Polyhydroxybutyrate Production Research Articles

The Hyperproduction of Polyhydroxybutyrate Using Bacillus mycoides ICRI89 through Enzymatic Hydrolysis of Affordable Cardboard.

Bioplastics are contemplated as remarkable substitutes for conventional plastics to accommodate green technological advancements. However, their industrial production has not been fully implemented owing to the cost of carbon resources. From another perspective, valorizing different paper mill wastes has become a prominent research topic. These materials may serve as an affording sustainable feedstock for bioplastic production. Adjustment of cardboard waste hydrolysate as suitable fermentation media for production of bacterial polyhydroxyalkanoates (PHAs) has been investigated. Cardboard samples were defibered and dried before enzymatic hydrolysis. The enzymatic degradation of commercial cellulase was monitored over 15 days. Interestingly, 18.2 ± 0.2 g/L glucose yield was obtained from 50 g cardboard samples using a 1.5% (v/v) enzyme concentration. The samples exhibited maximum weight loss values of 69–73%. Meanwhile, five soil samples were collected from local sites in Lodz, Poland. A total of 31 bacterial isolates were screened and cultured on Nile blue plates. Analysis of the 16S rRNA gene sequence of the most potent producer revealed 100% similarity to Bacillus mycoides. Cardboard hydrolysates whole medium, modified MSM with cardboard hydrolysate and nitrogen depleted MSM with cardboard hydrolysate were utilized for PHA production, followed by PHA productivity and cell dry weight (CDW) estimation compared to glucose as a standard carbon source. An impressive PHA accumulation of 56% CDW was attained when the waste hydrolysate was used as a carbon source. FTIR and NMR analysis of the isolated PHA indicated that functional groups of the polymer were related to PHB (polyhydroxybutyrate). Thermal analysis demonstrates that PHB and PHB-CB (PHB produced from cardboard hydrolysate) have degradation temperatures of 380 and 369 °C, respectively, which reflect the high thermal stability and heat resistance compared to the same properties for a standard polymer. This is the first demonstration of full saccharification of corrugated cardboard paper waste for high-level production of PHA. In addition, the attained PHB productivity is one of the highest levels achieved from a real lignocellulosic waste.

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.

Monitoring PHB production in Synechocystis sp. with hyperspectral images.

Microalgae wastewater treatment systems have the potential for producing added-value products. More specifically, cyanobacteria are able to accumulate polyhydroxybutyrates (PHBs), which can be extracted and used for bioplastics production. Nonetheless, PHB production requires proper culture conditions and continue monitoring, challenging the state-of-the-art technologies. The aim of this study was to investigate the application of hyperspectral technologies to monitor cyanobacteria population growth and PHB production. We have established a ground-breaking measurement method able to discern spectral reflectance changes from light emitted to cyanobacteria in different phases. All in all, enabling to distinguish between cyanobacteria growth phase and PHB accumulation phase. Furthermore, first tests of classification algorithms used for machine learning and image recognition technologies had been applied to automatically recognize the different cyanobacteria species from a complex microbial community containing cyanobacteria and microalgae cultivated in pilot-scale photobioreactors (PBRs). We have defined three main indicators for monitoring PHB production: (i) cyanobacteria specific-strain density, (ii) differentiate between growth and PHB-accumulation and (iii) chlorosis progression. The results presented in this study represent an interesting alternative for traditional measurements in cyanobacteria PHB production and its application in pilot-scale PBRs. Although not directly determining the amount of PHB production, they would give insights on the undergoing processes.

Polyhydroxybutyrate (PHB) production by Halomonas boliviensis isolated from waste water

Plastic materials are often utilized as plastic packaging and have various uses since their longevity and consistency. Plastic products are generally not biodegradable or persist in our surroundings, posing major harm to the ecology. Biopolymers, or bioplastics, are developing as a blessing in the fight against waste buildup. Polyhydroxybutyrate (PHB) is a biopolymer that can be used instead of manufactured polymers. PHB is a fatty storage substance that accumulates inside microbes' cell walls when stressed. Halophilic microbes can be highly useful in manufacturing PHB since they are cheap, and PHB extraction is considerably easy in halo-tolerant species. As a result, our research emphasizes the identification of PHB-generating halotolerant species of microbes using untreated wastewater. Nile blue A and Sudan Black B staining were used to identify PHB favourable strains. The effective microbe generated PHB on a large scale utilizing sewage as the medium.

Direct production of polyhydroxybutyrate and alginate from crude glycerol by Azotobacter vinelandii using atmospheric nitrogen

While biodiesel is drawing attention as an eco-friendly fuel, the use of crude glycerol, a byproduct of the fuel production process, has increasingly become a concern to be addressed. Here we show the development of a low-cost fermentation technology using an atmospheric nitrogen-fixing bacterium to recycle crude glycerol into functional biopolymers. Azotobacter vinelandii showed substantial growth on tap water-diluted crude glycerol without any pretreatment. The number of viable A. vinelandii cells increased over 1000-fold under optimal growth conditions. Most of the glycerol content (~ 0.2%) in the crude glycerol medium was completely depleted within 48 h of culture. Useful polymers, such as polyhydroxybutyrate and alginate, were also produced. Polyhydroxybutyrate productivity was increased ten-fold by blocking the alginate synthesis pathway. Although there are few examples of using crude glycerol directly as a carbon source for microbial fermentation, there are no reports on the use of crude glycerol without the addition of a nitrogen source. This study demonstrated that it is possible to develop a technology to produce industrially useful polymers from crude glycerol through energy-saving and energy-efficient fermentation using the atmospheric nitrogen-fixing microorganism A. vinelandii.

Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production.

An antibiotic- and inducer-free culture condition was proposed for polyhydroxybutyrate (PHB) production in recombinant Escherichia coli. First, antibiotic-free vectors were constructed by installing the plasmid maintenance system, alp7, hok/sok, and the hok/sok and alp7 combination into the pUC19 vector. The plasmid stability test showed that pVEC02, the pUC19 vector containing the hok/sok system, was the most effective in achieving antibiotic-free cultivation in the E. coli B strain but not in the K strain. Second, the putative phaCAB operon derived from Caldimonas manganoxidans was inserted into pVEC02 to yield pPHB01 for PHB production in E. coli BL21 (DE3). The putative phaCAB operon was first shown function properly for PHB production and thus, inducer-free conditions were achieved. However, the maintenance of pPHB01 in E. coli requires antibiotics supplementation. Finally, an efficient E. coli ρ factor-independent terminator, thrLABC (ECK120033737), was inserted between the phaCAB operon and the hok/sok system to avoid possible transcriptional carry-over. The newly constructed plasmid pPHB01-1 facilitates an antibiotic- and inducer-free culture condition and induces the production of PHB with a concentration of 3.0 on0.2 g/L, yield of 0.26 /L0.07 g/g-glucose, and content of 44 /g3%. The PHB production using E. coli BL21 (DE3)/pPHB01-1 has been shown to last 84 and 96 h in the liquid and solid cultures.

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.

Accumulation of polyhydroxybutyrate in Synechocystis sp. isolated from wastewaters: Effect of salinity, light, and P content in the biomass

Cyanobacteria are of particular interest as they can produce Polyhydroxybutyrate (PHB), which is a promising alternative to oil-based plastics, using only inorganic carbon and solar light. However, new strategies should be found to further increase their PHB content. In this study, the effect of salinity, light intensity, and internal phosphorus content in the inoculated biomass, on the production of PHB with the cyanobacteria Synechocystis sp. were investigated. Results indicated that a sudden increase in salinity (together with nutrients starvation) stimulated the accumulation of PHB. In a first experiment where the cells were adapted to an electrical conductivity of 12 mS cm−1, a maximum content of PHB of a 7.5%VSS was achieved by adding NaCl and NaHCO3- (electrical conductivity of 28 mS cm−1). While in a second experiment, where cells were not adapted, similar PHB content (7.2%VSS) was achieved by adding only NaCl and C-HCO3-, providing only 11.4 mS cm−1. Furthermore, it was observed a reduced molecular weight of the polymer (38 kDa with respect to around 100 kDa), when really high salinities were used. Eventually, it was observed an increased production of glycogen (maximum of a 32%VSS) when high illumination (500 µmol m−2 s−1) was applied. Similar PHB content was obtained independently of the illumination. However, the PHB productivity (15.7 mgPHB L−1 d−1) was increased with light intensity. These results contribute to decipher the effect of important parameters in the PHB production and bring research a step forward towards the optimization of PHB production with Synechocystis sp.

A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications.

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.

Process optimization of the polyhydroxybutyrate production in the cyanobacteria Synechocystis sp. and Synechococcus sp.

The effect of four parameters (acetate, NaCl, inorganic carbon and days in darkness) affecting the polyhydroxybutyrate (PHB) production were tested and optimized for Synechococcus sp. and Synechocystis sp. using a Box-Behnken design. The optimal conditions for Synechocystis sp. were found to be 1.2 g L-1 of acetate, 4 gC L-1 of NaHCO3, 18 g L-1 of NaCl and 0 days in darkness. For Synechococcus sp., equal acetate concentration and days in darkness, and lower inorganic carbon and NaCl concentrations than those for Synechocystis sp. were needed (0.05 g L-1 inorganic carbon and 9 g L-1 NaCl). Optimal conditions were scaled up to 3 L photobioreactors. Using Synechocystis sp., 5.6 %dcw of PHB was obtained whether adding or not acetate. In opposition, a maximum of 26.1 %dcw by using acetate was reached with Synechococcus sp. These results provide an easy method to optimize the cultivation conditions to enhance PHB production with cyanobacteria.

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.

Polyhydroxybutyrate production by Chlorella sorokiniana SVMIICT8 under Nutrient-deprived mixotrophy

Polyhydroxybutyrates (PHBs) are naturally occurring biopolymeric compounds that accumulate in a variety of microorganisms, including microalgae as energy and carbon storage sources. The present study was designed to evaluate nature-based PHB production using microalgae (Chlorella sorokiniana SVMIICT8) in biphasic (growth (GP) and stress phase (SP)) nutritional mode of cultivation. Microalgal PHB accumulation was driven by nutrient constraint, with a maximal production of 29.5% of PHB from 0.94 gm L−1 of biomass. Fluorescence microscopy revealed PHB granules in the cell cytoplasm, while NMR (1H and 13C), XRD and TGA analysis confirmed the structure. The biopolymer obtained was homopolymer of PHB with carbonyl (C=O) stretch of the aliphatic ester moiety. In GC-MS analysis, major peak representing butyric acid methyl ester also confirmed the PHB. Chlorophyll a fluorescence transients inferred through OJIP, exhibited significant variation in photosynthetic process during growth and nutrient limiting conditions. Mining of bio-based products from microalgae cultivation embrace nature-based approach addressing climate change and sustainability inclusively.

Poly-3-hydroxybutyrate production in acetate minimal medium using engineered Methylorubrum extorquens AM1

Acetate is regarded as a sustainable microbial feedstock that is synthesized from biowastes such as synthesis gas (syngas), carbon dioxide, lignocellulose, or organic waste. In this study, Methylorubrum extorquens AM1 was engineered to improve the production of bioplastic poly-3-hydroxybutyrate (PHB) using acetate as the sole carbon source. To utilize acetate as a carbon source and methanol as an energy source, acs encoding acetyl-CoA synthetase and fdh from Burkholderia stabilis were overexpressed, while ftfL involved in the assimilation of methanol into formyl-tetrahydrofolate was deleted. The yields of biomass and PHB from acetate significantly improved, and the growth rate and PHB content of the bacteria increased. In addition, sustainability of the PHB production was demonstrated using acetate derived from carbon dioxide and syngas. This study shows that biopolymers could be synthesized efficiently using acetate as the sole carbon source through metabolic engineering and the supply of energy cofactors.

Optimization of nitrogen feeding strategies for improving polyhydroxybutyrate production from biogas by Methylocystis parvus str. OBBP in a stirred tank reactor

The design of efficient cultivation strategies to produce bioplastics from biogas is crucial for the implementation of this biorefinery process. In this work, biogas-based polyhydroxybutyrate (PHB) production and CH4 biodegradation performance was investigated for the first time in a stirred tank bioreactor inoculated with Methylocystis parvus str. OBBP. Decreasing nitrogen loading rates in continuous mode and alternating feast:famine regimes of 24 h-cycles, and alternating feast:famine regimes of 24 h:24 h and 24 h:48 h were tested. Continuous N feeding did not support an effective PHB production despite the occurrence of nitrogen limiting conditions. Feast-famine cycles of 24 h:24 h (with 50% stoichiometric nitrogen supply) supported the maximum PHB production (20 g-PHB m−3 d−1) without compromising the CH4-elimination capacity (25 g m−3 h−1) of the system. Feast:famine ratios ≤1:2 entailed the deterioration of process performance at stoichiometric nitrogen inputs ≤60%.

Finding of novel polyhydroxybutyrate producer Loktanella sp. SM43 capable of balanced utilization of glucose and xylose from lignocellulosic biomass

Polyhydroxybutyrate (PHB) is a potential substitute for plastics derived from fossil fuels, owing to its biodegradable and biocompatible properties. Lignocellulosic biomass could be used to reduce PHB production costs; however, the co-utilization of sugars, such as glucose and xylose, without catabolite repression is a difficult problem to be solved. Here, we selected a novel Loktanella sp. SM43 from a marine environment and optimized the conditions for PHB production. Loktanella sp. SM43 showed high PHB production (66.5% content) from glucose. When glucose and xylose were used together, this strain showed high utilization of both substrates compared to other high PHB-producers such as Halomonas sp. and Cupriavidus necator, which showed glucose preference. Loktanella sp. SM43 showed high growth and PHB production with lignocellulosic hydrolysates. When pine tree hydrolysates were used, PHB production was the highest at 3.66 ± 0.01 g/L, followed by Miscanthus (3.46 ± 0.09 g/L) and barley straw hydrolysate (3.36 ± 0.36 g/L). Overall, these results reveal the potential of Loktanella sp. SM43 to produce PHB using various lignocellulosic hydrolysates as feedstock and the first systematic study for PHB production with Loktanella sp. The approach of screening novel strains is a strategy to overcome co-utilization of sugars without genetic engineering.

The Carbon Source Effect on the Production of Ralstonia eutropha H16 and Proteomic Response Underlying Targeting the Bioconversion with Solar Fuels.

Chemoautotrophic bacterium Ralstonia eutropha H16 can fix CO2 to bioplastic and is potentially useful for CO2 neutralization. Targeting the solar fuel-based plastic biomanufactory, the polyhydroxybutyrate (PHB) production between heterotrophy and chemoautotrophy conditions was evaluated and the proteomic responses of the R. eutropha H16 cells to different carbon and energy sources were investigated. The results show that the chemoautotrophic mode hardly affected the cellular PHB accumulation capacity. Benefited from the high coverage proteome data, the global response of R. eutropha H16 to different carbon and energy sources was presented with a 95% KEGG pathway annotation, and the genome-wide location-related protein expression pattern was also identified. PHB depolymerase Q0K9H3 was found as a key protein responding to the low carbon input while CO2 and H2 were used, and will be a new regulation target for further high PHB production based on solar fuels.

Inorganic carbon stimulates the metabolic routes related to the polyhdroxybutyrate production in a Synechocystis sp. strain (cyanobacteria) isolated from wastewater

Cyanobacteria are capable of transforming CO2 into polyhydroxybutyrate (PHB). In this study, different inorganic carbon concentrations (0–2 gC L−1) were evaluated for a Synechocystis sp. strain isolated from wastewater. Quantitative RT-qPCR was also performed to decipher the links between inorganic carbon and PHB and glycogen metabolism. 2 gC L−1 of bicarbonate stimulated cell growth, nutrients consumption and production of PHB. Using this concentration, a 14%dcw of PHB and an average productivity of 2.45 mgPHB L−1 d−1 were obtained. Gene expression analysis revelated that these conditions caused the overexpression of genes related to glycogen and PHB synthesis. Moreover, a positive correlation between the genes codifying for the glycogen phosphorylase, the acetyl-CoA reductase and the poly(3-hydroxyalkanoate) polymerase was found, meaning that PHB synthesis and glycogen catabolism are strongly related. These results provide an exhaustive evaluation of the effect of carbon on the PHB production and cyanobacterial metabolism.

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.