Liliana Model Sets 1-100
Items 1 - 10 of 10 - A scale model is most generally a physical representation of an object, which maintains. Static aircraft scale modeling falls broadly into three.
This paper presents a systematic investigation into monomer development during mixed culture Polyhydroxyalkanoates (PHA) accumulation involving concurrent active biomass growth and polymer storage. A series of mixed culture PHA accumulation experiments, using several different substrate-feeding strategies, was carried out. The feedstock comprised volatile fatty acids, which were applied as single carbon sources, as mixtures, or in series, using a fed-batch feed-on-demand controlled bioprocess.
A dynamic trend in active biomass growth as well as polymer composition was observed. The observations were consistent over replicate accumulations. Metabolic flux analysis (MFA) was used to investigate metabolic activity through time. It was concluded that carbon flux, and consequently copolymer composition, could be linked with how reducing equivalents are generated. This paper presents a systematic investigation into monomer development during mixed culture Polyhydroxyalkanoates (PHA) accumulation involving concurrent active biomass growth and polymer storage. A series of mixed culture PHA accumulation experiments, using several different substrate-feeding strategies, was carried out.
The feedstock comprised volatile fatty acids, which were applied as single carbon sources, as mixtures, or in series, using a fed-batch feed-on-demand controlled bioprocess. A dynamic trend in active biomass growth as well as polymer composition was observed. The observations were consistent over replicate accumulations. Metabolic flux analysis (MFA) was used to investigate metabolic activity through time. It was concluded that carbon flux, and consequently copolymer composition, could be linked with how reducing equivalents are generated. IntroductionPolyhydroxyalkanoates (PHAs) are biobased and biodegradable polyesters. PHA copolymers, such as poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV), are of particular interest as they are the basis for biomaterials with desirable mechanical properties.
These copolymers can be produced in mixed microbial cultures. However, predicting and controlling the copolymer composition can be challenging.PHAs are most typically synthesized in mixed microbial cultures from volatile fatty acids (VFAs), through well-described metabolic pathways ,. In the specific case of PHBV, short chain acids such as acetic and propionic acids are transported though the cell membrane and converted into acetyl-CoA and propionyl-CoA respectively. PHA synthesis then takes place in three steps. Firstly, two acyl-CoA molecules are condensed in a reaction catalyzed by a thiolase to produce various intermediates. For example, two acetyl-CoA monomers form acetoacetyl-CoA (a 3-hydroxybutyrate (3HB) precursor), while one acetyl-CoA and one propionyl-CoA combine to form ketovaleryl-CoA (a 3-hydroxyvalerate (3HV) precursor) , Escapa et al. In addition, it has been observed that a portion of the propionyl-CoA produced is converted into acetyl-CoA though different pathways.
Secondly, a reduction, catalyzed by a reductase, produces 3-hydroxyalkanoate (3HA) monomers, with the reducing power to support PHA production being generated during anabolic pathways for cell growth, as well as in reactions related to the tricarboxylic acid (TCA) cycle. Finally, a polymerase adds 3HA monomers to the PHA polymer. As such, the flux of carbon through the acyl-CoA intermediates influences the resulting polymer composition.The fraction of 3HV monomer units in the final PHBV copolymer can be manipulated by adjusting the proportion of even-chain (i.e., acetic acid) to odd-chain (i.e., propionic acid) fatty acids in the feed composition , since odd-chain fatty acids are generally required for the formation of propionyl-CoA, which is the precursor of 3HV monomer. Diverse monomer compositions and sequence distributions of PHBV copolymers produced by mixed microbial cultures have been achieved using different feeding strategies with acetic and propionic acid mixtures as model substrates ,.Most mixed microbial culture accumulation studies have been applied under conditions of some form of nutrient starvation to inhibit cell growth and favor PHA synthesis ,.
In contrast, a recent study has shown that PHA storage can occur concurrently with active biomass growth. Valentino et al. achieved a consistent improvement of PHA productivity when N and P were supplied in an optimal C:N:P ratio. It is important to consider that shifts in the active biomass growth rates may influence carbon flux through the acyl-CoA intermediates and the availability of reducing equivalents for PHA synthesis, and therefore affect polymer production and composition.Literature on mixed microbial culture PHA production coupled with high rate cell growth is scant. Simulations of existing models have successfully fitted data on PHA productivity and even monomer composition evolution in some cases ,; however, these models apply only for scenarios of negligible growth. In addition, in these experiments the feedstock composition was kept constant during the accumulation process resulting in a polymer with a constant ratio of 3HB:3HV.
These existing model frameworks are in contrast to some published experimental data that do show shifts in copolymer composition during fed-batch mixed culture PHA accumulation, even under non-growing conditions. Such data indicate that the 3HB:3HV ratio during accumulation is not simply dependent on the feedstock but is also affected by the history of the accumulation and the resulting metabolic activity in the biomass. The potential for biomass growth and other processes to directly influence the composition of intracellular acyl-CoA reservoirs and hence copolymer composition has not been examined.The aim of this paper is to examine 3HB and 3HV monomer evolution through PHA accumulation, giving consideration to the effect of biomass growth and alternating feedstocks on this process. To this end, monomer development through four sets of PHA accumulation experiments (based on the feeding regime) is investigated: Set 1: acetic acid (HAc) feed; Set 2: propionic acid (HPr) feed; Set 3: mixed HAc and HPr feed; and Set 4: alternating HAc/HPr feed. Concurrent biomass growth and carbon storage is encouraged in each set. Metabolic Flux Analysis (MFA) is used to quantify metabolic pathway activity through the accumulations.
Experimental Set-UpPHA was produced at pilot scale at AnoxKaldnes AB (Lund, Sweden) using a three stage process that had been in continuous operation from 2008 to 2013. The first stage (acidogenic fermentation) was performed in a 200 L continuous stirred tank reactor under anaerobic conditions and fed with cheese whey permeate, producing a mixture comprising 35% ± 4% acetic, 4% ± 1% propionic, 49% ± 4% butyric, 4% ± 1% valeric and 8% ± 3% caproic acids. The second stage was carried out in a sequential batch reactor (SBR) operated under Aerobic Dynamic Feeding (ADF) conditions with nutrient addition (COD:N of 200:5). The excess biomass with a high PHA storage capacity (as enriched in stage two) was used to produce PHA-rich biomass in the third stage, in a reactor operated in fed batch mode. The details of this process and analytical methods can be found in Janarthanan et al.
Fed-Batch PHA ProductionPHA was accumulated in batches of 100 L harvested SBR mixed liquor by means of a 150 L (working volume) aerated reactor. Aeration provided mixing as well as oxygen supply. Acetic and propionic acids (HAc and HPr, respectively) were fed using different HAc:HPr ratios and feeding strategies. The microbial community was dominated by the genera Flavisolibacter and Zoogloea.The carbon source concentration for pulse-wise substrate addition was 100 gCOD/L (see ), with pH adjusted to 4 and additions of nitrogen and phosphorus for nutrient limitation to give COD:N:P of 200:2:1. N and P additions were 3.82 g/L NH 4Cl and 0.22 g/L KH 2PO 4, respectively. For the fed-batch accumulations, a pulse-wise feedstock addition was applied for feed-on-demand controlled by the biomass respiration response as measured by dissolved oxygen (DO) trends ,.
Semi-continuous (pulse-wise) additions of feedstock aliquots were made targeting peak COD concentrations of between 100 and 200 mg-COD/L. Feedstock additions were triggered by a measured relative decrease in biomass respiration rate. PH was monitored but not controlled. The fed-batch accumulations were run over 20–25 h with samples taken at selected times for analyses, including VSS (volatile suspended solids), TSS (total suspended solids), PHA content and composition, soluble COD, volatile fatty acids (VFAs), and nutrients (nitrogen and phosphorus). Experiment setSubstrate Composition and Feeding Strategy (gCOD Basis)Experiment LabelProcess Time (h)Initial VSS (gL −1)Total substrate added (gCOD)Feed Concentration (gCODL −1)Total Number of PulsesHAcHPrHAcHPr1100% Acetic acidExp 14-96138-Exp 1′4-% Propionic acidExp 223.31.2-939106-77Exp 2′24.61.7-1450% Acetic/50% propionic acidExp 36009895Exp 3′00% Acetic acid—100% propionic acid (alternating)Exp 49906105Exp 4′56. Analytical MethodsTotal concentrations were analyzed from well-mixed grab samples and soluble concentrations were analyzed after filtering the aqueous samples with 1.6 μm pore size (Ahlstrom Munktell, Falun, Sweden) filters. Volatile fatty acid concentrations were quantified by gas chromatography.
Solids analyses (total and volatile suspended solids, or TSS/VSS) were performed according to Standard Methods.Hach Lange™ kits were used for the determination of soluble COD (sCOD) (LCK 114), NH 4–N (LCK 303), NO 3–N (LCK 339), soluble total phosphorus (LCK 349) and soluble total nitrogen (LCK 138). PHA content and monomeric composition (3HB and 3HV) of samples was determined using the gas chromatography method described in using a Perkin-Elmer gas chromatograph (GC) (Perkin Elmer, Inc., Waltham, MA, USA). Quantitative 13C high resolution NMR spectra were acquired on a Bruker Avance 500 spectrometer (Bruker, Billerica, MA, USA) as described by Arcos-Hernandez et al. to determine polymer microstructure (details of polymer structure can be found in ). Experimental Design for PHA AccumulationsThe full set of PHA accumulation (third stage) experiments are summarized in. For this work, four experiments (replicated, with replicates denoted using the symbol ’) were considered.
Experiment set 1 used a single acetic acid (HAc) substrate; Experiment set 2 used a single propionic acid (HPr) substrate; Experiment set 3 used a mixed HAc and HPr substrate fed simultaneously in equal COD ratios; while in Experiment set 4 the acids were supplied in alternating pulses. Rate and Yield CalculationsFor PHA concentration at a given time (in g PHA/ L), only the PHA produced during the accumulation process was considered.
Therefore, the measured PHA concentration ( PHA) was corrected by subtracting the initial measured PHA content ( PHA 0). Typically the initial biomass PHA content (% PHA 0 in wt%) was between 0% and 4%. Active biomass (CH 1.4O 0.4N 0.2) at a given time ( X, recorded in g/L) was determined from the total concentration of biomass, measured as volatile suspended solids ( VSS in g VSS/ L), subtracting the produced PHA concentration ( PHA). F P H A = P H A ( i n g C O D P H A / L ) X ( i n m C O D X / L )Experimental data for the total amount of VFA consumed, PHA polymer ( PHA) produced and active biomass ( X) produced were fitted using global nonlinear regression in GraphPad Prism (v.6.0.5). This analysis was performed using an exponential growth model (one phase association).
The batch process mass balance accounted for input feed dosing volumes as well as sampling withdrawal volumes. Kinetic rates and yields were calculated from fitted data as follows:Acetic ( q H A c) and propionic acid ( q H P r) specific consumption rates and specific monomer 3HB and 3HV production rates: q H B and q H V, respectively, for the ith uptake of each acid or production of each monomer were calculated with reference to active biomass ( X) concentration. % 3 H V i n s t = q H V ( m o l 3 H V h − 1 X − 1 ) q P H A ( m o l P H A h − 1 X − 1 )As previously reported by Janarthanan et al. , a linear correlation was obtained between gCOD PHA produced versus total substrate consumed (also in gCOD) and the yield ( Y PHA / S) in gCOD PHA/gCOD S at 20 h was determined (0.968.
Metabolic Flux Analysis (MFA)MFA was performed in order to investigate the effect of VFA composition and the feeding strategy on active biomass growth and PHA (3HV and 3HB) monomer formation kinetics assuming a pseudo-steady state. The metabolic network used in this work is based on previously published models , and summarized in. The reactions R 9 and R 10 describe the conversion of acetyl-CoA and propionyl-CoA into PHA precursors, where acetyl-CoA.
and propionyl-CoA. are representations of molecules which have undergone the first two steps of PHA synthesis (condensation and reduction) ,. Subsequently, PHA precursors are polymerized to form the biopolymer (PHB and PHV), with two units of acetyl-CoA. forming one 3HB molecule, and one unit of acetyl-CoA. and one of propionyl-CoA. forming one molecule of 3HV. The cells obtain energy from adenosine triphosphate (ATP), which is generated by the oxidation of NADH, and the efficiency of ATP production is represented by the phosphorylation efficiency (P/O) ratio (δ).
Model Cars
The maximum theoretical P/O ratio is 3 mol-ATP/mol-NADH 2 in bacteria growing under aerobic conditions. Metabolic network for PHBV synthesis and biomass production, adapted from , with permission from © 2013 Elsevier. Light blue dotted squares represent external metabolites; white dotted squares represent internal metabolites.The metabolic model consists of 12 reactions, 6 intracellular metabolites (acetyl-CoA, propionyl-CoA, acetyl-CoA., propionyl-CoA., ATP, and NADH), 4 substrates (HAc, HPr, O 2, and NH 4), and 4 end products (3HB, 3HV, X, and CO 2). The system of equations has six degrees of freedom , and a total of seven rates were measured (VFA consumption rates, PHA monomers storage rate, oxygen uptake rate, active biomass synthesis rate, and ammonium consumption). Therefore, the system is overdetermined, with one degree of redundancy, which made it possible to estimate the experimental errors in measurements.The following constraints and assumptions were set for MFA:. PHA depolymerization was not considered.MFA was performed using the CellNetAnalyzer (v.
2014.1, Max Planck Institute, Magdeburg, Germany) toolbox for Matlab. To evaluate the consistency of experimental data with the assumed biochemistry and the pseudo-steady state assumption a chi-squares-test was carried out. The flux distributions calculated were found to be reliable given that the consistency index ( h) values were below a reference chi-squared test function (χ 2 = 3.84 for a 95% confidence level and 1 degree of redundancy).
Liliana Model Sets 1-15
The stoichiometry of the metabolic reactions is provided in the. Biomass Growth and PHA ContentThe experiments were designed to follow the time evolution of PHA storage and active biomass growth during the third stage of the PHA-production system and representative Experiment sets 3 and 4 are shown in (sets 1 and 2 can be seen in the ). The extent of production of active biomass was variable between the experiments, but higher maximum specific growth rates were achieved for accumulations where acetic acid was fed (Experiment set 1 with 100% HAc, Experiment set 3 with 50%/50% HAc/HPr, and Experiment set 4 with alternating substrates). However, active biomass growth rates attenuated sooner for those accumulations where acetic acid was present at all times (Experiment set 1 and Experiment set 3), while the highest biomass production (X/X 0) was achieved in Exp 4 (, ). With regard to PHA fraction evolution, a similar PHA content at plateau was achieved for all experiments.
Imgchili Liliana Model Sets 1-100
However, PHA content and yield tended to be higher in those accumulations with alternating substrates (Experiment set 4), with one experiment (Exp 4) maintaining an increasing PHA fraction even after 22 h of accumulation. This observation fits with the interpretations from other works that it is possible to stimulate PHA storage with concurrent cellular growth by supplying an optimal nutrient ratio ,.