properties of PHB, which is an important aspect for its applications.

Polyhydroxybutyrate (PHB) production from crude glycerol by genetic engineering of Rhodotorula glutinis

Polyhydroxybutyrate (PHB) is a member of the Polyhydroxyalkanoates (PHAs) family, which is generally produced through microbial biosynthesis. The synthetic biology tools are applied to transform PHB genes (PhaA, PhaB and PhaC) into an oleaginous yeast (Rhodotorula glutinis) genome. Western blot analysis confirms the expression of PhaA, PhaB and PhaC in the candidate transformants. The strain of R. glutinis #100–29 was selected to be the best PHB production candidate according to the shaker results. As compared to glucose, crude glycerol was known to be a suitable carbon source for R. glutinis cultivation. The PHB concentration, yield and content obtained in a 5-L fermenter using 30 g/L of crude glycerol were 2.87 g/L, 95.53 mg/g and 62% respectively. To the best of our knowledge, these should be the highest numbers obtained in yeast for PHB production, which indicates that strain #100–29 could be a potential PHB producer from crude glycerol.

High-Solid Loading Enzymatic Hydrolysis of Waste Office Paper for poly-3-hydroxybutyrate Production Through Simultaneous Saccharification and Fermentation

Waste paper holds great potential as a substrate for the microbial production of bioplastic (Poly-3-hydroxybutyrate (PHB)). This study aimed to produce PHB by utilizing office paper as a substrate using Cupriavidus necator through batch and fed-batch simultaneous saccharification and fermentation (SSF) approach. For the batch experiment, different loadings of shredded office paper (3, 5 and 10%) with two different pretreatments H2O2 (OPH) and H2O2 and Triton X-100 (OPTH) were carried out. For the fed-batch experiment, paper loading started with 3% and two more additions were made at 36 and 84 h. Both experiments were conducted at 30 °C, 200 rpm and pH 7 using 55.5 FPU/g of cellulase and 37.5 CBU/g of β-glucosidase with a fixed amount of nitrogen source. High PHB yield was observed with OPH in all loadings, though the OPHT showed a better hydrolysis. Maximum PHB yield (4.27 g/L) was achieved with 10% OP on the sixth day of fermentation in batch SSF. Whereas, maximum PHB yield (4.19 g/L) was obtained within a shorter time (66 h)with OPH in the fed-batch experiment. The extracted PHB showed well-matched characteristic features to the standard PHB. Finally, this study proves the feasibility of employing the SSF process for PHB production using waste paper as an alternative approach to overcome the shortcoming of the separate hydrolysis and fermentation (SHF) process.

Improved polyhydroxybutyrate production by Cupriavidus necator and the photocatalyst graphitic carbon nitride from fructose under low light intensity

The photocatalyst graphitic carbon nitride (g-C3N4) is known to photostimulate the production of the bioplastic polyhydroxybutyrate (PHB) by Cupriavidus necator. In previous studies, the combination of C. necator and g-C3N4 increased PHB yield from either an organic or inorganic carbon substrate under a light intensity of 4200 lx. Here, different parameters including light intensity, pH, temperature, nitrogen and carbon concentrations, aeration, and inoculum size were explored to maximize PHB production by hybrid photosynthesis from fructose and visible light. A g-C3N4/C. necator culture grown with a lower light intensity of 2100 lx, an inoculum size of 128.30 × 106 CFU ml−1, and constant aeration produced 7.16 g l−1 d−1 PHB with a product yield from fructose of 60.94%. Furthermore, the ratio of incident photons harvested by g-C3N4 converted into NADPH+H+ by C. necator for PHB production was improved to 19.74% after the process optimization. In comparison, the PHB production rate of a non-optimized g-C3N4/C. necator system exposed to 4200 lx was only 2.94 g l−1 d−1 with a product yield from fructose of 33.29%. These results demonstrate that hybrid photosynthesis productivity can be significantly augmented by decreasing light intensity and adjusting other parameters, which is promising for future bioproduction applications.

PHB into PHB: Recycling of polyhydroxybutyrate by a tandem “thermolytic distillation-microbial fermentation” process

The recycling of polyhydroxybutyrate (PHB) via depolymerization-polymerization is here proposed and assessed for the first time. Effective depolymerization of PHB to crotonic acid through a thermolytic distillation at mild conditions (170 °C and 150 mbar), and without the need of any catalyst, gives a crotonic acid-enriched condensate (94% yield and 98% selectivity towards trans-crotonic acid) that was used without any further purification as a substrate for producing renewed PHB with a culture of Cupriavidus necator. The yield of PHB (0.6 g PHB per g of crotonic acid), final PHB content (63%), and PHB-molecular weight (1.5 MDa) confirmed the technical feasibility of this tandem approach, with a 55% overall yield. A preliminary evaluation of the energy consumption of various depolymerization approaches indicated that the thermolytic distillation consumes 20–25% less energy than previously reported methods; the energy requirement for producing crotonic acid from PHBwaste with 50% moisture has a still acceptable energy demand (4 MJ/kgcrotonic acid), comparable to the energy required for obtaining fermentable sugars (4.4 MJ/kgsugar) to be used for feeding bacteria that accumulate PHB.

Harnessing molasses as a low-cost carbon source for production of poly-hydroxy butyrate (PHB) using Burkholderia sp. B73 bacteria

Burkholderia sp. has been reported as a poly-hydroxy-butyrate (PHB) producer. PHB is a natural polyester class with a wide range of applications in foods, medicines, and biomedicines. However, the high production cost of PHB may limit its potential. Molasses, a by-product of the sugarcane industry available abundantly, may be used as an alternative carbon source of PHB production. In this research, we aimed to evaluate PHB production by Burkholderia sp. B73 in fermentation media using molasses as an alternative carbon source. Small-scale experiments were performed in Erlenmeyer flasks on a shaker at 150 rpm and 30 °C to evaluate the best initial C/N ratio for biomass accumulation and PHB production. A set of parameters including bacterial growth, dry cell weight, yield, and FTIR spectrum of PHB were observed. The results showed that molasses could be used to grow Burkholderia sp. B73 and the highest PHB production was obtained when a 20:1 C/N ratio of molasses was applied in the fermentation medium. In addition, when the initial pH was adjusted to 7.0, the highest PHB yield was also produced. More importantly, the use of molasses as a carbon source improved the PHB yield by nearly 2-fold compared with our previous report using a synthetic Ramsay’s minimal medium. In conclusion, the experiment results showed that molasses could be used as a low-cost carbon source for PHB production by Burkholderia sp. B73 bacteria.

Enhanced polyhydroxybutyrate production from acid whey through determination of process and metabolic limiting factors

To sustainably produce biodegradable polyhydroxybutyrate (PHB), this study investigated effects of process and metabolic limiting factors during bioconversion of acid whey (AW) to PHB, offering economic and environmental advantages for dairy industry. Recombinant Escherichia coli LSBJ was used to achieve high PHB yields by utilizing both lactose and lactic acid as carbon source. Up to 85% PHB accumulation was achieved during growth on the synthetic AW. Growth on raw AW had the highest PHB yield of 4 g/L and a high substrate utilization efficiency (95%). Notably, ratios of lactate: lactose and C/N impacted metabolic flux and PHB yields. Maintaining the fermentation pH enhanced PHB production. Furthermore, additives of inorganic nitrogen sources, minerals and trace metals promoted PHB production from AW. The study improves the understanding of factors affecting utilization of AW and demonstrated the high PHB yields using recombinant E. coli that could be leveraged to design a sustainable process.

Bacillus cereus saba.zh, a novel bacterial strain for the production of bioplastic (polyhydroxybutyrate).

The identification of novel bacterial strains with a high production potential of polyhydroxybutyrate (PHB) to substitute the bioplastics with non-biodegradable plastics and reducing environmental pollution is really effective. The present study was done with the purpose of PHB bioplastic production using a novel bacterial strain. Twenty-one different bacterial isolates were obtained from petrochemical wastewater, which among them, 10 isolates were PHB positive. The presence of PHB granules was detected in the isolates using Sudan Black B staining. The most excellent PHB-accumulating bacterium with a maximum yield of PHB (61.53%) was selected and identified as Bacillus cereus saba.zh, based on morphological, biochemical, and molecular techniques. 16S rRNA nucleotide sequence of the bacterium was assigned accession number: MT975245 in the NCBI database. The phylogenetic tree data showed that the closest type strain to the Bacillus cereus saba.zh is the Bacillus cereus SDB4 (91%). The three genes (phaA, phaB, and phaC) responsible for the PHB biosynthesis were amplified using the specific oligonucleotide primers by PCR technique. The highest PHB yield was achieved when the culture medium was supplemented with 3% sugarcane molasses as a carbon source, ammonium sulfate as the nitrogen source, at pH 7, and temperature of 30°C. The characterization of the extracted polymer by FTIR and 1H NMR spectroscopy proves the presence of methyl, methylene, methine, hydroxyl, and ester carbonyl groups and confirmed the structure of biopolymer as PHB. The novel strain Bacillus cereus saba.zh has good potential as an appropriate candidate for low-cost industrial production of bioplastic.

Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

Polyhydroxybutyrate (PHB) is a sustainable bioplastic produced by bacteria that is a potential replacement for conventional plastics. This study delivers an integrated experimental and computational modeling approach to decipher metabolic factors controlling PHB production and offers engineering design strategies to boost production. In the metabolically robust Rhodopseudomonas palustris CGA009, PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (C9H8O3) compared to virtually no PHB titer from acetate (C2H3NaO2). The maximum yield did not improve further when grown on coniferyl alcohol (C10H12O3), but comparison of the PHB profiles showed that coniferyl alcohol's higher carbon content resulted in a higher rate of PHB production. Combined experimental results revealed that cytoplasmic space may be a limiting factor for maximum PHB titer. In order to obtain a systems-level understanding of factors driving PHB yield, a model-driven investigation was performed. The model yielded several engineering design strategies including utilizing reduced, high molecular weight substrates that bypass the thiolase reaction (phaA). Based on these strategies, utilization of butyrate was predicted and subsequently validated to produce PHB. Model analysis also explained why nitrogen starvation was not essential for PHB production and revealed that renewable and abundant lignin aromatics are ideal candidates for PHB production. Most importantly, the generality of the derived design rules allows them to be applied to any PHB-producing microbe with similar metabolic features.

Simultaneous biosynthesis of bacterial polyhydroxybutyrate (PHB) and extracellular polymeric substances (EPS): Process optimization and Scale-up

Owing to their biodegradability and renewability, biopolymers are being employed in industrial and bio-medical sectors as sustainable alternatives to chemical based polymers. In the present study, isolated Providencia sp. depicted dual production of intra and extracellular biopolymers, polyhydroxybutyrate (PHB) and extracellular polymeric substances (EPS), respectively. The polymer production process was optimised by varying process parameters such as carbon load (20, 30 and 40 g L-1) and pH (6, 7 and 8) for enhancing PHB and EPS productivity. Maximum yield of both PHB (2.62 g L-1) and EPS (3.92 g L-1) was observed with carbon load of 30 g L-1 at pH 7. Scale-up studies were performed with optimized conditions and PHB and EPS production of 2.62 g L-1 and 3.91 g L-1, respectively was observed. The extracted EPS and PHB were characterized using FT-IR, FE-SEM-EDX, H1 and C13 NMR and fluorescence microscopy.

Effects of different sodium salts and nitrogen sources on the production of 3-hydroxybutyrate and polyhydroxybutyrate by Burkholderia cepacia

The effects of NaCl, Na2SO4, Na2HPO4, and Na3C6H5O7 on the production of 3-hydroxybutyrate, polyhydroxybutyrate, and by-products by Burkholderia cepacia. Proper addition of Na3C6H5O7 can significantly promote the production of 3-hydroxybutyric acid and polyhydroxybutyrate. The concentration, productivity, and yield of 3-hydroxybutyrate were increased by 48.2%, 55.6%, and 48.3% at 16 mM Na3C6H5O7. The increases of 80.1%, 47.1%, and 80.0% in the concentration, productivity, and yield of polyhydroxybutyrate were observed at 12 mM Na3C6H5O7. Na2SO4 and Na2HPO4 also have positive effects on the production capacity of 3-hydroxybutyrate and polyhydroxybutyrate within a certain range of concentration. NaCl is not conducive to the improvement of fermentation efficiency. Compared with a single nitrogen source, a mixed nitrogen source is more conducive to enhancing the production of 3-hydroxybutyrate and polyhydroxybutyrate.

Co-production of polyhydroxybutyrate (PHB) and coenzyme Q10 (CoQ10) via no-sugar fermentation\u2014a case by Methylobacterium sp. XJLW

PurposeTo explore a competitive PHB-producing fermentation process, this study evaluated the potential for Methylobacterium sp. XJLW to produce simultaneously PHB and coenzyme Q10 (CoQ10) using methanol as sole carbon and energy source.MethodsThe metabolic pathways of PHB and CoQ10 biosynthesis in Methylobacterium sp. XJLW were first mined based on the genomic and comparative transcriptomics information. Then, real-time fluorescence quantitative PCR (RT-qPCR) was employed for comparing the expression level of important genes involved in PHB and CoQ10 synthesis pathways’ response to methanol and glucose. Transmission electron microscope (TEM), gas chromatography/mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), Fourier transformation infrared spectrum (FT-IR), and liquid chromatography/mass spectrometry (LC-MS) methods were used to elucidate the yield and structure of PHB and CoQ10, respectively. PHB and CoQ10 productivity of Methylobacterium sp. XJLW were evaluated in Erlenmeyer flask for medium optimization, and in a 5-L bioreactor for methanol fed-batch strategy according to dissolved oxygen (DO) and pH control.ResultsComparative genomics analysis showed that the PHB and CoQ10 biosynthesis pathways co-exist in Methylobacterium sp. XJLW. Transcriptomics analysis showed that the transcription level of key genes in both pathways responding to methanol was significantly higher than that responding to glucose. Correspondingly, strain Methylobacterium sp. XJLW can produce PHB and CoQ10 simultaneously with higher yield using cheap and abundant methanol than using glucose as sole carbon and energy source. The isolated products showed the structure characteristics same to that of standard PHB and CoQ10. The optimal medium and cultural conditions for PHB and CoQ10 co-production by Methylobacterium sp. XJLW was in M3 medium containing 7.918 g L-1 methanol, 0.5 g L-1 of ammonium sulfate, 0.1% (v/v) of Tween 80, and 1.0 g L-1 of sodium chloride, under 30 °C and pH 7.0. In a 5-L bioreactor coupled with methanol fed-batch process, a maximum DCW value (46.31 g L-1) with the highest yields of PHB and CoQ10, reaching 6.94 g L-1 and 22.28 mg L-1, respectively.ConclusionMethylobacterium sp. XJLW is potential for efficiently co-producing PHB and CoQ10 employing methanol as sole carbon and energy source. However, it is still necessary to further optimize fermentation process, and genetically modify strain pathway, for enhanced production of PHB and CoQ10 simultaneously by Methylobacterium sp. XJLW. It also suggests a potential strategy to develop efficiently co-producing other high-value metabolites using methanol-based bioprocess.

Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB)

BackgroundPlastic plays a crucial role in everyday life of human living, nevertheless it represents an undeniable source of land and water pollution. Polyhydroxybutyrate (PHB) is a bio-based and biodegradable polyester, which can be naturally produced by microorganisms capable of converting and accumulating carbon as intracellular granules. Hence, PHB-producing strains stand out as an alternative source to fossil-derived counterparts. However, the extraction strategy affects the recovery efficiency and the quality of PHB. In this study, PHB was produced by a genetically modified Escherichia coli strain and successively extracted using dimethyl carbonate (DMC) and ethanol as alternative solvent and polishing agent to chloroform and hexane. Eventually, a Life Cycle Assessment (LCA) study was performed for evaluating the environmental and health impact of using DMC.ResultsExtraction yield and purity of PHB obtained via DMC, were quantified, and compared with those obtained via chloroform-based extraction. PHB yield values from DMC-based extraction were similar to or higher than those achieved by using chloroform (≥ 67%). To optimize the performance of extraction via DMC, different experimental conditions were tested, varying the biomass state (dry or wet) and the mixing time, in presence or in absence of a paper filter. Among 60, 90, 120 min, the mid-value allowed to achieve high extraction yield, both for dry and wet biomass. Physical and molecular dependence on the biomass state and solvent/antisolvent choice was established. The comparative LCA analysis promoted the application of DMC/ethanol rather than chloroform/hexane, as the best choice in terms of health prevention. However, an elevated impact score was achieved by DMC in the environmental-like categories in contrast with a minor contribution by its counterpart.ConclusionThe multifaceted exploration of DMC-based PHB extraction herein reported extends the knowledge of the variables affecting PHB purification process. This work offers novel and valuable insights into PHB extraction process, including environmental aspects not discussed so far. The findings of our research question the DMC as a green solvent, though also the choice of the antisolvent can influence the impact on the examined categories.

Impact of exoD gene knockout on the polyhydroxybutyrate overaccumulating mutant Mt_a24

ABSTRACT New possibilities for sustainable and climate-friendly plastic production are urgently needed. One degradable alternative could be polyhydroxybutyrate naturally produced out of CO2 via photosynthesis within cyanobacteria. Therefore, the polyhydroxybutyrate production of the so far little investigated polyhydroxybutyrate overproducing Synecho-cystis sp. PCC6714 mutant Mt_a24 should be further increased. The impact of reducing the exopolysaccharide production on the polyhydroxybutyrate yield was examined by knocking the exoD gene out. The generated transformants KOexoD#4 and KOexoD#65 were compared to the control strain under standard and nutrient limiting cultivation conditions regarding their growth, glycogen, polyhydroxybutyrate and exopolysaccharide production. Whereas the growth of all three strains was comparable under standard conditions, only minor differences could be detected under limited conditions. An increase of polyhydroxybutyrate could be observed in both transformants (KOexoD#4: 1.17-fold, KOexoD#65: 1.26-fold) under limited conditions. The exopolysaccharide production was increased in both transformants under standard cultivation conditions, showing a strong increase of soluble exopolysaccharides. In contrast, during limitation, the soluble exopolysaccharides decreased in all cultures whereas the tightly bound exopolysaccharides fraction strongly increased. Hence, the influence of limiting medium, and connection to the energy storage pathways could be shown as well as the great potential of Mt_a24 as a sustainable production system for polyhydroxybutyrate.