Specific Product Formation Rate Research Articles

Direct control of recombinant protein production rates in E. coli fed-batch processes by nonlinear feedback linearization

Biotechnological cultivation processes aim for high and long lasting productivities. In this paper, a method to directly control the productivity of a recombinantly produced protein in an E. coli fed-batch process is introduced. After modeling the process using a nonlinear kinetic model, which includes the degeneration of the product formation capacities, a controller was derived by feedback linearization. The derived control law presents an improved and novel approach to directly influence the process parameter associated with the biomass specific product formation rate. In order to deal with observed model-plant mismatches a Two-Degrees-of-Freedom controller was implemented. A simulation study using different model parameters derived from calibration and validation data sets and two realistic measurement scenarios was carried out to demonstrate the potential of the presented method in comparison to a constant substrate addition. Compared to optimized, constant glycerol feed rates the simulations with controlled productivities led to significantly higher specific titers with the same amount of fed glycerol. The feeding rates given by the developed controller minimize the metabolic load as well as product release and therefore stabilizes the productivity for a prolonged, potentially continuous, production process.

Effect of the cerium modification on acid–base properties of Mg–Al hydrotalcite-derived oxide system and catalytic performance in ethanol conversion

The prospects of using cerium as a modifying additive of Mg–Al hydrotalcite-derived oxide system have been estimated by low-temperature nitrogen ad(de)sorption, X-ray diffraction, scanning electron microscopy, 1H and 27Al solid-state nuclear magnetic resonance (NMR), X-ray photoelectron and UV–Vis diffuse reflectance spectroscopy techniques. The effect of such tuning of structural-textural characteristics and acid–base properties of the surface on its catalytic performance in ethanol conversion has been studied. Solid-state NMR spectroscopic study of the probe molecule adsorption and temperature-programmed desorption of NH3 and CO2 with mass-spectrometry control are used to determine the nature and strength distribution of acidic and basic sites of Mg–Al(–Ce) oxide systems. It has been found that the selectivity towards 1-butanol up to 68.1% (548 K) is achieved in ethanol conversion over Mg–Al–Ce sample at time-on-stream ≤ 2 h. At higher time-on-stream, the specific rates of the target product (1-butanol) formation are comparable for both modified and un-modified catalysts, but the products distribution differs. Cerium modification of Mg–Al oxide catalyst leads to increase in specific rate of acetaldehyde formation due to increase in the number of basic sites and their surface density. Wherein, the rate of ethanol dehydration products (ethylene and diethyl ether) is reduced, apparently, due to decrease in a number of Lewis acidic sites formed by Al3+ cations octahedrally coordinated to oxygen.

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris.

Random mutagenesis of super Koji (Aspergillus oryzae): improvement in production and thermal stability of \u03b1-amylases for maltose syrup production

BackgroundAlpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto-oligosaccharides. It is an important enzyme generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought after properties for industrial use. The Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and safe for use in food industry. Hence, Koji strain’s development for the screening of potent mutants, hyper producer of thermostable α-amylases, with desired attributes is the need of the time.ResultsA process has been developed to improve super Koji (A. oryzae cmc1) strain through γ-rays treatment. The doses i.e. 0.60, 0.80, 1.00, 1.20 & 1.40 KGy gave more than 3.0 log kill. Initially, 52 Koji mutants resistant to 1% (w/v) Triton X-100 were selected. 2nd screening was based on α-amylases hyper production and 23 mutants were sorted out by measuring clearing zones index (CI). Afterwards nine potent mutants, resistant to 2-deoxy D-glucose, were screened based on CI. These were further analyzed for thermal stability and productivity of α-amylase under submerged conditions. The mutants’ M-80(10), M-100(6) & M-120(5) gave about four fold increases in α-amylases productivity. The half life of M-100(6) α-amylase at 55 °C was 52 min and was highest among the mutants. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia depicted that exposure to gamma rays increased rigidity of the mycelium. The potent Koji mutant M-100(6) was grown on soluble starch in 10L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1 protein. Growth kinetic parameters were: μ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1.ConclusionIt was concluded that the developed five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ-rays mediated physical mutagenesis. The developed thermostable α-amylases of super Koji mutantM-100(6) has immense potential for application in saccharification process for maltose syrup production. Moreover, the developed five step strain’s development process may be used for the simultaneous improvement in productivity and thermal stability of other microbial enzymes.

Enzyme/whole-cell biotransformation of plant oils, yeast derived oils, and microalgae fatty acid methyl esters into n-nonanoic acid, 9-hydroxynonanoic acid, and 1,9-nonanedioic acid

Oils and fatty acids are important renewable resources provided by nature. Therefore, biotransformation of renewable oils and fatty acids into industrially relevant C9 chemicals was investigated in this study. Olive oil, soybean oil, yeast derived oil, and microalgae fatty acid methyl esters were converted into n-nonanoic acid, 9-hydroxynonanoic acid, and 1,9-nonanedioic acid by a lipase and a recombinant Escherichia coli expressing oleate hydratase, long chain secondary alcohol dehydrogenase, Baeyer-Villiger monooxygenase, long chain primary alcohol dehydrogenase, and aldehyde dehydrogenase. It was found that n-nonanoic acid and azelaic acid could be produced to a concentration of 4.3 mM from 3 g/L olive oil with a specific product formation rate of 3.1 U/g dry cells. Biotransformation rates were influenced by compositions of fatty acids and purity of the starting material. This study may contribute to the production of industrially relevant C9 chemicals from renewable oils and fatty acids by simultaneous enzyme/whole-cell biotransformation.

Double promoter expression systems for recombinant protein production by industrial microorganisms.

Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more effective and adaptive processes to meet the increasing demand of r-protein industry.

Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations.

Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of200-400U(gxh)-1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01-0.35 with smooth change of D at a rate of 0.003 h-2. EPG production was found to be comparable with a qp of 400 U (gx h)-1 at D = 0.27 h-1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.

Semi-Continuous Fermentation of Onion Vinegar and Its Functional Properties.

For the fermentation of vinegar using onion, acetic acid bacteria and yeast strains with high fermentation ability were screened. Among them, Saccharomyces cerevisiae 1026 was selected as a starter for ethanol production and Acetobacter orientalis MAK88 was selected as a vinegar producer. When the two-stage fermentation of onion vinegar was performed at 28 °C, the titratable acidity reached 4.80% at 24 h of fermentation. When semi-continuous fermentation proceeded to charge-discharge consisting of three cycles, the acetic acid content reached 4.35% at 48 h of fermentation. At this stage, the fermentation efficiency, acetic acid productivity, and specific product formation rate were 76.71%, 17.73 g/(L·d), and 20.58 g/(g·h), respectively. The process in this study significantly reduced the fermentation time and simplified the vinegar production process. The content of total flavonoids and total polyphenols in onion vinegar were 104.36 and 455.41 μg/mL, respectively. The antioxidant activities of onion vinegar in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS+) radical scavenging activity, and reducing power were 75.33%, 98.88%, and 1.28, respectively. The nitrite scavenging abilities of onion vinegar were 95.38 at pH 1.2. The onion vinegar produced in this study showed higher organoleptic acceptability than commercial onion vinegar.

Mathematical modeling of biotechnological process of lactic acid production by batch fermentation: A review

The results of analyzing mathematical models of the process of batch fermentation for the production of lactic acid have been presented. Three groups of mathematical models have been considered. The first two groups are based exclusively on the notion of the specific growth rate, dependent or independent on substrate concentration. The third group uses the notions of the specific rate of substrate consumption and specific rate of product formation in additions to the notion of specific growth rate. The numeric estimates of constants are presented for the majority of models as well as comparison with the experimental studies. Mathematical models of the processes where the formation of side products is possible have been discussed, which may or may not be of value on their own. The possible practical applicability of the models has been evaluated based on the results of the analysis.

Unstructured mathematical models of lactic acid biosynthesis kinetics: A review

The publications on the kinetics of lactic acid biosynthesis were analyzed. The review mainly considers foreign publications. The equations formed using the unstructured approach were given. Special emphasis was laid on the equations that received the best experimental rationale. The equations include substrate and product inhibition. The effect of medium acidity on the kinetic constants was evaluated. A nearly neutral medium was shown to be most acceptable for the synthesis of lactic acid. The kinetic equations including the specific product formation and substrate utilization rates in addition to the specific biomass formation rate were presented. The values of kinetic constants were given.

Utilization of Agro-Industry Residue for Rhamnolipid Production by P. aeruginosa AMB AS7 and Its Application in Chromium Removal.

Coconut oil sludge and oil cake was utilized as carbon source for biosurfactant production by Pseudomonas aeruginosa AMB AS7. The results of optimization study revealed that 1.5% (w/v) of coconut oil cake, 2% (w/v) of coconut oil sludge, pH7.2, 37°C, and 120rpm were the optimum conditions for biosurfactant production. The yield coefficient of biosurfactant on biomass (Y P/X ) was 1.29g/g. Besides, the results indicated that aeration of 0.5vvm and agitation of 450rpm in bioreactor resulted in high volumetric productivity of biosurfactant (r p ) and specific product formation rate (q p ) of 0.115g/(Lh) and 0.0131g/(gh), respectively in medium containing 2% (w/v) coconut oil sludge. The maximum biosurfactant concentration of 5.53g/L was obtained during 60h of cultivation. The emulsification index (EI24) against coconut oil was found to be 88.42±0.5%, and cell surface hydrophobicity of P. aeruginosa AMB AS7 was obtained 32.4±0.9%. FTIR and GC-MS analysis revealed that the biosurfactant is rhamnolipid with anionic charge. The critical micelle concentration (CMC) of rhamnolipid was found to be 50mg/L. It was found that 66.95% of chromium from aqueous solution can be removed using rhamnolipid at its CMC.

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (qp) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l−1) and at a growth rate close to μmax (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of qp > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated qp occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the qp(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in qp were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

High titer heterologous rhamnolipid production.

Heterologous mono-rhamnolipid production by Pseudomonas putida KT2440 pSynPro8oT_rhlAB using glucose as the single carbon source was characterized in fed-batch bioreactor cultivations. For the described experiments, a defined mineral salt medium was used, and a two phase glucose feeding profile was applied, which yielded a final rhamnolipid concentration of 14.9 g/L. Applying the feeding profile, glucose stayed almost constant until 28 h of cultivation and decreased afterwards to limiting levels. Until the end of cultivation 253.0 ± 0.1 g glucose was added to the bioreactor of which a total of 252.0 ± 0.6 g glucose was metabolized. By modeling the fed-batch bioreactor cultivations the time courses of generated biomass, rhamnolipid and consumed glucose were described. The model was furthermore used to derive key process parameters from the collected data. The obtained values for the specific product formation rates (qRL) reached 18 mg/(g h) and yield coefficients (YRL/S) 10 mg/g respectively.

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells.

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg(-1) ). Hyperosmotic culture conditions caused a ∼2.6-fold increase of specific ATP formation rates together with a ∼1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance qp by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj etal., 2010; Andrew etal., 2011, Parashar etal., 2014 & Venkatachalam etal., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.

Analysis of reaction kinetics during chemostat cultivation of Saccharomyces cerevisiae using a multiphase microreactor

This paper presents the determination of reaction kinetic parameters and the kinetic analysis of a chemostat cultivation performed using a multiphase microreactor (mMR). Saccharomyces cerevisiae was cultivated aerobically in continuous chemostat mode using an mMR. Steady-state biomass, substrate and ethanol concentrations were determined at dilution rates between 0.14≤D≤0.42h−1 with a glucose-feed concentration of 10gL−1. Modelling the aerobic chemostat culture was based on stationary balance equations. Maximal specific growth rate and Monod constant were determined using different linearization methods. The aerobic yeast metabolism was considered using two validity ranges of the model: (a) with purely oxidative metabolism in which glucose was converted into biomass or was used for maintenance metabolism, and no ethanol was generated; (b) with oxido-reductive metabolism employing an active Crabtree effect, in which ethanol was generated at the expense of biomass production. The different yield coefficients could be determined using the plots of the specific substrate consumption rate qS=f(D) and the specific product formation rate qP=f(D), respectively. Using this model, the reaction kinetic parameters were determined from the stationary concentrations of the biomass, glucose and ethanol that were determined during aerobic cultivation in the mMR. Finally, the kinetic parameters were compared with those reported in the literature that had been obtained using laboratory-scale reactors.

Utilization of waste engine oil by Ochrobactrum pseudintermedium strain C1 that secretes an exopolysaccharide as a bioemulsifier

Abstract Waste engine oil rich in toxic hydrocarbon pollutants was used in this study as cheap alternative substrate for bioemulsifier production by Ochrobactrum pseudintermedium sp. C1. During growing on waste engine oil as sole carbon source, the strain C1 released an exopolysaccharide exhibiting bioemulsifying activity into the surrounding BH medium. Optimization of production medium by response surface methodology enhanced the bioemulsifier yield from 1.26 g/L to 2.54 g/L. The maximum emulsifier production (2.54±0.3 g/L) and emulsifying activity (EI=85±4%) in cell free optimized culture medium were recorded at 120 h of incubation. Kinetics of growth and production revealed that the bioemulsifier product is mixed growth associated. The maximum specific growth rate and specific product formation rate were calculated to be 0.29 h −1 and 0.075 g g −1 h −1 respectively. The bioemulsifier was characterized by FT-IR, NMR and SEM, found to be a glycoprotein composed of 61.5% carbohydrates, 29.2% proteins and 9.3% lipids. The emulsifying agent maintained its properties over a wide range of pH (2–8), at moderate salinity (4–6% NaCl), and during exposure to elevated temperatures (100 °C). The bioemulsifier was effective at these extreme environmental conditions and was able to emulsify various petroleum fractions which suggest its potential application in petroleum industries that require the use of effective compounds at low cost.

Improved Dynamic System of Microbial Continuous Fermentation and its Parameter Identification

In this paper, the nonlinear dynamic system of microbe continuous fermentation products 1,3-propanediol (1,3-PD) is rewritten by improving the specific cellular growth rate, specific substrate consumption rate and specific product formation rate. Firstly, under the condition of substrate glycol excess and active trans-membrane transport, according to the dynamic behavior the fermentation process, we consider the glycerol and 1,3–PD concentration within the cell, and improve the specific cellular growth rate, specific substrate consumption rate and specific product formation rate, then rewrite the dynamic system of microbial continuous fermentation process. Secondly, taking the dynamic system as main constraint condition, we establish the parameter identification model and prove the existence of the optimal solution. Lastly, the numerical results calculated by particle swarm algorithm show that the improved model is suitable for describe the dynamic behavior of 1,3-PD, but is not accurate enough for by-products.

Important physiological features of the lactic acid bacterium employed in lactic acid production

One of the key characteristics of industrial bioprocesses is near maximum specific product formation rate and it can be attained as result of focused metabolic activity of employed microorganism under operational conditions. Most of the current knowledge about the metabolic activity of traditional industrial microorganisms - lactic acid bacteria includes relatively simple metabolic network based on carbohydrate catabolism and lactate production. However, recent investigations of metabolic flexibility of the lactic acid bacteria showed need for further investigation of their metabolic potential which can significantly improve efficiency of the industrial bioprocesses. Physiology of the lactic acid bacterium Lactobacillus coryniformis was investigated in a chemically defined medium containing glucose as main carbon and energy source. Batch cultivations were carried out at relatively low temperature and effect of dissolved oxygen and carbon dioxide concentration on bacterial growth was investigated. Limitation with carbon and energy source and accumulation of biomass was connected with D-lactic acid and other end-products formation.

Biosynthesis of Poly-3-Hydroxybutyrate (PHB) from Glycerol by Paracoccus denitrificans in a Batch Bioreactor: Effect of Process Variables

In this study, the kinetics of poly-3-hydroxybutyrate (PHB) biosynthesis from glycerol by Paracoccus denitrificans DSMZ 413 were explored in a batch bioreactor. Effects of inorganic and organic nitrogen source, carbon to nitrogen ratio, and other process variables such as pH, aeration, and initial glycerol concentration on PHB production were investigated in a 2.5-L bioreactor. Yeast extract was found to be the best nitrogen source compared to several organic nitrogen sources tested. At pH 6, specific growth rate, product formation rate, and accumulation of PHB within the cell were maximum. Specific growth rate increased with increase in oxygen transfer rate, but moderate oxygen transfer rate promoted PHB production. High glycerol concentration inhibited specific product formation rate but not growth. High initial carbon/nitrogen (C/N) ratio favored PHB accumulation and its productivity. At a C/N ratio of 21.4 (mol mol−1), 10.7 g L−1 of PHB corresponding to 72% of cell dry weight was attained.