Polyhydroxybutyrate Yield Research Articles

Valorizing waste liquor from dilute acid pretreatment of lignocellulosic biomass by Bacillus megaterium B-10

Dilute acid pretreatment liquor (DAPL) of lignocellulose contains abundant waste sugar resource for further upgrade. But the inhibitors, produced from sugar dehydrogenation, impedes the waste sugar valorization. In this study, an efficient biostrategy was developed to upgrade the waste sugar in DAPL. The concentration of sugar and phenolics in DAPL reached 19.61 g/L and 0.57 g/L, respectively, after pretreatment of rice straw with 0.5 % (v/v) sulfuric acid for 40 min at 121 °C. Bacillus megaterium B-10 could tolerate the furfural toxicity products in DAPL and convert the waste sugar into high-valuable products, polyhydroxyalkanoate (PHA). This polymer was characterized by GC–MS and 1H-NMR, and the as-synthesized PHA was identified as poly-3-hydroxybutyrate (PHB), a typical PHA. A demonstrated yield of PHB (1.496 g/L) was achieved, which accounted for 32.56 % of dry cell weight. Moreover, characterizations of PHB demonstrated that the biosynthetic PHB exhibited favorable thermoplastic properties. This bioconversion of B-10 provides an efficient strategy not only for waste pollution treatment, but also for lignocellulose valorization.

Utilization of Noxious Weed Water Hyacinth Biomass as a Potential Feedstock for Biopolymers Production: A Novel Approach

This study aims to utilize a noxious weed water hyacinth biomass (WH) for polyhydroxybutyrate (PHB) production. Alkaline and peracetic acid pretreatment was employed for the hydrolysis of WH and consequently enzymatic saccharification to produce fermentable sugars for PHB production. The pretreatment competence was determined using various operational parameters. By applying ambient conditions, alkaline pretreatment gave higher lignin removal of 65.0%, with 80.8% hydrolysis yield, and on enzyme hydrolysis (40 FPU/g of dry WH), produced total reducing sugar of about 523 mg/g of WH. The resulted WH enzymatic hydolysates were evaluated for the production of PHB by Ralstonia eutropha (ATCC 17699). The WH hydrolysates cultivation was compared to synthetic hydrolysates that contain a similar carbon composition in terms of bacterial growth and PHB synthesis. The effects of various supplements to enhance PHB production were estimated. Supplementation of corn steep liquor (CSL) as a cheap nitrogen source with WH hydrolysates favored a higher amount of PHB synthesis (73%), PHB titer of 7.30 g/L and PHB yield of 0.429 g/g of reducing sugar. Finally, using standard analytical tools, the physical and thermal characteristics of the extracted PHB were evaluated. The findings revealed WH was a promising and technically feasible option for transforming biomass into sustainable biopolymer conversion on a large scale.

Enhanced isobutanol production by co-production of polyhydroxybutyrate and cofactor engineering

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon into Escherichia coli conferred a metabolic advantage for alcohol production, generating 1.14-fold more isobutanol. Furthermore, following nitrogen source optimization and cofactor engineering, the engineered E. coli strain produced 2-fold more isobutanol and 0.25 g/L PHB. Moreover, E. coli cells showed higher tolerance to isobutanol with the overexpression of PHB biosynthesis genes. This co-production system resulted in an increased biomass, higher glucose utilization, and lower acetate maintenance, leading to higher productivity regarding PHB and isobutanol yield. Thus, this novel system is applicable to future fermentation studies for the co-production of PHB and isobutanol.

Bioplastic (poly-3-hydroxybutyrate) production by the marine bacterium Pseudodonghicola xiamenensis through date syrup valorization and structural assessment of the biopolymer

Biobased degradable plastics have received significant attention owing to their potential application as a green alternative to synthetic plastics. A dye-based procedure was used to screen poly-3-hydroxybutyrate (PHB)-producing marine bacteria isolated from the Red Sea, Saudi Arabia. Among the 56 bacterial isolates, Pseudodonghicola xiamenensis, identified using 16S rRNA gene analyses, accumulated the highest amount of PHB. The highest PHB production by P. xiamenensis was achieved after 96 h of incubation at pH 7.5 and 35 °C in the presence of 4% NaCl, and peptone was the preferred nitrogen source. The use of date syrup at 4% (w/v) resulted in a PHB concentration of 15.54 g/L and a PHB yield of 38.85% of the date syrup, with a productivity rate of 0.162 g/L/h, which could substantially improve the production cost. Structural assessment of the bioplastic by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy revealed the presence of methyl, hydroxyl, methine, methylene, and ester carbonyl groups in the extracted polymer. The derivative products of butanoic acid estimated by gas chromatography-mass spectrometry [butanoic acid, 2-amino-4-(methylseleno), hexanoic acid, 4-methyl-, methyl ester, and hexanedioic acid, monomethyl ester] confirmed the structure of PHB. The present results are the first report on the production of a bioplastic by P. xiamenensis, suggesting that Red Sea habitats are a potential biological reservoir for novel bioplastic-producing bacteria.

Proteome profile changes during poly-hydroxybutyrate intracellular mobilization in gram positive Bacillus cereus tsu1

BackgroundBacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12 h, and depleted after 48 h. Fore-spore and spore structure were observed after 24 h culture.ResultsQuantitative proteomic analysis of B. cereus tsu1 identified 2952 quantifiable proteins, and 244 significantly changed proteins (SCPs) in the 24 h:12 h pair of samples, and 325 SCPs in the 48 h:12 h pair of samples. Based on gene ontology classification analysis, biological processes enriched only in the 24 h:12 h SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress, carboxylic acid catabolism, and cellular amino acid catabolism. The 48 h:12 h SCPs were enriched into processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, and formation of translation ternary structure. A key enzyme for PHB metabolism, poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) accumulated significantly higher in 12 h-culture. Sporulation related proteins SigF and SpoEII were significantly higher in 24 h-samples. Enzymes for nitrate respiration and fermentation accumulated to the highest abundance level in 48 h-culture.ConclusionsChanges in proteome of B. cereus tsu1 during PHB intracellular mobilization were characterized in this study. The key enzyme PhaC for PHB synthesis increased significantly after 12 h-culture which supports the highest PHB accumulation at this time point. The protein abundance level of SpoIIE and SigF also increased, correlating with sporulation in 24 h-culture. Enzymes for nitrate respiration and fermentation were significantly induced in 48 h-culture which indicates the depletion of oxygen at this stage and carbon flow towards fermentative growth. Results from this study provide insights into proteome profile changes during PHB accumulation and reuse, which can be applied to achieve a higher PHB yield and to improve bacterial growth performance and stress resistance.

Development of ultrasound aided chemical pretreatment methods to enrich saccharification of wheat waste biomass for polyhydroxybutyrate production and its characterization

Development of an effective pretreatment method is a vital stage to enhance biomass-based biopolymers production. The present study aims on the pretreatment of wheat waste biomass (WS) with alkaline and in combination with ultrasound (US) treatment to enhance enzymatic saccharification of WS into monomeric sugars. Effects of chemical dosage, substrate loading, US power and US pretreatment time on the pretreatment efficiency were systematically investigated. Under optimized conditions; US + NaOH pretreatment attained maximum 70% delignification of WS, with 84.5% hydrolysis yield, 90% glucose yield and 65% xylose yield after enzymatic hydrolysis (30 FPU/g of dry WS) which is substantially higher than the individual pretreatment. Analytical studies (XRD, FTIR, and SEM) revealed that the combined US + NaOH pretreatment can effectively destroyed the ultrastructure of biomass and improved the accessibility to hydrolyzing enzymes. The resulted enzymatic hydrolysates were utilized as a possible biomass feedstock for polyhydroxybutyrate (PHB) production using Ralstonia eutropha. Effects of supplementation of nutrients, stressing agents and initial concentration of US + NaOH pretreated WS hydrolysates (20 to 40 g/L) on PHB production were evaluated. The maximum 74% PHA accumulation, PHB titer of 7.85 g/L and yield of PHB about 0.441 g/g of reducing sugar production was obtained. Analytical characterization and thermal characteristics of extracted biopolymer showed the poperies of standard PHB. The foregoing results suggested the potential of WS as an alternate renewable resource for sustainable biopolymer production and enables WS waste biomass disposal concerns.

A closed-loop biorefinery approach for polyhydroxybutyrate (PHB) production using sugars from carob pods as the sole raw material and downstream processing using the co-product lignin

A novel closed-loop biorefinery model using carob pods as the feed material was developed for PHB production. The carob pods were delignified, and as the second step, sugars present in the delignified carob pods were extracted using water. Ralstonia eutropha and Bacillus megaterium were cultivated on the carob pod extract and its performance was evaluated using Taguchi experimental design. R. eutropha outperformed the B. megaterium in terms of its capability to grow at a maximum initial sugar concentration of 40 g L−1 with a maximum PHB production of 12.2 g L−1. Finally, the concentrated lignin from the first step was diluted with different proportion of chloroform to extract PHB from the bacterial biomass. The PHB yield and purity obtained were more than 90% respectively using either R. eutropha or B. megaterium. Properties of the PHB produced in this study were examined to establish its application potential.

Bioconversion of Sugarcane Molasses into Bioplastic (Polyhydroxybutyrate) using Bacillus cereus 2156 under Statistically Optimized Culture Conditions

The polyhydroxybutyrate (PHB) macromolecule production using cheap substrate is need of today’s life since rate of accumulate of plastic in the environment is million tonnes per year, leading to life threatening pollution problems. In this account, Response Surface Methodology (RSM) was implemented for optimization of low cost substrate (molasses) and method parameters for the manufacturing of PHB granules using Bacillus cereus and further refurbishing it into bioplastic. Primary screening of PHB manufacturing was done using Sudan black staining. 67.4 % of PHB yield of dry weight with the predicted percentage yield of 68.78 % was obtained using Box Behnken Design. Present results demonstrated that carbon source and nitrogen source were found to be the critical control factors that affected the PHB production. FTIR was performed for their functional group determination. The existing work demonstrates that produced PHB using low cost molasses is eco-friendly, economically feasible, easily available and promising alternative substrate for commercial production.

In situ based surface-enhanced Raman spectroscopy (SERS) for the fast and reproducible identification of PHB producers in cyanobacterial cultures.

The production of polyhydroxybutyrate (PHB) by autotrophic fermentation of cyanobacteria has received increasing interest in the light of carbon emission reducing process strategies. Biotechnological approaches are in development to optimize the yield of PHB, including adapted cultivation media, characterized by a limitation of key nutrients: cyanobacteria accumulate PHB as energy storage molecules under limited growth conditions. Since there is an increasing demand for fast, simple and reliable analytics, we report the establishment of surface enhanced Raman spectroscopy (SERS) as a suitable monitoring tool for up scaled PHB production processes. Both, pure Ag-colloids mixed with bacterial culture, and in situ prepared colloids (Ag-Synechocystis), generated on the cell surface directly, were successfully applied and evaluated for this purpose. SERS measurements with in situ prepared Ag-colloids improved the reproducibility of Raman signals from 54.8% to 93.9%. The measurement time could be reduced significantly, completing our secondary goal. The quality of classically and in situ prepared Ag-colloids was monitored by zeta potential measurements and scanning electron microscopy (SEM) respectively. For data interpretation and statistical model-building an in house written code in the open source software RStudio was implemented. It was applied for the differentiation of PHB producers at the cellular level, revealing heterogeneities within sample groups regarding the PHB amount accumulated. The results obtained using the statistical model were validated as well and were complementary to the reference HPLC analysis. Therefore, a fast and reliable identification in situ SERS tool for the selection of the most promising cyanobacterial PHB production was established.

Biosynthesis and Characterization of Bioplastic Poly-3-hydroxybutyrate from Hydrolyzate of Ulva using Bacillus subtilis rnM

Biopolymers are a new generation biopolymers, which have wide range of applications. Poly-3-hydroxybutyrate (PHB) is one of the best biopolymers for replacement of non-biodegradable petroleum-based plastic and can be produced from organic wastes source by some bacterial strains under imbalance growth conditions. In present study, hydrolyzate was prepared from biomass of seaweed Ulva by acid pre-treated and used as a feedstock for PHB biosynthesis by Bacillu subtilis rnM. The pre-treatment was carried out by two different pre-treatment conditions such as room temperature and high temperature and pressure (HTP). The hydrolysate prepared by 2 % HCl at HTP supported for the maximum PHB biosynthesis than the other pre-treated conditions. The yield of PHB obtained by B. subtilis rnM when tested with laboratory grade sugars was lower to that achieved with hydrolysate of Ulva sp. The biosynthesized PHB was characterized by TGA, DSC, FTIR, XRD and NMR techniques.

Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system.

Polyhydroxybutyrate (PHB) is biodegradable and renewable and thus considered as a promising alternative to petroleum-based plastics. However, PHB production is costly due to expensive carbon sources for culturing PHB-accumulating microorganisms under sterile conditions. We discovered a hyper PHB-accumulating denitrifying bacterium, Zobellella denitrificans ZD1 (referred as strain ZD1 hereafter) capable of using non-sterile crude glycerol (a waste from biodiesel production) and nitrate to produce high PHB yield under saline conditions. Nevertheless, the underlying genetic mechanisms of PHB production in strain ZD1 have not been elucidated. In this study, we discovered a complete pathway of glycerol conversion to PHB, a novel PHB synthesis gene cluster, a salt-tolerant gene cluster, denitrifying genes, and an assimilatory nitrate reduction gene cluster in the ZD1 genome. Interestingly, the novel PHB synthesis gene cluster was found to be conserved among marine Gammaproteobacteria. Higher levels of PHB accumulation were linked to higher expression levels of the PHB synthesis gene cluster in ZD1 grown with glycerol and nitrate under saline conditions. Additionally, a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas type-I-E antiviral system was found in the ZD1 genome along with a long spacer list, in which most of the spacers belong to either double-stranded DNA viruses or unknown phages. The results of the genome analysis revealed strain ZD1 used the novel PHB gene cluster to produce PHB from non-sterile crude glycerol under saline conditions.

Pretreatment of kenaf (Hibiscus cannabinus L.) biomass feedstock for polyhydroxybutyrate (PHB) production and characterization

Kenaf biomass (KB) was employed as feedstock for the synthesis of polyhydroxybutyrate (PHB) using Ralstonia eutropha to replace conventional petroleum-derived polymers. Various pretreatments followed by enzymatic saccharification were applied to release monomeric sugars from KB for PHB production. The effects of increasing concentration of Na2CO3 + Na2SO3 (NaC + NaS) pretreated KB hydrolysates (20–40 g/L) on PHB production were investigated. NaC + NaS pretreated KB hydrolysates (30 g/L) exhibited maximum 70.0% PHA accumulation, with PHB titers of 10.10 g/L and PHB yields of about 0.488 g/g of reducing sugar produced within 36 h of fermentation. PHA accumulation, PHB yield and R. eutropha growth performance using KB hydrolysates were found to be comparable with those of synthetic sugar mixture. Characterization of the produced PHB in terms of crystalline structure, and thermal properties was done using various analytical techniques and results coincide with standard PHB. Thus, green liquor pretreated KB hydrolysates deliver a promising and economically feasible carbon substrate for PHB production.

Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB

Polyhydroxybutyrates (PHBs) are energy reserves synthesized by different micro-organisms such as Alcaligenes, Pseudomonas, Staphylococcus, Algae, in excess of carbon and limitation of nutrients like nitrogen. These biopolymers are suitable alternate to synthetic carbon-based polymers. However, the high production cost limits their commercialization. The aim of this study was thus, focused on optimization of culture condition for maximum PHB production in an attempt to reduce the production cost. The micro-organisms for this purpose were isolated from 4 different soil samples and screened for PHB production. Culture conditions for these organisms were optimized by changing the parameters, viz., incubation time, pH, carbon source and NaCl concentration. Thus, optimized culture condition was used to culture the isolates for extraction of PHB and its analysis. The extracted compounds on FTIR-analysis gave characteristic C=O peak of PHB, thus, confirming the seven isolates to be PHB producers. Results for optimized parameters for the isolated PHB positive species showed that synthesis of PHB was maximum at 48 hours i.e. during the early stages of stationary phase. However, different isolates favored different culture conditions. Highest PHB accumulation and growth of isolates were seen at pH 7 and 9. Similarly, it was observed that glucose was favored by 4 isolates and sucrose was favored by 3 isolates. Interestingly, NaCl concentration did not cause significant effect on neither the bacterial growth nor the PHB production. During the extraction of PHB from the optimized culture conditions, extraction of PHB from broth gave significant yield than that from agar. A good PHB yield from broth amounting to 36.41% and 34.59% was observed for Bacillus pasteurii and Micrococcus luteus respectively, showing a potential for their exploitation in industrial PHB production. At optimized conditions, 7 isolates exhibited significant PHB yields, thus showing a potential for further exploitation.

Simultaneous pollutant removal and PHB accumulation in simple anaerobic treatment of oily wastewater

Oil and grease is one of the complicated pollutants to be removed due to oil-in-water emulsions content. This research is a treatment of oily wastewater from an edible oil factory. Addition to pollutant removal, accumulation of PHB (Polyhydroxybutyrate) is investigated simultaneously. Objectives of this research are to obtain the optimum operating conditions for removal of COD and oil, grease, and accumulation of PHB. Operating parameters are acclimatization period (3-7 days), pH (6-8) and sludge concentration (25-75%). Anaerobic treatment was conducted in a bottle and put on a shaker. The wastewater from the edible oil factory was fed to reactors as the substrate and the sludge was added. The result shows that this treatment removed 59.4-90.4% of COD and 65.3-85.2% of oil and grease. Overall PHB accumulation was 0.67-6.00% of PHB/VSS, which is much lower than the pure culture. The influence of pH (6-8) is less significant to both pollutant removal and PHB accumulation. The COD removal efficiency increased with the period of acclimatization and 5 days of acclimatization shows the highest yield of PHB. 50% sludge concentration shows the highest yield of PHB, which is 6.0% PHB/VSS.

From reconstruction to C4 metabolic engineering: A case study for overproduction of polyhydroxybutyrate in bioenergy grasses

The compartmentalization of C4 plants increases photosynthetic efficiency, while constraining how material and energy must flow in leaf tissues. To capture this metabolic phenomenon, a generic plant metabolic reconstruction was replicated into four connected spatiotemporal compartments, namely bundle sheath (B) and mesophyll (M) across the day and night cycle. The C4 leaf model was used to explore how amenable polyhydroxybutyrate (PHB) production is with these four compartments working cooperatively. A strategic pattern of metabolite conversion and exchange emerged from a systems-level network that has very few constraints imposed; mainly the sequential two-step carbon capture in mesophyll, then bundle sheath and photosynthesis during the day only. The building of starch reserves during the day and their mobilization during the night connects day and night metabolism. Flux simulations revealed that PHB production did not require rerouting of metabolic pathways beyond what is already utilised for growth. PHB yield was sensitive to photoassimilation capacity, availability of carbon reserves, ATP maintenance, relative photosynthetic activity of B and M, and type of metabolites exchanged in the plasmodesmata, but not sensitive towards compartmentalization. Hence, the compartmentalization issues currently encountered are likely to be kinetic or thermodynamic limitations rather than stoichiometric.

Controlling cell volume for efficient PHB production by Halomonas

Bacterial morphology is decided by cytoskeleton protein MreB and cell division protein FtsZ encoded by essential genes mreB and ftsZ, respectively. Inactivating mreB and ftsZ lead to increasing cell sizes and cell lengths, respectively, yet seriously reduce cell growth ability. Here we develop a temperature-responsible plasmid expression system for compensated expression of relevant gene(s) in mreB or ftsZ disrupted recombinants H. campaniensis LS21, allowing mreB or ftsZ disrupted recombinants to grow normally at 30°C in a bioreactor for 12h so that a certain cell density can be reached, followed by 36h cell size expansions or cell shape elongations at elevated 37°C at which the mreB and ftsZ encoded plasmid pTKmf failed to replicate in the recombinants and thus lost themselves. Finally, 80% PHB yield increase was achieved via controllable morphology manipulated H. campaniensis LS21. It is concluded that controllable expanding cell volumes (widths or lengths) provides more spaces for accumulating more inclusion body polyhydroxybutyrate (PHB) and the resulting cell gravity precipitation benefits the final separation of cells and product during downstream.

Acid pretreatment and enzymatic saccharification of brown seaweed for polyhydroxybutyrate (PHB) production using Cupriavidus necator

The brown seaweed Sargassum sp. was used as a feedstock to produce polyhydroxybutyarte (PHB) using Cupriavidus necator PTCC 1615. In order to release monomeric sugars, dilute acid hydrolysis of Sargassum sp. biomass was followed by enzymatic saccharification. In addition, the effect of different nitrogen sources was evaluated for PHB production. The fermentation of hydrolysate with the ammonium sulfate as selected nitrogen source resulted PHB yield of 0.54±0.01g/g reducing sugar. Then, NaCl was used as external stress factor which was added to the media. Addition of 8g/L NaCl had a positive impact on high PHB yield of 0.74±0.01g/g reducing sugar. Increasing trend of NaCl concentration to 16g/L was found to inhibit the production of PHB. Based on obtained results using 20g/L of reducing sugar, at desired condition the highest cell dry weight and PHB concentrations were 5.36±0.22 and 3.93±0.24g/L, respectively. The findings of this study reveal that Sargassum sp. is a promising feedstock for biopolymer production. The characteristics of produced PHB were analyzed by FTIR, differential scanning calorimetry and 1H NMR.

Microbial production, ultrasound-assisted extraction and characterization of biopolymer polyhydroxybutyrate (PHB) from terrestrial (P. hysterophorus) and aquatic (E. crassipes) invasive weeds

This study reports synthesis of biodegradable poly(3-hydroxybutyrate) (PHB) polymer from two invasive weeds, viz. P. hysterophorus and E. crassipes. The pentose and hexose-rich hydrolyzates obtained from acid pretreatment and enzymatic hydrolysis of two biomasses were separately fermented using Ralstonia eutropha MTCC 8320 sp. PHB was extracted using sonication and was characterized using FTIR, 1H and 13C NMR and XRD. PHB content of dry cell mass was 8.1–21.6% w/w, and the PHB yield was 6.85×10−3–36.41×10−3% w/w raw biomass. Thermal properties of PHB were determined by TGA, DTG and DSC analysis. PHB obtained from pentose-hydrolyzate had glass transition temperatures of 6°–9°C, while PHB from hexose-rich hydrolyzate had maximum thermal degradation temperatures of 370°–389°C. These thermal properties were comparable to the properties of commercial PHB. Probable causes leading to differences in thermal properties of pentose and hexose-derived PHB are: extent of crystallinity and presence of impurity in the polymer matrix.

Development of polyhydroxybutyrate biosynthesis in Bacillus subtilis with combination of PHB-associated genes derived from Ralstonia eutropha and Bacillus megaterium

Abstract Polyhydroxybutyrate (PHB) is a biodegradable polymer synthesized by bacteria for carbon storage in response to environmental stress. B. subtilis is generally recognized as a safe Gram-positive bacterium and is widely used for industrial-scale production of proteins and chemicals. Owing to lack of toxic lipopolysaccharides, B. subtilis is a superior alternative to the Gram-negative bacteria presently used for PHB production. In this study, we introduced PHB synthesis genes from R. eutropha and B. megaterium into B. subtilis for PHB production. The highest yield of PHB was achieved by coexpression of phaA from R. eutropha and phaR, phaB , and phaC from B. megaterium (up to 13.02% ± 1.67% of total cell dry weight in LB medium containing 1% glucose). The results indicate that B. subtilis is potentially used as a safe vehicle for producing PHB.

Production and characterization of polyhydroxybutyrate fromVibrio harveyiMCCB 284 utilizing glycerol as carbon source

Production and characterization of polyhydroxybutyrate (PHB) from moderately halophilic bacterium Vibrio harveyi MCCB 284 isolated from tunicate Phallusia nigra. Twenty-five bacterial isolates were obtained from tunicate samples and three among them exhibited an orange fluorescence in Nile red staining indicating the presence of PHB. One of the isolates, MCCB 284, which showed rapid growth and good polymer yield, was identified as V. harveyi. The optimum conditions of the isolate for the PHB production were pH 8·0, sodium chloride concentration 20gl-1 , inoculum size 0·5% (v/v), glycerol 20gl-1 and 72h of incubation at 30°C. Cell dry weight (CDW) of 3·2gl-1 , PHB content of 2·3gl-1 and final PHB yield of 1·2gl-1 were achieved. The extracted PHB was characterized by FTIR, NMR and DSC-TGA techniques. An isolate of V. harveyi that could effectively utilize glycerol for growth and PHB accumulation was obtained from tunicate P. nigra. PHB produced was up to 72% based on CDW. This is the first report of an isolate of V. harveyi which utilizes glycerol as the sole carbon source for PHB production with high biomass yield. This isolate could be of use as candidate species for commercial PHB production using glycerol as the feed stock or as source of genes for recombinant PHB production or for synthetic biology.