PHB Accumulation Research Articles

Poly(3-hydroxybutyrate) production for food packaging from biomass derived carbohydrates by cupriavidus necator DSM 545

The extensive utilization of conventional plastics has resulted in a concerning surge in waste. A potential solution lies in biodegradable polymers mostly derived from renewable sources. Cupriavidus necator DSM 545 is a microorganism capable, under stress conditions, of intracellularly accumulating Poly(3-hydroxybutyrate) (PHB), a bio-polyester. This study aimed to identify optimal conditions to maximize the intracellular accumulation of PHB and its global production using natural media obtained by processing lignocellulosic residues of cardoon, a low-cost feedstock. An intracellular PHB accumulation was observed in all of the tested media, indicating a metabolic stress induced by the lack of macronutrients. Increasing C/N ratios led to a significant decrease in cellular biomass and PHB production. Furthermore C. necator DSM 545 was incapable of consuming more than 25 g/L of supplied monosaccharides. Surprisingly, in the samples supplied with 60 % of the pentose-rich liquid fraction, complete consumption of xylose was observed. This result was also confirmed by subsequent tests using Medium 1 growth media containing xylose as the sole carbon source. Using a diluted medium with a C/N ratio of 5, a PHB production of 5.84 g/L and intracellular PHB accumulation of 77 % w/w were respectively achieved. Finally, comparative shelf-life tests conducted against conventional pre-packaging materials in PP suggested that PHB films performed similarly in preserve ready-to-eat products.

Two-step polyhydroxybutyrate production from hydrogenic effluent by freshwater microalgae Coelastrella sp. KKU-P1 and Acutodesmus sp. KKU-P2 under mixotrophic cultivation

This study aimed to produce PHB using hydrogenic effluent discharged from the biohydrogen production process with freshwater microalgae including Coelastrella sp. KKU-P1, and Acutodesmus sp. KKU-P2. Batch experiments explored the influence of initial pH and hydrogenic effluent concentration, revealing optimal conditions at 10 % (v/v) effluent concentration and a pH of 6.5 for both KKU-P1 and KKU-P2. Subsequently, medium formulation and photoperiods were optimized to maximize biomass and PHB accumulation. The results showed that the optimal condition for PHB accumulation with KKU-P1 and KKU-P2 was nitrogen phosphorus (NP)-limited Bold's Basal Medium (BBM) under dark conditions. A two-step PHB accumulation in the upscale bioreactor was investigated under optimal conditions. The results showed that KKU-P1 achieved maximum PHB, protein, carbohydrate, and lipid contents of 4.57 %, 29.37 %, 24.76 %, and 13.21 %, respectively, whereas KKU-P2 achieved 6.35 %, 31.53 %, 16.16 %, and 4.77 %, respectively. Based on these findings, it appears that a mixotrophic approach under nutrient-limiting conditions is effective for PHB production in both KKU-P1 and KKU-P2 strains.

Methylosinus trichosporium OB3b drives composition-independent application of biogas in poly(3-hydroxybutyrate) synthesis

Biological conversion of biogas into poly(3-hydroxybutyrate) (PHB) via Type II methanotrophs holds great promise. However, a prerequisite for widespread biogas utilization is a comprehensive understanding of the effects of biogas composition on methanotrophic PHB synthesis. Herein, we explore the effects of biogas composition on the microbial growth and PHB accumulation of Methylosinus trichosporium OB3b under five different artificial biogas conditions with varying the ratio of methane (CH4) to carbon dioxide (CO2), the two main components of raw biogas. Stoichiometric and kinetic analyses revealed that increased CO2 concentrations correlated with higher maximum specific growth rates (0.021–0.029 h−1) and maximum CH4 utilization rates (0.029–0.042 mg CH4/mg TSS-h). Besides, no significant correlation was found between the CH4-to-CO2 ratio and other growth parameters. Consistent PHB contents (20.2–25.3 wt%) were achieved, irrespective of the CH4-to-CO2 ratio. Furthermore, the ability of M. trichosporium OB3b to grow and synthesize PHB (13.2 wt%) was confirmed using pilot-scale biogas. Our findings highlight the potential of biogas as a feedstock for producing valuable biodegradable polymers, thus contributing to waste-derived fuel (WDF) innovation and environmental sustainability.

Valorization of Dextrose from Cassava Starch and Sugarcane Vinasse as Polyhydroxyalkanoates by Submerged Cultures of Cupriavidus necator: A Physicochemical–Biotechnological Approach

The production of polyhydroxyalkanoates using submerged cultures of Cupriavidus necator DSM 428 was evaluated using low-cost substrates from agroindustry: (i) dextrose from cassava starch and (ii) a mixture of sugarcane vinasse from the bioethanol industry and dextrose from cassava starch. The effects of vinasse composition (2.5, 5.0, 7.5, 25, 50, and 75% v/v) and the use of raw and activated carbon-pre-treated vinasse were assessed. The results indicate that cultivations using only cassava starch dextrose reached 4.33 g/L of biomass as the dry cell weight and a poly(3-hydroxybutyrate) (PHB) production of 47.1%. Raw vinasse proportions of 25, 50, and 75% in the culture medium resulted in total inhibition. Vinasse treated at the same ratios led to biomass production in the range 1.7–4.44 g/L. The higher PHB production scenario was obtained in a medium containing dextrose and treated vinasse (7.5%), yielding 5.9 g/L of biomass and 51% of PHB accumulation. The produced PHB was characterized by XRD and FTIR for an analysis of crystalline structure and chemical functional groups, respectively. EDS was employed for a semi-quantitative analysis of the chemical composition, and SEM was used to analyze the morphology of the microgranules. The results of DSC and TGA analyses demonstrated the thermal stability of the obtained PHB.

A newly isolated strain for poly(3-hydroxybutyrate) production under anaerobic conditions and the key enzyme analysis

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with great possibility to replace petroleum-based plastics, but the high costs of production associated with energy inputs have been an obstacle to their commercial application. The production of PHAs under anaerobic environments without aeration appears to be more attractive but has not been adequately investigated. A strain capable of accumulating poly(3-hydroxybutyrate) (PHB) under anaerobic conditions was isolated. The strain anaerobically accumulated PHB reached 38.7 % of cell dry weight (CDW), which was 1.9 times higher than under aerobic conditions. It was also found that the concentrations of key enzymes for PHB synthesis were higher under anaerobic conditions than under aerobic conditions. The highest PHB accumulation was 77.9 % obtained at oxygen-limited conditions and the highest PHB yield was 0.13 g PHB/g carbon obtained at anaerobic conditions. It suggests that the conditions should be well controlled according to the purpose of PHB production. This study offers a new alternative of PHAs formation.

A Comparison of the Effects of Continuous Illumination and Day/Night Regimes on PHB Accumulation in Synechocystis Cells.

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable polymer with properties comparable to polypropylene and therefore has the potential to replace conventional plastics. PHB is intracellularly accumulated by prokaryotic organisms. For the cells PHB functions manly as carbon and energy source, but all possible functions of PHB are still not known. Synechocystis (cyanobacteria) accumulates PHB using light as energy and CO2 as carbon source. The main trigger for PHB accumulation in cyanobacteria is nitrogen and phosphorous depletion with simultaneous surplus of carbon and energy. For the above reasons, obtaining knowledge about external factors influencing PHB accumulation is of highest interest. This study compares the effect of continuous light exposure and day/night (16/8 h) cycles on selected physiology parameters of three Synechocystis strains. We show that continuous illumination at moderate light intensities leads to an increased PHB accumulation in Synechocystis salina CCALA 192 (max. 14.2% CDW - cell dry weight) compared to day/night cycles (3.7% CDW). In addition to PHB content, glycogen and cell size increased, while cell density and cell viability decreased. The results offer new approaches for further studies to gain deeper insights into the role of PHB in cyanobacteria to obtain bioplastics in a more sustainable and environmentally friendly way.

Impact of the environmental parameters on single cell protein production and composition by Cupriavidus necator

Due to the rapid increase in the world's population, many developing countries are facing malnutrition problems, including famine and food insecurity. Particularly, the deficiency of protein sources becomes a serious problem for human and animal nutrition. In this context, Single Cell Proteins, could be exploited as an alternative source of unconventional proteins. The aim of the study was to investigate SCP production and composition by Cupriavidus necator under various environmental conditions, temperature and pH values. A mono-factorial approach was implemented using batch bioreactor cultures under well-controlled conditions. Results were compared in terms of bacterial growth and SCP composition (proteins, nucleic acids, amino acids and elemental formula). Complementary analyses were performed by flow cytometry to study cell morphology, membrane permeability and the presence of Poly(3-hydroxybutyrate) (PHB) production. Our data confirmed the ability of C. necator to produce high amount of proteins (69 %DW at 30 °C and pH7). The results showed that temperature and pH independently impact SCP production and composition. This impact was particularly observed at the highest temperature (40 °C) and also the lowest pH value (pH5) providing lower growth rates, cell elongation, changes in granularity and lower amounts of proteins (down to 44 %DW at pH5) and nucleic acids. These low percentages were related to the production of PHB production (up to 44 %DW at 40 °C) which is the first report of a PHB accumulation in C. necator under nutrient unlimited conditions.

The fundamental role of pH in CH4 bioconversion into polyhydroxybutyrate in mixed methanotrophic cultures

Climate change and plastic pollution are likely the most relevant challenges for the environment in the 21st century. Developing cost-effective technologies for the bioconversion of methane (CH4) into polyhydroxyalkanoates (PHAs) could simultaneously mitigate CH4 emissions and boost the commercialization of biodegradable polymers. Despite the fact that the role of temperature, nitrogen deprivation, CH4:O2 ratio or micronutrients availability on the PHA accumulation capacity of methanotrophs has been carefully explored, there is still a need for optimization of the CH4-to-PHA bioconversion process prior to becoming a feasible platform in future biorefineries. In this study, the influence of different cultivation broth pH values (5.5, 7, 8.5 and 10) on bacterial biomass growth, CH4 bioconversion rate, PHA accumulation capacity and bacterial community structure was investigated in a stirred tank bioreactor under nitrogen deprivation conditions. Higher CH4 elimination rates were obtained at increasing pH, with a maximum value of 50.4 ± 2.7 g CH4·m−3·h−1 observed at pH 8.5. This was likely mediated by an increased ionic strength in the mineral medium, which enhanced the gas-liquid mass transfer. Interestingly, higher PHB accumulations were observed at decreasing pH, with the highest PHB contents recorded at a pH 5.5 (43.7 ± 3.4 %w·w−1). The strong selective pressure of low pH towards the growth of Type II methanotrophic bacteria could explain this finding. The genus Methylocystis increased its abundance from 34 % up to 85 and 90 % at pH 5.5 and 7, respectively. On the contrary, Methylocystis was less abundant in the community enriched at pH 8.5 (14 %). The accumulation of intracellular PHB as energy and carbon storage material allowed the maintenance of high CH4 biodegradation rates during 48 h after complete nitrogen deprivation. The results here obtained demonstrated for the first time a crucial and multifactorial role of pH on the bioconversion performance of CH4 into PHA.

Integrated biorefinery strategy for poly(3-hydroxybutyrate) accumulation in Cupriavidus necator DSM 545 using a sugar rich syrup from cereal waste and acetate from gas fermentation

Poly(3-hydroxybutyrate) (PHB) is one of the most well-known biodegradable and biocompatible biopolymers produced by prokaryotic microorganisms. It belongs to the family of polyhydroxyalkanoates (PHAs), and it has gained significant attention in recent years due to its potential as a sustainable alternative to traditional petroleum-based plastics. Cupriavidus necator has been identified as a potential producer of PHB for industrial applications due to its ability to produce high amounts of the polymer under controlled conditions, using a wide range of waste substrates. In this study, the ability of Cupriavidus necator DSM 545 strain to produce PHB was tested in a fed-batch strategy providing two different organic substrates. The first is a sugar-based syrup (SBS), derived from cereal waste. The second is an acetate-rich medium obtained through CO2 -H2 fermentation by the acetogenic bacterium Acetobacterium woodii. The carbon sources were tested to improve the accumulation of PHB in the strain. C. necator DSM 545 proved to be able to grow and to perform high accumulation of biopolymer on waste substrates containing glucose, fructose, and acetate, reaching about 10 g/L of PHB, 83% of biopolymer accumulation in cell dry mass, in 48 h of fed-batch fermentation in 0.6 L working volume in a bioreactor. Moreover, a Life Cycle Assessment analysis was performed to evaluate the environmental impact of the process converting the sugar syrup alone and the integrated one. It demonstrated that the integrated process is more sustainable and that the most impactful step is the PHB production, followed by the polymer extraction.

Production of poly (3-hydroxybutyrate) and extracellular polymeric substances from glycerol by the acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic, and metallotolerant bacteria able to use dissolved oxygen or Fe(III) as an electron sink. The ability of this extremophile to accumulate poly(3-hydroxybutyrate) (PHB) and secrete extracellular polymeric substances (EPS) has also been reported. Hence, the aim of this work is to characterize the production of PHB and EPS by the wild strain DSM2389 using glycerol in shaken flasks and bioreactor. Results showed that maximum PHB accumulation (37-42% w/w) was obtained using glycerol concentrations of 9 and 15gL-1, where maximum dry cell weight titers reached 3.6 and 3.9gL-1, respectively. The culture in the bioreactor showed that PHB accumulation takes place under oxygen limitation, while the redox potential of the culture medium could be used for online monitoring of the PHB production. Recovered EPS was analyzed by Fourier-transform infrared spectroscopy and subjected to gas chromatography-mass spectrometry after cleavage and derivatization steps. These analyses showed the presence of sugars which were identified as mannose, rhamnose and glucose, in a proportion near to 3.2:2.3:1, respectively. Since glycerol had not been used in previous works, these findings suggest the potential of A. cryptum to produce biopolymers from this compound at a large scale with a low risk of microbial contamination due to the low pH of the fermentation process.

Assessing the performance of synthetic co-cultures during the conversion of methane into Poly(3-hydroxybutyrate)

Synthetic co-cultures can enhance pollutant bioconversion performance through synergistic effects among co-existing species. In this study, the potential of Methylocystis hirsuta and Methylocystis parvus to support poly(3-hydroxybutyrate) (PHB) production in co-cultivation with Rhodococcus opacus and Pseudomonas putida under a CH4:O2 atmosphere was assessed batchwise. The metabolic activation of almost all co-cultures studied was faster than that of single strain cultures, bringing higher methane and oxygen consumption rates. Higher PHB yields were achieved when coupling M. hirsuta with R. opacus (63 % w w−1) or with R. opacus and P. putida (64.4 % w w−1) compared to M. hirsuta alone (38.5 % w w−1). Interestingly, the combination of both R. opacus and P. putida with M. parvus reduced PHB accumulation to 42.2 % w w−1 compared to the content observed in M. parvus monocultures (62.2 % w w−1) and M parvus + R. opacus co-cultures (66.6 % w w−1).

Enrichment strategies for mixed cultures in valorisation of crude glycerol into polyhydroxyalkanoate bioplastics

This study investigated two different strategies for enrichment using crude glycerol and mixed culture. Coupled feeding allowed simultaneous supplies of glycerol and nutrients at start of feast, whereas uncoupled feeding postponed nutrient supply until start of famine. Adaptation of culture enriched by the uncoupled feeding strategy was faster with higher operational stability. This however resulted in low PHB content of 20% and yield of 0.137 mg PHB/mg glycerol in feast phase, cell growth at yield of 0.298 mgX/mg PHB in famine. With more abundance (29.2%) of PHA storing bacteria in culture enriched by the coupled feeding strategy as revealed from 16 S rRNA analysis, results showed that polymer storage was dominated in feast phase (max. PHB content of 68%, yield of 0.563 mg PHB/mg glycerol), leading to high cell growth yield of 0.745 mg X/mg PHB. PHB accumulation by the enriched culture from coupled feeding has achieved 82% PHB content with 1094 mg PHB/L.h volumetric productivity. Thus, the coupled feeding was more effective to enrich PHA storing culture for PHA production using crude glycerol via two-step process.

Erythromycin treatment under a specific nitrogen supply affects carbon metabolism and increases poly(3-hydroxybutyrate) and glycogen accumulation in cyanobacterium Synechocystis sp. PCC 6803

Under an adequate nitrate supply (BG11), cyanobacterium Synechocystis sp. PCC 6803 has high protein content but a low storage of glycogen and poly(3-hydroxybutyrate) (PHB). In contrast, when deprived of nitrogen (N), protein synthesis was repressed and the accumulation of glycogen and PHB was remarkably increased. The repressed protein synthesis caused by sulfur deprivation or treatment with chloramphenicol was also found to slightly enhance PHB accumulation in Synechocystis. Thus, the repressed protein synthesis is associated with the increased PHB and glycogen storage. Here, we examined two other antibiotics that inhibit protein synthesis, erythromycin (EM) and streptomycin (SM) for their effect on the accumulation of PHB and glycogen in Synechocystis. Under N cultivation, 10 μM EM treatment significantly increased the PHB level to 18 % (w/w cell dry weight [DW]) compared to 3 % (w/w DW) in the untreated cells; however, SM treatment did not increase the PHB level. Under BG11 cultivation, exposure to 0.7 μM SM and 5 μM EM enhanced glycogen accumulation up to 15 % and 41 % (w/w DW) respectively, compared to only 7 % (w/w DW) in the untreated cells. The EM treatment also remarkably reduced the total protein and phycobiliprotein contents. Results from metabolomic analysis suggested that EM treatment caused protein degradation into amino acids, some of which were metabolized into substrates that can be used for PHB and glycogen synthesis. This was substantiated by the result showing that EM treated cells had significantly increased levels of both the PHB-synthesizing substrates (acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA) when cultured under N, and the glycogen-synthesizing substrates (fructose-1,6-BP, fructose-6-P, glucose-6-P, glucose-1-P, and ADP-glucose) when cultured in BG11. Therefore, EM treatment might be applicable for enhancing the accumulation of these two biopolymers in other cyanobacteria.

Fermentation development using fruit waste derived mixed sugars for poly(3-hydroxybutyrate) production and property evaluation

Free sugars from fruit wastes were evaluated for the production of poly(3-hydroxybutyrate) (PHB) in Paraburkholderia sacchari fed-batch bioreactor fermentations. Different initial sugar concentration, carbon to inorganic phosphorus (C/IP) ratio, IP addition during feeding and volumetric oxygen transfer coefficient (kLa) were evaluated to promote PHB production. The highest intracellular PHB accumulation (66.6%), PHB concentration (108.3 g/L), productivity (3.28 g/(L·h)) and yield (0.33 g/g) were achieved at 40 g/L initial sugars, C/IP 26.5, 202.6 h−1kLa value and 20% IP supplementation in the feeding solution. The effect of different microbial mass harvesting time on PHB properties showed no influence in weight average molecular weight and thermal properties. The harvest time influenced the tensile strength that was reduced from 28.7 MPa at 22 h to 13.3 MPa at 36 h. The elongation at break and Young’s modulus were in the range 3.6–14.8% and 830–2000 MPa, respectively.

Optimization-based control strategy for a large-scale polyhydroxyalkanoates production in a fed-batch bioreactor using a coupled PDE–ODE system

Abstract Control strategy development for fed-batch bioreactor (FBBR) plays an important role in the improvement of polyhydroxyalkanoate (PHA) production. To develop a feeding strategy for PHA production in a large-scale FBBR, an optimization-based control scheme that considers nutrient dispersion is proposed in this work. A coupled partial differential equations and ordinary differential equation model is proposed to describe the axial-dispersed nutrient and well-dispersed microbial dynamics with process constraints. An analytical model predictive control (AMPC) method that applies integrated variables of nutrients is employed to develop the real-time control system. The control objective is to regulate the PHA concentration at the updated set points by adjusting the nutrient feed rates; a process disturbance is introduced to evaluate the control robustness. Simulation experiments of a fed-batch operation are conducted to investigate the performance of the developed controller; the controlled output is designed to track the updated set points corresponding to the biomass concentration. Results of closed-loop and regulatory systems showed that the proposed control strategy could provide more productivity (33–38%) compared to the applied PI controller. The performance test demonstrates that the developed control system could apply the biomass concentration for updating set points, provide the optimal control actions that promote PHB accumulation and handle the disturbance effectively.

Cultivation of the PHB-producing cyanobacterium Synechococcus leopoliensis in a pilot-scale open system using nitrogen from waste streams

PHB-producing cyanobacteria may provide the raw material for bio-based and biodegradable plastics. To commercialize photoautotrophic PHB production, their cultivation needs to be scaled up in open systems and to reduce costs and increase sustainability, nutrients must be obtained from waste streams. Here, the feasibility of these steps was verified. Different PHB-producing cyanobacteria were compared in laboratory-scale cultivations using either water from recirculating aquaculture systems or pre-processed liquid digestate as nutrient sources. Then, Synechococcus leopoliensis was cultivated in an open thin-layer photobioreactor (18 m2, 200 L), were growth in mineral Z-medium was again compared to said waste streams. All cultivations were successful. Cultivation in mineral medium resulted in both the highest final biomass yield (6 g L−1) and productivity (0.7 g L−1 d−1). Both waste stream-based media showed lower biomass yields and productivities (2 g L−1 and 0.25–0.3 g L−1 d−1). However, due to differences in the cultivation conditions (e.g., temperature, nutrient supply), final biomass yield and productivity do not represent the performance of the cultivations adequately. Relative parameters such as nitrogen and energy conversion ratios indicate that cultivation with aquaculture water suffered from insufficient nitrogen supply to the culture, whereas the use pre-processed liquid digestate resulted in a substantial loss of nitrogen due to volatilization. All cultivations in the open system were continued in the laboratory, where cultures were starved for ten days under nutrient-depleted conditions (without nitrogen, phosphorus, or both). While PHB accumulation occurred, concentrations were comparatively low (< 1%dw). The comparison of the results suggests that PHB yields were influenced more by the initial cultivation condition than by the specific type of nutrient depletion. Thus, while the cultivation with waste streams in an open system is feasible, environmental parameters seem to influence PHB yields considerably and must be considered for the optimization of the complete process.

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

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

Influence of environmental conditions on accumulated polyhydroxybutyrate in municipal activated sludge

Poly(3-hydroxybutyrate) (PHB) was accumulated in full-scale municipal waste activated sludge at pilot scale. After accumulation, the fate of the PHB-rich biomass was evaluated over two weeks as a function of initial pH (5.5, 7.0 and 10), and incubation temperature (25, 37 and 55°C), with or without aeration. PHB became consumed under aerobic conditions as expected with first order rate constants in the range of 0.19 to 0.55 d−1. Under anaerobic conditions, up to 63 percent of the PHB became consumed within the first day (initial pH 7, 55°C). Subsequently, with continued anaerobic conditions, the polymer content remained stable in the biomass. Degradation rates were lower for acidic anaerobic incubation conditions at a lower temperature (25°C). Polymer thermal properties were measured in the dried PHB-rich biomass and for the polymer recovered by solvent extraction using dimethyl carbonate. PHB quality changes in dried biomass, indicated by differences in polymer melt enthalpy, correlated to differences in the extent of PHB extractability. Differences in the expressed PHB-in-biomass melt enthalpy that correlated to the polymer extractability suggested that yields of polymer recovery by extraction can be influenced by the state or quality of the polymer generated during downstream processing. Different post-accumulation process biomass management environments were found to influence the polymer quality and can also influence the extraction of non-polymer biomass. An acidic post-accumulation environment resulted in higher melt enthalpies in the biomass and, consequently, higher extraction efficiencies. Overall, acidic environmental conditions were found to be favourable for preserving both quantity and quality after PHB accumulation in activated sludge.

Untargeted metabolomics elucidated biosynthesis of polyhydroxyalkanoate by mixed microbial cultures from waste activated sludge under different pH values

Waste activated sludge has been frequently used as mixed substrate to produce polyhydroxyalkanoate (PHA). However, insufficient research on microbial metabolism has led to difficulties in regulating PHA accumulation in mixed microbial cultures (MMCs). To explore the variation of functional genes during domestication and the effect of different pH conditions on metabolic pathways during PHA accumulation, MMCs were domesticated by adding acetate and propionate with aerobic dynamic feeding strategy for 60 days. As the domestication progressed, the microbial community diversity declined and PHA-producing bacteria, Brevundimonas, Dechloromonas and Hyphomonas, were enriched. Through bacterial function prediction by PICRUSt the gene rpoE involved in starvation resistance of bacteria was enriched after the domestication. The pH value of 8.5 was the best condition for PHA accumulation in MMCs, under which a maximum PHA content reached 23.50% and hydroxybutyric (HB)/hydroxyvaleric (HV) reached 2.22. Untargeted metabolomics analysis exhibited that pH conditions of 7 and 8.5 could promote the up-regulation of significant differential metabolites, while higher alkaline conditions caused the inhibition of metabolic activity. Functional annotation showed that pH condition of 8.5 significantly affected Pyrimidine metabolism, resulting in an increase in PHA production. Regarding the pathways of PHA biosynthesis, acetoacetate was found to be significant in the metabolism of hydroxybutyric, and the alkaline condition could restrain the conversion from hydroxybutyric (HB) to the acetoacetate to protect PHB accumulation in MMCs compared with neutral condition. Taken together, the present results can advance the fundamental understanding of metabolic function in PHA accumulation under different pH conditions.

Effect of acute vs chronic stress on Polyhydroxybutyrate production by indigenous cyanobacterium

Fossil-based plastic has become a global-threat due to its high stability and transformation into more lethal forms such as micro plastics with time. An alternative should be found to combat this global enemy. Polyhydroxybutyrate or PHB can be such an alternative to plastic. Present study explores the synthesis of PHB in Neowollea manoromense, using two different cultivation-approaches: acute and chronic stress. This study has used 6 carbon sources and 3 different level of phosphate to study the accumulation of PHB along with lipid, carbohydrate, and proteins. Highest PHB in chronic-stress was achieved under glucose supplementation without phosphate at 21st day (156.5 ± 22.5 μg/mg), whereas in acute-stress, it was achieved under acetate without phosphate (91.0 ± 2.7 μg/mg). Despite higher accumulation in chronic-stress, high PHB productivity was achieved in acute-stress. Principal Component Analysis suggests that all the variables were positively correlated with each other. Here we first report PHB accumulation in Neowollea manoromense. This study highlights that acute-stress can be a powerful tool in establishment of a sustainable cyanobacteria based bio refinery for PHB production.