Modeling growth,substrate consumption and product formation of <Emphasis Type="Italic">Penicillium nalgiovense</Emphasis> grown on meat simulation medium in submerged batch culture

Penicillium nalgiovense is the most widely used starter mold for cured and fermented meat products. The development of a biomass film on the surface of these products prevents a large degree undesirable growth of various fungal contaminants and contributes to the ripening process with production of metabolites. This work presents an attempt to model the growth of P. nalgiovense and to relate it to substrate consumption and product release. Because of the extremely complex nature of the meat product fermentation, submerged culture was employed in a bioreactor system that enabled on-line monitoring, using a meat simulation medium, which contained peptones and lactate as carbon, nitrogen and energy sources. The unstructured model presented is based on a partial association of substrate assimilation and product formation with growth. Experimentally derived values for peptones and lactate were compared with model-derived values and their proportions corresponding to growth associated parts, used for biosynthesis, and non-growth associated parts, used for maintenance. The model was applied for the products ammonia, carbon dioxide and protons. Both peptones and lactate were used mainly for biosynthesis (85 and 80% of the total amounts provided, respectively). Assimilation of lactate and ammonia formation from amino acid metabolism resulted in a proton exchange, which was mainly growth associated. The contribution of the growth associated mechanism to the total proton exchange was estimated to be 75% while the contribution of the non-growth associated mechanism increased during the growth phase and reached a maximum of 25%. For carbon dioxide production, the contribution of a maintenance mechanism was evident at 40 h, while production was growth-associated and remained such even at the end of fermentation at 168 h when growth rate was very low. The partially growth associated model showed good agreement with the experimental data and allows accurate determination of the proportions of substrates or products related to biosynthesis and cell maintenance.     

Mathematical model of Chlorella minutissima UTEX2341 growth and lipid production under photoheterotrophic fermentation conditions

To reduce the cost of algal biomass production, mathematical model was developed for the first time to describe microalgae growth, lipid production and glycerin consumption under photoheterotrophic conditions based on logistic, Luedeking-Piret and Luedeking-Piret-like equations. All experiments were conducted in a 2 L batch reactor without considering CO(2) effect on algae's growth and lipid production. Biomass and lipid production increased with glycerin as carbon source and were well described by the logistic and Luedeking-Piret equations respectively. Model predictions were in satisfactory agreement with measured data and the mode of lipid production was growth-associated. Sensitivity analysis was applied to examine the effects of certain important parameters on model performance. Results showed that S(0), the initial concentration of glycerin, was the most significant factor for algae growth and lipid production. This model is applicable for prediction of other single cell algal species but model testing is recommended before scaling up the fermentation of process.     

Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures

The kinetics of batch anaerobic hydrogen production by mixed anaerobic cultures was systemically investigated in this study. Unstructured models were used to describe the substrate utilization, biomass growth and product formation in the hydrogen production process. The relationship between the substrate, biomass and products were also evaluated. Experimental results show that the Michaelis-Menten equation, Logistic model and modified Gompertz equation all could be adopted to respectively describe the kinetics of substrate utilization, biomass growth and product formation. Furthermore, the relationship between the acidogenic products and biomass was simulated by Luedeking-Piret model very well. The experimental results suggest that the formation of hydrogen and the main aqueous products, i.e., butyrate and acetate, was all growth-associated.     

Growth and acid production by Lactobacillus (L) delbrueckii in a dialysis culture system

Growth and lactic acid production by L. delbrueckii was studied in a dialysis culture system and the inhibitory effect of lactate confirmed by removing lactate from the culture medium by dialysis. It has been shown that lactate inhibits growth after the log phase and that the maintenance of low lactate concentrations after this point permits higher specific growth rates and higher maximum cell concentrations. Acid production is also significantly higher in a dialysis culture system. Finally, a modification of the Luedeking-Piret model, incorporating the lactate inhibition effect, is proposed.     

黑曲霉过氧化氢酶发酵过程的数学模型

研究了黑曲霉发酵生产过程氧化氢酶的分批发酵动力学,并建立了发酵过程菌体生长,基质消耗及酶合成的随时间变化的数学模型。Logistic方程,Luedekin-Piret方程及与Luedeking-Piret方程相似的基质消耗方程能够很好地分别描述黑曲霉细胞的生长,发酵产酶过程及葡萄糖的消耗,过氧化氢酶的发酵合成是生长耦联的,研究中还将3个动力学模型的预测值和实验值进行了比较。     

Growth and sporulation stoichiometry and kinetics of Coniothyrium minitans on agar media

Coniothyrium minitans was cultivated on agar media with different concentrations of starch, urea, and trace elements. By means of elemental balances, the stoichiometry of growth and sporulation was established. C. minitans produced byproducts on all media, especially in the medium with high urea concentrations, where 30% of the starch was converted into byproducts. Simple empirical models were used to describe the kinetics of growth, sporulation, CO(2) production, and substrate consumption on all media. Total biomass and mycelium could be described reasonably well with the logistic law. Starch, urea, and oxygen consumption and CO(2) production could be described as a function of total biomass by the linear-growth model of Pirt. There were almost no differences between media for the estimates of yield coefficients and maintenance coefficients. Only at high urea concentrations were maintenance coefficients much higher. Similar to substrate consumption and CO(2) production, the kinetics of sporulation could be described as a function of mycelium production with the linear-growth model. It is shown that sporulation of C. minitans is growth-associated. Based on kinetics, the process costs for producing spores are roughly calculated. In addition, it is shown that fermentor costs represent the majority of production costs.     

Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.     

Matrix notation for efficient development of first-principles models within PAT applications: integrated modeling of antibiotic production with Streptomyces coelicolor

A matrix notation coupled to macroscopic principles is introduced as a means to develop first- principles models in an efficient and structured way within PAT applications. The notation was evaluated for developing an integrated biological, chemical (pH modeling) and physical (gas-liquid exchange) model for describing antibiotic production with Streptomyces coelicolor in batch fermentations. The model provided statistically adequate fits to all the monitored macroscopic biological, chemical and physical data of the process, except the phosphate uptake dynamics. This phosphate discrepancy is hypothesized to result from the internal storage of phosphate as polyphosphate prior to the exponential growth phase. The antibiotic production was associated with the stationary phase and its kinetics was adequately described using a modified Luedeking-Piret equation. Further, the maintenance was best described by employing a combination of Pirt and Herbert models, a result that was supported by a model-based hypothesis testing. Overall the process knowledge currently incorporated in the model is believed to be useful both for process optimization purposes and for further testing of hypotheses aiming at improving the mechanistic understanding of antibiotic production with S. coelicolor. Last but not least, the matrix notation is believed to be a promising supporting tool for efficient development and communication of complex dynamic models within a PAT framework.     

Energetics of growth of a defined mixed culture of Desulfovibrio vulgaris and Methanosarcina barkeri: maintenance energy coefficient of the sulfate-reducing organism in the absence and presence of its partner

The maintenance energy coefficient of Desulfovibrio vulgaris was studied by using a chemostat, with Methanosarcina barkeri or sulfate as the electron acceptor; lithium lactate or sodium pyruvate served as the electron donor. The experiments showed that the growth energetics of D. vulgaris or M. barkeri were greatly affected by maintenance energy coefficients. When D. vulgaris grew on lactate or pyruvate medium with sulfate, these coefficients reached 4.40 and 2.80 mM g-1 h-1, respectively; on lactate medium in the presence of M. barkeri the same coefficient reached a value of 2.90 mM g-1 h-1. Results also showed that the increase of the value of the maintenance energy coefficient corresponded to a decrease of the biomass produced. D. vulgaris maximal growth yield values calculated by use of the Pirt equation were slightly higher with M. barkeri (maximal growth yield, 10 g/mol) than with sulfate (maximal growth yield, 7.5 g/mol). This finding could be interpreted by reference to the ATP-generating reactions involved in D. vulgaris growth in the presence of sulfate or M. barkeri.     

Pirt, a TRPV1 modulator, is required for histamine-dependent and -independent itch

Itch, or pruritus, is an important clinical problem whose molecular basis has yet to be understood. Recent work has begun to identify genes that contribute to detecting itch at the molecular level. Here we show that Pirt, known to play a vital part in sensing pain through modulation of the transient receptor potential vanilloid 1 (TRPV1) channel, is also necessary for proper itch sensation. Pirt(-/-) mice exhibit deficits in cellular and behavioral responses to various itch-inducing compounds, or pruritogens. Pirt contributes to both histaminergic and nonhistaminergic itch and, crucially, is involved in forms of itch that are both TRPV1-dependent and -independent. Our findings demonstrate that the function of Pirt extends beyond nociception via TRPV1 regulation to its role as a critical component in several itch signaling pathways.     

Modeling of exo-inulinase biosynthesis by Kluyveromyces marxianus in fed-batch mode: correlating production kinetics and metabolic heat fluxes

A metabolic heat-based model was used for estimating the growth of Kluyveromyces marxianus, and the modified Luedeking-Piret kinetic model was used for describing the inulinase production kinetics. For the first time, a relationship was developed to relate inulinase production kinetics directly to metabolic heat generated, which corroborated well with the experimental data (with R ² values of above 0.9). It also demonstrated the predominantly growth-associated nature of the inulinase production with Luedeking-Piret parameters α and β, having values of 0.75 and 0.033, respectively, in the exponential feeding experiment. MATLAB was used for simulating the inulinase production kinetics which demonstrated the model’s utility in performing real-time prediction of inulinase concentration with metabolic heat data as input. To validate the model predictions, a biocalorimetric (Bio RC1e) experiment for inulinase production by K. marxianus was performed. The inulinase concentration (IU/mL) values acquired from the model in were validated with the experimental values and the metabolic heat data. This modeling approach enabled the optimization, monitoring, and control of inulinase production process using the real-time biocalorimetric (Bio RC1e) data. Gas chromatography and mass spectrometry analysis were carried out to study the overflow metabolism taking place in K. marxianus inulinase production.

     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

Actinorhodin production by Streptomyces coelicolor A3(2): kinetic parameters related to growth,substrate uptake and production

The dynamics of an Streptomyces coelicolor A3(2) culture in a 20-l computer-controlled batch bioreactor was investigated both experimentally and theoretically. In defined medium, depending on the initial conditions, the calculated value of some of the kinetic parameters were: maximum specific growth rate, 0.03 h^–1; death rate constant, 1.4–6.3 × 10^–3 h^–1; observed biomass yield, 0.21 g cells g^–1 glucose and the maintenance coefficient for the cells, 0.0448 g glucose g^–1 cells h^–1. According to both experimental observations and the Luedeking-Piret model, actinorhodin production was found to be growth-associated. This paper provides the first published quantitative information on the main kinetic parameters describing the activity of S. coelicolor in batch culture. Correspondence to: F. Mavituna     

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

Precursors of the major end products of aerobic metabolism of Brochothrix thermosphacta

To further our understanding of off-odour production by Brochothrix thermosphacta , the nature and origins of its end products have been compared during aerobic growth in complex and in minimal, defined medium. In complex medium glucose is the major precursor of acetoin and acetic acid but alanine may be an additional minor source. Iso butyric, iso valeric (3-methylbutyric) and 2-methylbutyric acids are derived exclusively from valine, leucine and iso leucine, respectively. In minimal defined medium although the same end products are produced they are all derived from glucose.     

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of energetics and product yields of the acidogenesis

An assessment of both the growth and the metabolism of acidogenic cells Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a CSTR under controlled pH conditions. Cultures were carried out using a semi-synthetic medium supplemented with lactose as carbon source. Acids and solvents, that represent products of the ABE process, have been purposely added in controlled amounts to the culture medium to investigate their effects on the product yields. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. The maximum ATP yield and the maintenance resulted 29.1 g(DM)/mol(ATP) and 0.012 mol(ATP)/g(DM)h, respectively. Quantitative features of the C. acetobutylicum growth model were in good agreement with experimental results. The model proposes as a tool to estimate the mass fractional yield even for fermentations carried out under conditions typical of the solventogenesis.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

Metabolism of isolated kidney tubules. Interactions between lactate, glutamine and oleate metabolism.

Kidney-cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L-lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations. All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process. Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75% of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased. The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell.