PHB Production Research Articles

Microbial symbiotic electrobioconversion of carbon dioxide to biopolymer (poly (3-hydroxybutyrate)) via single-step microbial electrosynthesis cell

This study proposes an novel single-stage microbial electrosynthesis system (MES) that bioelectrochemically converts CO2 into poly(3-hydroxybutyrate) (PHB). This innovative approach utilizes a symbiotic co-culture of electroactive acetogenic bacteria and PHB-accumulating bacteria within a single reactor, bypassing the need for separate acetate production and extraction. Systematic optimization of key operational parameters, including applied voltage, carbon source, and cathode material, was conducted to maximize PHB production. Results demonstrate that using a voltage of 2.5 V yielded a 7.14-fold increase in PHB content compared to open circuit conditions. Furthermore, functionalizing the cathode with a conductive and hydrophilic PEDOT: PSS polymer coating significantly enhanced the system’s performance, resulting in a 1.5-fold increase in both acetate and PHB production compared to unmodified carbon felt electrodes. The long-term stability and effectiveness of the co-culture system were validated through comprehensive microbial and metagenomic analyses. Results revealed a significant enrichment of CO2-utilizing electrotrophs within the cathode biofilm, including Acetobacterium and Clostridium. Concurrently, a 4 to 14-fold increase in the relative abundance of PHB-biosynthesizing bacteria, such as Pseudomonas, Rhodobacter and Caulobacter was observed in the planktonic phase. This study offers a promising pathway towards a circular bioeconomy by enabling the valorization of CO2 into valuable bio-based products via single-stage MES, utilizing a symbiotic co-culture.

Genetic Diversity and Functional Potential of Streptomyces spp. Isolated from Pachmarhi Biosphere Reserve, India.

Streptomyces is a diverse genus, well known for producing a wide array of metabolites that have significant industrial utilization. The present study investigates the genetic and functional diversity of Streptomyces spp. isolated from the Pachmarhi Biosphere Reserve (PBR), India, an unexplored site. The 16S rRNA gene sequencing and analysis revealed 96 isolates belonging to 40 different species indicating a substantial phylogenetic diversity. The strains were clustered into two groups: a major cluster with 94 strains and a small cluster with two strains. BOX- PCR analyses revealed an incredible genetic diversity existing among the strains of Streptomyces spp. in PBR. The analyses revealed the intra-species diversity and inter-species closeness within the genus Streptomyces in the study area. Qualitative screening for enzyme production has shown that 53, 42, 41, 11, and 54 strains tested positive for CMCase, xylanase, amylase, pectinase, and β-glucosidase, respectively. Additionally, 54 strains tested positive for PHB production. The strains were assayed quantitatively for the production of CMCase, xylanase, amylase, and pectinase. Streptomyces sp. MP9-2, Streptomyces sp. MP10-11, Streptomyces sp. MP10-18, and Streptomyces sp. MP10-6 recorded maximum CMCase (0.604 U/mL), xylanase (0.553 U/mL), amylase (1.714 U/mL), and pectinase (13.15 U/mL) activities, respectively. Furthermore, several strains demonstrated plant growth-promoting traits, viz. zinc and phosphate solubilization and production of ammonia, HCN (hydrogen cyanide), and IAA (Indole acetic acid), and nitrogen fixation. Fifty strains showed antifungal activity against Fusarium oxysporum f. sp. lycopersici with inhibitions ranging from 7.5 to 47.5%. Current findings underscore the ecological and biotechnological significance of Streptomyces spp. in the unexplored habitat of PBR.

Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes

Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9% lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5%) and purity (98.1%) were obtained with 1,3-dioxolane at 80°C and 6h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor cultures leading to the production of 78.7 gPHB/L, 56.9% (w/w) PHB content and 2.3g/(Lꞏh) productivity. Low global warming potential (1.3 CO2-eq/kgPHB) and abiotic depletion fossil (14.4 MJ/kgPHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.

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.

Bacterial potential of simultaneous PHB production and wastewater treatment through submerged fermentation in a sequencing batch reactor

Bacterial potential of simultaneous PHB production and wastewater treatment through submerged fermentation in a sequencing batch reactor

Pressure fermentation to boost CO2-based poly(3-hydroxybutyrate) production using Cupriavidus necator

CO2-based poly(3-hydroxybutyrate) (PHB) can be produced by the versatile bacterium Cupriavidus necator through chemolithoautotrophic fermentation, using a gas mixture consisting of CO2, H2, and O2. Despite offering a propitious route for carbon–neutral bioplastic manufacturing, its adoption is currently hampered by the wide explosive range of the required gas mixture, as well as the limited gas-to-liquid mass transfer rates. To address these challenges, pressure fermentation was applied as a robust and effective strategy, while ensuring safe operation by adhering to the limiting O2 concentration, utilizing state-of-the-art bioreactors. Consequently, exponential growth could be prolonged, boosting CO2-based PHB production from 10.8 g/L at 1.5 bar up to 29.6 g/L at 3 bar. The production gain closely aligns with the theoretical calculations, except for when the pressure was increased up to 4 bar. Overall, the demonstrated increase in PHB production underscores the potential of pressure fermentation to enhance aerobic gas fermentation.

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.

Biorefinery electrification of brewers’ spent grains using plasma bubbles for sustainable production of poly(3-hydroxybutyrate)

Biorefinery electrification could be implemented for sustainable refining of crude renewable resources eliminating the use of high temperatures and pressures, alkaline and acidic solutions and conventional solvents. A plasma bubble reactor using air as the feed gas was employed to enhance poly(3-hydroxybutyrate) (PHB) production from brewers’ spent grains (BSG) facilitating the fractionation of protein and lignin as value-added co-products. Plasma factors (voltage, pretreatment duration, duty cycle, initial solids concentration) were evaluated with the highest overall conversion yield of glucan and hemicellulose (73 %, 0.494 g sugars/gBSG) achieved for initial solids concentration of 150 g/L. The produced BSG hydrolysate led to higher PHB production efficiency (130 g/L total dry weight, 74.4 gPHB/L, 0.32 g/g yield, 2.64 g/L/h productivity) than conventional hydrolysates produced by enzymatic hydrolysis alone and enzymatic hydrolysis combined with hydrothermal treatment. The protein and lignin losses in the remaining BSG solids were 24.1 % and 29.2 % respectively. When renewable electricity is employed, the global warming potential was estimated at 0.59 kg CO2-eq per kgPHB and the abiotic depletion potential fossil at 11.12 MJ per kgPHB for PHB production based on plasma treated BSG. In the proposed biorefinery concept, 1 kg BSG could be converted into 147.6 g PHB with a solid fraction containing 137.4 g protein and 98.4 g lignin that could be subsequently fractionated as value-added co-products. The rationale of this study is to demonstrate that BSG plasma treatment can be used for the exploitation of the full potential of BSG for the production of PHB with improved environmental impact and minimal losses of protein and lignin.

Poly(3-hydroxybutyrate) production using supplemented corn-processing byproducts through Cupriavidus necator via solid-state fermentation: Cultivation on flask and bioreactor scale

The widespread adoption of Poly(3-hydroxybutyrate) (PHB) encounters challenges due to its higher production costs compared to conventional plastics. To overcome this obstacle, this study investigates the use of low-cost raw materials and optimized production methods. Specifically, food processing byproducts such as corn germ and corn bran were utilized as solid substrates through solid-state fermentation, enriched with molasses and cheese whey. Employing the One Factor at a Time technique, we examined the effects of substrate composition, temperature, initial substrate moisture, molasses, and cheese whey on PHB production at the flask scale. Subsequently, experiments were conducted at the bioreactor scale to evaluate the influence of aeration. In flask-scale experiments, the highest PHB yield, reaching 4.1 (g/kg Initial Dry Weight Substrate) (IDWS) after 72 hours, was achieved using a substrate comprising a 1:1 mass ratio of corn germ to corn bran supplemented with 20 % (v/w) cheese whey. Furthermore, PHB production in a 0.5-L packed-bed bioreactor yielded a maximum of 8.4 (g/kg IDWS), indicating a more than 100 % increase in yield after 72 hours, with optimal results achieved at an aeration rate of 0.5 l/(kg IDWS. h).

Paracoccus kondratievae produces poly(3-hydroxybutyrate) under elevated temperature conditions.

As part of ongoing efforts to discover novel polyhydroxyalkanoate-producing bacterial species, we embarked on characterizing the thermotolerant species, Paracoccus kondratievae, for biopolymer synthesis. Using traditional chemical and thermal characterization techniques, we found that P. kondratievae accumulates poly(3-hydroxybutyrate) (PHB), reaching up to 46.8% of the cell's dry weight after a 24-h incubation at 42°C. Although P. kondratievae is phylogenetically related to the prototypical polyhydroxyalkanoate producer, Paracoccus denitrificans, we observed significant differences in the PHB production dynamics between these two Paracoccus species. Notably, P. kondratievae can grow and produce PHB at elevated temperatures ranging from 42 to 47°C. Furthermore, P. kondratievae reaches its peak PHB content during the early stationary growth phase, specifically after 24 h of growth in a flask culture. This is then followed by a decline in the later stages of the stationary growth phase. The depolymerization observed in this growth phase is facilitated by the abundant presence of the PhaZ depolymerase enzyme associated with PHB granules. We observed the highest PHB levels when the cells were cultivated in a medium with glycerol as the sole carbon source and a carbon-to-nitrogen ratio of 10. Finally, we found that PHB production is induced as an osmotic stress response, similar to other polyhydroxyalkanoate-producing species.

Kinetics and synthesis of poly(3-hydroxybutyrate) by a putative-mutant of Bacillus licheniformis

The present study deals with the kinetics of improved poly(3-hydroxybutyrate) (PHB) production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis. Production of biodegradable polymers is to eliminate use of materials derived from petrochemicals and also because of their environmental impact. For the current study, mutant strain (NA-21) & wild-type (IIB-isl19) were used for PHB production. Submerged culture with two-stage fermentation technique was used for PHB production. Results indicated that PHB production was improved with 300 mM of –HNO2. The superior mutant strain (NA-21) resulted in 2-fold more PHB as compared to the wild-type (IIB-isl9). It was selected, and resistance against L-cysteine HCl was developed. At 4 ppm concentration of L-cysteine HCl, PHB production by mutant strain (NA-cys4) was higher than its wild counterpart by 5.7-fold. Kinetic study of parameters including specific growth rate (µ h− 1), growth (Yx/s,Ys/x), product yield coefficients (Yp/s,Yp/x), volumetric rate constants (Qp, Qs, Qx) and specific rate constants (qp, qs, qx), were also accomplished. Moreover, Yp/x, Qp and qp = µ × Yp/x were found to be very significant as 1.254 ± 0.06 (g/g biomass), 0.134 ± 0.01 (g/l/h) and 0.168 ± 0.01 (g/g/h), respectively. The effect of fatty acids on PHB production highlighted the improvement in PHB production by 1.94-fold. The highest PHB production during the study was 16.35 ± 3.12 g/l which highlighted its significance (p ≤ 0.05) and impact on the overall process. The variation in PBH yield between wild-type and mutant B. licheniformis is possibly because of induced DNA interstrand thus making unstable thymidine-thymidine dymers. From the results, it was concluded that improved PHB production on industrial scale is fairly possible and it holds the potential to contribute significantly to plastic circularity in the future.

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.

Genetic characterization of a novel Salinicola salarius isolate applied for the bioconversion of agro-industrial wastes into polyhydroxybutyrate

BackgroundPolyhydroxybutyrate (PHB) has emerged as a promising eco-friendly alternative to traditional petrochemical-based plastics. In the present study, we isolated and characterized a new strain of Salinicola salarius, a halophilic bacterium, from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further genome analysis of the isolated strain, ES021, was conducted to identify and elucidate the genes involved in PHB production.ResultsDifferent PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. Additionally, phaJ and fadB were identified as key genes involved in PHB production from fatty acids. Optimization of environmental factors such as shaking rate and incubation temperature, resulted in the highest PHB productivity when growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h. To maximize PHB production economically, different raw materials i.e., salted whey and sugarcane molasses were examined as cost-effective carbon sources. The PHB productivity increased two-fold (13.34 g/L) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/L, and PHB of 12.88 g/L. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC–MS), and nuclear magnetic resonance spectroscopy (NMR) analyses.ConclusionsHerein,Salinicola salarius ES021 strain was demonstrated as a robust natural producer of PHB from agro-industrial wastes. The detailed genome characterization of the ES021 strain presented in this study identifies potential PHB-related genes. However, further metabolic engineering is warranted to confirm the gene networks required for PHB production in this strain. Overall, this study contributes to the development of sustainable and cost-effective PHB production strategies.

PHB production by Bacillus megaterium strain MNSH1-9K-1 using low-cost media.

Plastics are widely used for diverse applications due to their versatility. However, their negative impact on ecosystems is undeniable due to their long-term degradation. Thus, there is a rising need for developing eco-friendlier alternatives to substitute fossil-based plastics, like biopolymers. PHA are synthesized intracellularly by microorganisms under stressful conditions of growth and have similar characteristics to conventional polymers, like their melting point, transition temperatures, crystallinity, and flexibility. Although it is feasible to use biopolymers for diverse industrial applications, their elevated production cost due to the supplies needed for microbiological procedures and the low productivity yields obtained have been the main limiting factors for their commercial success. The present study assessed the ability of Bacillus megaterium strain MNSH1-9K-1 to produce biopolymers using low-cost media from different kinds of fruit-peel residues. The results show that MNSH1-9K-1 can produce up to 58 g/L of PHB when grown in a medium prepared from orange-peel residues. The data obtained provide information to enhance the scalability of these kinds of biotechnological processes.

Identification and characterization of a potential strain for the production of polyhydroxyalkanoate from glycerol.

While poly (3-hydroxybutyrate) (PHB) holds promise as a bioplastic, its commercial utilization has been hampered by the high cost of raw materials. However, glycerol emerges as a viable feedstock for PHB production, offering a sustainable production approach and substantial cost reduction potential. Glycerol stands out as a promising feedstock for PHB production, offering a pathway toward sustainable manufacturing and considerable cost savings. The identification and characterization of strains capable of converting glycerol into PHB represent a pivotal strategy in advancing PHB production research. In this study, we isolated a strain, Ralstonia sp. RRA (RRA). The strain exhibits remarkable proficiency in synthesizing PHB from glycerol. With glycerol as the carbon source, RRA achieved a specific growth rate of 0.19 h-1, attaining a PHB content of approximately 50% within 30 h. Through third-generation genome and transcriptome sequencing, we elucidated the genome composition and identified a total of eight genes (glpR, glpD, glpS, glpT, glpP, glpQ, glpV, and glpK) involved in the glycerol metabolism pathway. Leveraging these findings, the strain RRA demonstrates significant promise in producing PHB from low-cost renewable carbon sources.

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.

Analysis, Optimization and Molecular Characterization of PHB Positive Bacteria Isolated from Agricultural Soil Sample

Polyhydroxybutyrates are the widely studied biopolymer because of its biodegradability and non-toxic effect in the environment. This work focused on the isolation, screening, characterization and optimization of the PHB producing bacteria from agricultural soil. About eighty percentage of the isolated bacteria were identified as PHB producing bacteria after Sudan Black Blue staining. Among them three colonies were randomly selected and their morphologically and biochemically characterized. The biopolymer was extracted using sodium hypochlorite method. Potent PHB producer (WJSP1) (13.5µg/ml) was identified by crotonic acid assay. The functional groups of the produced PHB were identified by Fourier Transform Infra-Red spectroscopy (FTIR). The presence of functional groups like carbon group, C=O ester group, CH group, carbonyl group, carboxyl group which was corelated with the peak values1454.33cm-1, 1724.36 cm-1, 1274.95 cm-1, 1049.28 cm-1, 1280-1053 cm-1 indicates the presence of PHB. The potent producer (WJSP1) was optimized under different conditions to achieve the best condition for the production of PHB. The maximum production was obtained at the temperature - 37˚C, the carbon source - glucose, pH - 7 and at a time period of 72 hours. 16SrRNA sequencing was performed and the organism was identified as Bacillus cereus from the BLST (Basic Local Alignment Search Tool). A phylogenetic tree was constructed and the sequence was submitted in GENBANK (Accession No: OR192860). The isolated bacteria Bacillus cereus is an efficient producer of PHB under laboratory condition in presence of glucose as a chief carbon source. Large scale production of PHB can be done in large scale fermenters in industries. Large scale production of PHB will help the environment to get rid of the petroleum-based bioplastics.

Construction of cascade circuits for dynamic temporal regulation and its application to PHB production

BackgroundTo maximize the production capacity and yield of microbial cell factories, metabolic pathways are generally modified with dynamic regulatory strategies, which can effectively solve the problems of low biological yield, growth retardation and metabolic imbalance. However, the strategy of dynamic regulating multiple genes in different time and order is still not effectively solved. Based on the quorum-sensing (QS) system and the principle of cascade regulation, we studied the sequence and time interval of gene expression in metabolic pathways.ResultsWe designed and constructed a self-induced dynamic temporal regulatory cascade circuit in Escherichia coli using the QS system and dual regulatory protein cascade and found that the time intervals of the cascade circuits based on the Tra, Las system and the Lux, Tra system reached 200 min and 150 min, respectively. Furthermore, a dynamic temporal regulatory cascade circuit library with time intervals ranging from 110 to 310 min was obtained based on this circuit using promoter engineering and ribosome binding site replacement, which can provide more selective synthetic biology universal components for metabolic applications. Finally, poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of the cascade circuit library. The content of PHB increased 1.5-fold. Moreover, circuits with different time intervals and different expression orders were found to have different potentials for application in PHB production, and the preferred time-interval circuit strain C2-max was identified by screening.ConclusionsThe self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.