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.

Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator.

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

Gram-scale production of the sesquiterpene α-humulene with Cupriavidus necator.

Terpenoids have an impressive structural diversity and provide valuable substances for a variety of industrial applications. Among terpenes, the sesquiterpenes (C15 ) are the largest subclass with bioactivities ranging from aroma to health promotion. In this article, we show a gram-scale production of the sesquiterpene α-humulene in final aqueous concentrations of 2 g L-1 with the recombinant strain Cupriavidus necator pKR-hum in a fed-batch mode on fructose as carbon source and n-dodecane as an extracting organic phase for in situ product removal. Since C. necator is capable of both heterotrophic and autotrophic growth, we additionally modeled the theoretically possible yields of a heterotrophic versus an autotrophic process on CO2 in industrially relevant quantities. We compared the cost-effectiveness of both processes based on a production of 10 t α-humulene per year, with both processes performing equally with similar costs and gains. Furthermore, the expression and activity of 3-hydroxymethylglutaryl-CoA reductase (hmgR) from Myxococcus xanthus was identified as the main limitation of our constructed C. necator pKR-hum strain. Thus, we outlined possible solutions for further improvement of our production strain, for example, the replacement of the hmgR from M. xanthus by a plant-based variant to increase α-humulene production titers in the future.

Transcriptome differences between Cupriavidus necator NH9 grown with 3-chlorobenzoate and that grown with benzoate.

RNA-seq analysis of Cupriavidus necator NH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis-related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semisolid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.

Rapid analysis of polyhydroxyalkanoate contents and its monomer compositions by pyrolysis-gas chromatography combined with mass spectrometry (Py-GC/MS).

Here, we report an analysis method for determining PHA (polyhydroxyalkanoates) contents and their monomer composition in microbial cells based on pyrolysis gas chromatography combined with mass spectrometry (Py-GC/MS). Various kinds of microbial cells accumulating different PHA contents and monomer compositions were prepared through the cultivation of Ralstonia eutropha and recombinant Escherichia coli. Py-GC/MS could analyse these samples in a short time without complicated pretreatment steps. Characteristic peaks such as 2-butenoic acid, 2-pentenoic acid, and hexadecanoic acid regarding PHA compositions and cell components were identified. Considering constituents of cells and ratios of peak areas of dehydrated monomers to hexadecanoic acid, a simple equation for estimation of PHA contents in microbial cells was derived. Also, monomer compositions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in R. eutropha could be successfully determined based on peak area of 2-butenoic acid and 2-pentenoic acid of Py-GC/MS, which are the corresponding species of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in PHBV. Correlation of results between GC-FID and Py-GC/MS could be fitted very well. This method shows similar results for the samples obtained from same experimental conditions, allowing rapid and reliable analysis. Py-GC/MS can be a promising tool to rapidly screen PHA-positive strains based on polymer contents along with monomer compositions.

Engineering the Calvin-Benson-Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha for improved autotrophic growth and polyhydroxybutyrate production.

BACKGROUND: CO2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin-Benson-Bassham (CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO2 fixation efficiency of cells is an important subject of biological research with significant application potential. RESULTS: In this work, the autotrophic microbe Ralstonia eutropha (Cupriavidus necator) was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha, we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were functional during autotrophic growth of R. eutropha. SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system(A quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins) and RbcX(A chaperone in the folding of Rubisco), the culture OD600 of engineered strain increased 89.2% after 72 h of autotrophic growth, although the difference was decreased at 96 h, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and lead to improved growth in comparison to the host specific enzyme. Meanwhile, expression of hydrogenases was optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD600 to 93.4% at 72 h. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.7%. CONCLUSIONS: To our best knowledge, this is the first report of successfully engineering the CBB pathway and hydrogenases of R. eutropha for improved activity, and is one of only a few cases where the efficiency of CO2 assimilation pathway was improved. Our work demonstrates that R. eutropha is a useful platform for studying and engineering the CBB for applications.

Cupriavidus necator B-10646 growth and polyhydroxyalkanoates production on different plant oils.

The study addresses the growth of the wild-type strain Cupriavidus necator B-10646 and synthesis of polyhydroxyalkanoates by this strain on media containing plant oils with different compositions of fatty acids: palm, Siberian oilseed, and refined and unrefined sunflower seed oils. The study showed that the best carbon substrate was palm oil. Comparison of fatty acid compositions of the starting oils and unutilized residual substrates showed that C. necator B-10646 cells consumed the fatty acids from palm oil evenly while in experiments with other oils, they utilized polyenoic fatty acids first. Higher production parameters of the culture were obtained by preparation of emulsified oil medium using Tween 80 and sodium cocoyl glutamate as emulsifiers. All polyhydroxyalkanoate specimens were terpolymers that contained 3-hydroxybutyrate as the major component and minor amounts of 3-hydroxyvalerate (0.9-1.9 mol%) and 3-hydroxyhexanoate (0.5-1.1 mol%). Molecular weight of polyhydroxyalkanoate specimens depended on the type of plant oil and emulsifier.

Applying Statistical Design of Experiments To Understanding the Effect of Growth Medium Components on Cupriavidus necator H16 Growth.

Cupriavidus necator H16 is gaining significant attention as a microbial chassis for range of biotechnological applications. While the bacterium is a major producer of bioplastics, its lithoautotrophic and versatile metabolic capabilities make the bacterium a promising microbial chassis for biofuels and chemicals using renewable resources. It remains necessary to develop appropriate experimental resources to permit controlled bioengineering and system optimization of this microbe. In this study, we employed statistical design of experiments to gain understanding of the impact of components of defined media on C. necator growth and built a model that can predict the bacterium's cell density based on medium components. This highlighted medium components, and interaction between components, having the most effect on growth: fructose, amino acids, trace elements, CaCl2, and Na2HPO4 contributed significantly to growth (t values of <-1.65 or >1.65); copper and histidine were found to interact and must be balanced for robust growth. Our model was experimentally validated and found to correlate well (r2 = 0.85). Model validation at large culture scales showed correlations between our model-predicted growth ranks and experimentally determined ranks at 100 ml in shake flasks (ρ = 0.87) and 1 liter in a bioreactor (ρ = 0.90). Our approach provides valuable and quantifiable insights on the impact of medium components on cell growth and can be applied to model other C. necator responses that are crucial for its deployment as a microbial chassis. This approach can be extended to other nonmodel microbes of medical and industrial biotechnological importance.IMPORTANCE Chemically defined media (CDM) for cultivation of C. necator vary in components and compositions. This lack of consensus makes it difficult to optimize new processes for the bacterium. This study employed statistical design of experiments (DOE) to understand how basic components of defined media affect C. necator growth. Our growth model predicts that C. necator can be cultivated to high cell density with components held at low concentrations, arguing that CDM for large-scale cultivation of the bacterium for industrial purposes will be economically competitive. Although existing CDM for the bacterium are without amino acids, addition of a few amino acids to growth medium shortened lag phase of growth. The interactions highlighted by our growth model show how factors can interact with each other during a process to positively or negatively affect process output. This approach is efficient, relying on few well-structured experimental runs to gain maximum information on a biological process, growth.

Plasmid expression level heterogeneity monitoring via heterologous eGFP production at the single-cell level in Cupriavidus necator.

A methodology for plasmid expression level monitoring of eGFP expression suitable for dynamic processes was assessed during fermentation. This technique was based on the expression of a fluorescent biosensor (eGFP) encoded on a recombinant plasmid coupled to single-cell analysis. Fluorescence intensity at single-cell level was measured by flow cytometry. We demonstrated that promoter evaluation based on single-cell analysis versus classic global analysis brings valuable insights. Single-cell analysis pointed out the fact that intrinsic fluorescence increased with the strength of the promoter up to a threshold. Beyond that, cell permeability increases to excrete the fluorescent protein in the medium. The metabolic load due to the increase in the eGFP production in the case of strong constitutive promoters leads to slower growth kinetics compared with plasmid-free cells. With the strain Cupriavidus necator Re2133, growth rate losses were measured from 3% with the weak constitutive promoter Plac to 56% with the strong constitutive promoter Pj5. Through this work, it seems crucial to find a compromise between the fluorescence intensity in single cells and the metabolic load; in our conditions, the best compromise found was the weak promoter Plac. The plasmid expression level monitoring method was tested in the presence of a heterogeneous population induced by plasmid-curing methods. For all the identified subpopulations, the plasmid expression level heterogeneity was significantly detected at the level of fluorescence intensity in single cells. After cell sorting, growth rate and cultivability were assessed for each subpopulation. In conclusion, this eGFP biosensor makes it possible to follow the variations in the level of plasmid expression under conditions of population heterogeneity.Key Points•Development of a plasmid expression level monitoring method at the single-cell level by flow cytometry.•Promoter evaluation by single-cell analysis: cell heterogeneity and strain robustness.•Reporter system optimization for efficient subpopulation detection in pure cultures.

A study on the effects of increment and decrement repeated fed-batch feeding of glucose on the production of poly(3-hydroxybutyrate) [P(3HB)] by a newly engineered Cupriavidus necator NSDG-GG mutant in batch fill-and-draw fermentation.

This study investigates the effect of strategies on poly(3-hydroxybutyrate) [P(3HB)] production in bioreactor. In the production of P(3HB), urea and glucose feeding streams were developed to characterize the fed-batch culture conditions for new Cupriavidus necator NSDG-GG mutant. Feeding urea in repeated fed-batch stage (RFB-I) at 6, and 12 h in cultivation led to insignificant kinetic effect on the cell dry mass (CDM) and P(3HB) accumulation. Feeding glucose in repeated fed-batch stage (RFB-II) demonstrated that the incremental feeding approach of glucose after urea in fill-and-draw (F/D) mode at 24, 30, 36, 42, and 48 h in fermentation increased CDM and P(3HB) concentration. In the 1st cycle in RFB-II, the cumulative CDM reached the value of 26.22 g/L and then it increased with the successive repeated fed-batches to attain biomass of 145 g/L at the end of 5th cycle of RFB-II. The final cumulative P(3HB) concentration at the end of 5th cycle of RFB-II reached 111 g/L with the overall yield of 0.50 g P(3HB) g gluc- 1; the CDM productivity from the RFB-II cycles was in the range of 0.84-1.3 g/(L·h). The RFB-II of glucose in an increment mode produced nearly 2.2 times more increase in CDM and P(3HB) productivities compared to the decrement RFB-II mode. Repeated cultivation had also the advantage of avoiding extra time required for innoculum preparation, and sterilization of bioreactor during batch, thereby it increased the overall industrial importance of the process.

Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity.

BACKGROUND: The chemolithoautotrophic ß-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used. RESULTS: Growth, bioenergetic properties, and oxidation-reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L-1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)-1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N'-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH. CONCLUSIONS: Our results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.

High cell density culture of Cupriavidus necator H16 and improved biological recovery of polyhydroxyalkanoates using mealworms.

The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68-70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value <90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight.

Synthesis of Polyhydroxyalkanoates by Hydrogen-Oxidizing Bacteria in a Pilot Production Process.

The synthesis of polyhydroxyalkanoates (PHAs) was scaled up to pilot production in a 150-L fermenter on sugars (fructose and glucose) and purified and crude glycerol in a culture of the wild-type strain Cupriavidus necator B-10646. Over 60 h of cultivation, a cell concentration of 150-160 g/L was obtained on purified glycerol and glucose; cultivation on fructose and crude glycerol resulted in a cell concentration of 130 ± 10 g/L. Polymer content and yield coefficients for the biomass were similar on all substrates (80-85 wt % and 0.29-0.33 kg biomass/kg carbon substrate, respectively). Copolymers poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and terpolymers poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) having a decreased degree of crystallinity (36-46%) were first synthesized in the scaled-up process using C. necator B-10646 cultivated on glycerol. These results will provide the basis for scaling-up PHA synthesis in an organotrophic C. necator B-10646 culture.

Comparison of acid and enzymatic hydrolysis of pectin, as inexpensive source to cell growth of Cupriavidus necator.

Abstract: The present work investigated what the appropriate methods of hydrolysis of pectin for reducing compounds (RCs) production, employed as a substrate for cell growth of Cupriavidus necator. This microorganism has great importance industrial, because besides potential single cell protein (SCP), is the most studied microorganism for production of polyhydroxybutyrate (PHB), and both processes require high cell concentration with inexpensive substrates For this, it was compared to acid and enzymatic hydrolysis procedures, through rotational central composite experimental design, using pectin concentration (1.0%). It was analyzed as a variable response for both experimental design, the RCs' production. The best conditions of each procedure were used in study kinetics of RCs' production and as a substrate for cell growth of C. necator. The results indicated that the enzymatic hydrolysis method was the most efficient, with a 93.0% yield of RCs, while the yield for acid hydrolysis was 60.0%. The optimum conditions for enzymatic hydrolysis were an enzyme concentration of 10.01 UI/g (International Unit of enzyme per gram of pectin) and an agitation speed of 230.3 rpm. C. necator showed satisfactory growth in the media containing pectin hydrolysates, with specific growth rates (µMax) similar to those reported for other substrates.

Production and recovery of poly-3-hydroxybutyrate bioplastics using agro-industrial residues of hemp hurd biomass.

The present study describes production and recovery of poly(3-hydroxybutyrate) P(3HB) from agro-industrial residues. Production was conducted using Ralstonia eutropha strain with hemp hurd biomass hydrolysates sugars as a carbon source and ammonium chloride as the nitrogen source. Results show that maximum hydrolysis yield of 72.4% was achieved with total sugar hydrolysate concentration (i.e., glucose and xylose) of 53.0 g/L. Sugar metabolism by R. eutropha showed preference for glucose metabolism over xylose. Under optimum conditions, cells can accumulate P(3HB) polymer in quantity up to 56.3 wt% of the dry cell weight. This corresponds to total production of 13.4 g/L (productivity of 0.167 g/L h). Nitrogen source showed no adverse effect on P(3HB) biosynthesis, but rather on cell growth. Among several examined recovery techniques, ultrasonic-assisted sodium dodecyl sulfate (SDS) recovered bioplastic directly from the broth cell concentrate with P(3HB) content of 92%. Number average molecular weights (Mn) of final recovered bioplastic were in the range of 150-270 kDa with polydispersity index (Mw/Mn) in the range of 2.1-2.4.

Discarded Egg Yolk as an Alternate Source of Poly(3-Hydroxybutyrate-co-3-hydroxyhexanoate).

Many poultry eggs are discarded worldwide because of infection (i.e., avian flu) or presence of high levels of pesticides. The possibility of adopting egg yolk as a source material to produce polyhydroxyalkanoate (PHA) biopolymer was examined in this study. Cupriavidus necator Re2133/pCB81 was used for the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) or poly(3HHx), a polymer that would normally require long-chain fatty acids as carbon feedstocks for the incorporation of 3HHx monomers. The optimal medium contained 5% egg yolk oil and ammonium nitrate as a nitrogen source, with a carbon/nitrogen (C/N) ratio of 20. Time course monitoring using the optimized medium was conducted for 5 days. Biomass production was 13.1 g/l, with 43.7% co-polymer content. Comparison with other studies using plant oils and the current study using egg yolk oil revealed similar polymer yields. Thus, discarded egg yolks could be a potential source of PHA.

Cometabolic degradation of bisphenol A by pure culture of Ralstonia eutropha and metabolic pathway analysis.

Bisphenol A (BPA) is a toxic compound emitting to the environment mainly by polycarbonate production facilities. In this research, BPA with the initial concentrations in the range of 1-40 mg l-1 was degraded by Ralstonia eutropha. The bacteria were unable to use BPA as the sole carbon source. Therefore, resting and growing cells of phenol-adapted R. eutropha were used for cometabolic biodegradation of BPA with phenol at the concentration of 100 mg l-1. The optimum initial concentrations of BPA were 20 mg l-1 in both approaches of cometabolism. By using resting cells, BPA removal efficiency (RE) reached to 57%, however, RE decreased to 37% by growing cells in the presence of phenol. BPA-degrading activity was inhibited at BPA concentrations >20 mg l-1. Liquid chromatography-mass spectrometry technique was used to identify some metabolic intermediates generated during BPA degradation process as 1,2-bis(4-hydroxyphenyl)-2-propanol, 4-(2-propanol)-phenol, 4-hydroxyacetophenone, 4-isopropenylphenol, and 4-hydroxybenzoic acid. Finally, metabolic pathways for BPA degradation were proposed in this study.

Experimentation and CFD modeling of continuous degradation of formaldehyde by immobilized Ralstonia eutropha in a semi-pilot-scale plug flow bioreactor.

This study focuses on continuous formaldehyde (FA) biodegradation by Ralstonia eutropha immobilized on polyurethane foam in a semi-pilot-scale plug flow packed-bed bioreactor. The stepwise increasing of the influent FA concentration from 43.9 to 1325.1 mg L-1 was studied in the bioreactor during 70 days of operation. A complete removal of FA was achieved for inlet concentration up to 425.5 mg L-1 and the initial specific biodegradation rate reached to its maximum value about 44.3 mg gcell-1 h-1 at 425.5 mg L-1. However, further increase of inlet concentration resulted in decrease of the biodegradation performance of the immobilized cells due to the inhibitory effect of FA on the enzymatic system involved in the biodegradation process. Based on kinetic modeling results, the Luong equation with the following constants could best describe the behavior of the bio-system: maximum specific FA biodegradation rate (qmax) of 124 mg gcell-1 h-1, half-saturation constant (KS) of 337.2 mg L-1, maximum degradable FA concentration (Smax) of 1582 mg L-1, and shape factor (n) of 1.49. Also, three-dimensional simulation of the bioreactor was performed using an integrated computational fluid dynamics (CFD) approach that takes into account both the biokinetic constants of the immobilized system as well as the fluid properties under steady-state condition. Eulerian computations successfully anticipated the concentration gradients through the reactor for different inlet FA concentrations, and uniform vertical velocity pathlines and non-dispersed plug flow inside the reactor were verified by the presented velocity distribution and flow streamlines.

Polyhydroxyalkanoate synthesis based on glycerol and implementation of the process under conditions of pilot production.

The present study addresses the synthesis and properties of polyhydroxyalkanoates (PHA) of different composition synthesized by Cupriavidus eutrophus B-10646 using glycerol as a carbon substrate. Poly(3-hydroxybutyrate) [P(3HB)] was effectively synthesized in fed-batch culture in a 30-L fermenter on glycerol of various purification degrees, with 99.5, 99.7, and 82.1% content of the main component. Purified glycerol (99.7%) was used for 150-L pilot scale fermentation. The total biomass and P(3HB) concentration reached 110 and 85.8 g/L, respectively, after 45 h of fed-batch fermentation. An average volumetric productivity of P(3HB) was 1.83 g/(L h). The degree of crystallinity and molecular weight of P(3HB) synthesized on glycerol were lower than and temperature characteristics were the same as those of P(3HB) synthesized on sugars.

Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 of Ralstonia eutropha Are Phosphorylated In Vivo.

In this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase.IMPORTANCE Poly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation, Cupriavidus necator) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conducted in vitro and in vivo analyses of PHB synthase activity and PHB contents.