An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

A hidden square-root boundary between growth rate and biomass yield

Although the theoretical value of biomass yield can be calculated from metabolic network stoichiometry, the growth rate is difficult to predict. Since the rate and yield can vary independently, no simple relationship has been discovered between these two variables. In this work, we analyzed the well-accepted enzyme kinetics and uncovered a hidden boundary for growth rate, which is determined by the square-root of three physiological parameters: biomass yield, the substrate turnover number, and the maximum synthesis rate of the turnover enzyme. Cells cannot grow faster than the square-root of the product of these parameters. This analysis is supported by experimental data and involves essentially no assumptions except (i) the cell is not undergoing a downshift transition, (ii) substrate uptake enzyme activity is proportional to its copy number. This simple boundary (not correlation) has escaped notice for many decades and suggests that the yield calculation does not predict the growth rate, but gives an upper limit for the growth rate. The relationship also explains how growth rate is affected by the yield and sheds lights on strain design for product formation.     

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as k_S, is a crucial parameter for characterizing growth within bioreactors. State-of-the-art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate k_S. In this work, a recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method is applied for the estimation of k_S values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high-frequency biomass measurement (HFB) and substrate-limited respiratory activity monitoring (sl-RA). As a model system, the substrate affinity k_S of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod-type growth response. A k_S of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl-RA, and 2.66 ± 0.99 mg/L with sl-MSCC was obtained. Whereas HFB and sl-RA are suitable for a fast initial k_S estimation, sl-MSCC allows an affinity estimation by determining t_D at concentrations less or equal to the k_S value. Thus, sl-MSCC lays the foundation for strain-specific k_S estimations under defined environmental conditions with additional insights into cell-to-cell heterogeneity.     

Analyzing Microbial Population Heterogeneity—Expanding the Toolbox of Microfluidic Single-Cell Cultivations

Recent research on population heterogeneity revealed fascinating insights into microbial behavior. In particular emerging single-cell technologies, image-based microfluidics lab-on-chip systems generate insights with spatio-temporal resolution, which are inaccessible with conventional tools. This review reports recent developments and applications of microfluidic single-cell cultivation technology, highlighting fields of broad interest such as growth, gene expression and antibiotic resistance and susceptibility. Combining advanced microfluidic single-cell cultivation technology for environmental control with automated time-lapse imaging as well as smart computational image analysis offers tremendous potential for novel investigation at the single-cell level. We propose on-chip control of parameters like temperature, gas supply, pressure or a change in cultivation mode providing a versatile technology platform to mimic more complex and natural habitats. Digital analysis of the acquired images is a requirement for the extraction of biological knowledge and statistically reliable results demand for robust and automated solutions. Focusing on microbial cultivations, we compare prominent software systems that emerged during the last decade, discussing their applicability, opportunities and limitations. Next-generation microfluidic devices with a high degree of environmental control combined with time-lapse imaging and automated image analysis will be highly inspiring and beneficial for fruitful interdisciplinary cooperation between microbiologists and microfluidic engineers and image analysts in the field of microbial single-cell analysis.     

Misconceptions About the Relation Between Plant Growth and Respiration

Abstract: The relation between plant growth rate and respiration rate is readily derived from the overall chemical reaction for aerobic metabolism. The derived relation can be used to show that separation of respiration into growth (g) and maintenance (m) components is not a useful concept. g and m cannot be unambiguously measured or defined in terms of biochemical processes. Moreover, because growth yield calculations from biochemical pathway analysis, from biomass molecular composition, from biomass heat of combustion, and from biomass elemental composition have not included all of the energy costs for biosynthesis, they are not accurate measures of the carbon cost for plant growth. Improper definitions of growth-respiration relations are impeding the use of physiological properties for prediction of plant growth as a function of environmental variables.     

Experimentelle Untersuchung,Modellierung und optimale Berechnung eines mehrstufigen Säulenfermentors II. Experimentelle Bestimmung der Parameter und Koeffizienten des mathematischen Modells

Using a system analysis for the investigation of all processes which occur in a biochemical reactor on the micro and macro level, a mathematical model was worked out. It characterizes the model of the kinetics and stoichiometry of the growth of microorganisms and the rules of hydrodynamics and mass transfer in form of blocks. Relating to the discussed mathematical total model [11], experimental data on which the calculation of the model parameters is based are described in this second part of the paper. They were determined not only directly from the cultivation process, but also from experiments with model media.     

Determination of the Ks values during the growth of Alcaligenes eutrophus on phenol, 2,4-dichlorophenoxyacetic acid and fructose

The determination of the K_S values presented here is based on the estimation of the stationary substrate concentrations in continuous cultivation experiments. The separation of biomass from the suspension was performed by an ultrafiltration step which succeeded within one second. The decay of substrate concentration during sampling was calculated to amount to less than 6% of the stationary substrate concentration at relevant growth rates. The K_S values derived from these reduced substrate concentrations deviated by only 10% from the theoretical values at a biomass concentration of about 1 g/1. Thus relevant kinetic parameters can be calculated from the data obtained by this procedure. Values of 11, 59 and 14 μM were obtained with 2,4-dichlorophenoxyacetic acid (2,4-D), phenol and fructose, respectively. Similar K_S values were derived with 2,4-D and fructose by using a respirationbased determination for reasons of comparison. With phenol this value was only 7 μM which is as cribed to a physiological background.     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

Effect of starvation length upon microbial activity in a biomass recycle reactor

The kinetics of substrate degradation and bacterial growth was determined in a microbial community from a biomass recycle reactor that had been deprived of substrate feed for 0–32 days. Starvation caused changes in bacterial numbers, community composition, and physiological state. Substrate starvation for less than 1 day resulted in modest (less than threefold) changes in endogenous respiration rate, ATP content, and biomass level. During a starvation period of 32 days, there were substantial changes in microbial community composition, as assessed by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR amplicons of a portion of the 16S rDNA or by phospholipid fatty acid (PLFA) analysis. When the starved communities were stimulated with organic nutrients, the growth kinetics was a function of the length of the starvation period. For starvation periods of 2–8 days prior to nutrient addition, there was a phase of suboptimal exponential growth (S-phase) in which the exponential growth rate was about 30% of the ultimate unrestricted growth rate. S-phase lasted for 2–8 h and then unrestricted growth occurred at rates of 0.3–0.4 h⁻¹. At starvation times of 12 and 20 days, a lag phase preceded S-phase and the unrestricted growth phase. Received 04 January 2002/ Accepted in revised form 08 August 2002     

Contrasting effects of the cyanobacterium Microcystis aeruginosa on the growth and physiology of two green algae,Oocystis marsonii and Scenedesmus obliquus,revealed by flow cytometry

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Dynamics and stability analysis of the growth and astaxanthin production system of Haematococcus pluvialis

This paper investigated high cell density cultivation of Haematococcus pluvialis for astaxanthin production in 3.7-L bioreactors. A biomass concentration of 2.74 g L⁻¹and an astaxanthin yield of 64.4 mg L⁻¹ were obtained. Based on the experimental results, a new and simple dynamic model is proposed, differing from Monod kinetics, to describe cell growth, product formation and substrate consumption. Good agreement was found between the model predictions and experimental data. The model revealed that there was cell growth inhibition on product formation and product feedback compensation for substrate consumption, but no substrate inhibition or product inhibition of cell growth. Stability analysis demonstrated that no multiplicity of steady states was observed; the unique positive steady state was locally asymptotically stable; and the effect of dilution rate on steady states was greater than that of the initial substrate concentration. Received 23 February 1999/ Accepted in revised form 08 June 1999     

Influence of intraspecific interaction and substrate type on initial growth and establishment of Hydrilla verticillata

Both substrate type and plant–plant interaction can greatly influence the growth and establishment of plants. In order to assist the re-vegetation of submerged macrophytes, the growth of Hydrilla verticillata with increasing equi-distance neighboring plant density on two substrate types (sediment and sand, representing high- and low-nutrient level, respectively) was assessed in monoculture stands. The results showed that substrate type greatly changed the biomass allocation patterns of the target plants, with a smaller root mass ratio on sediment compared to sand (0.70 vs. 3.11%). However, interaction between substrate type and neighboring density was observed. At low density, growth on sediment greatly increased plant height (43.90 vs. 22.10 cm), leaf biomass (216.63 vs. 68.41 mg), and total biomass (298.39 vs. 121.77 mg) when compared to growth on sand. However, at high density, no significant effect of the substrate type was found in those parameters. On sediment, high neighboring density greatly decreased the height, root number, total root length, root mass, and total biomass, implying large intraspecific plant–plant competition. However, such competition can be greatly reduced in infertile environments. Therefore, when the plants were grown on sand, neighboring density showed little effect on the height (22.10–26.53 cm), total root length (21.34–40.50 cm), and root biomass (3.14–6.27 mg). Total biomass and root number significantly increased by 50% and 115%, respectively, at high density compared to low density on sand, suggesting that facilitation rather than competition was occurring. Therefore, plant–plant interaction can vary from competition in fertile environments to facilitation in infertile environments. In summary, neighboring density should be manipulated according to the environmental nutrient level, so as to reduce intraspecific competition or increase intraspecific facilitation, and finally enhance the initial growth and establishment of H. verticillata in re-vegetation activities.     

Utilization of Collagenous By-Products from the Meat Packing Industry: Production of Single-Cell Protein by the Continuous Cultivation of Bacillus megaterium

The conditions for continuous cultivation of Bacillus megaterium on a collagen-derived substrate (SP-100) were determined. The optimum conditions of temperature, pH, and dilution rate were 34 C, pH 7.0, and 0.25/hr, respectively. Increasing the substrate concentration in plain tap water resulted in proportional increases in the productivity of cell mass from 0.6 g per liter per hr at 1% substrate to 1.8 g per liter per hr at 10% substrate; however, the protein content of the biomass decreased from 60 to 36%, and the protein yield decreased from 91 to 50% at substrate concentrations of 1 and 10%, respectively. These effects (decreases) were reversed up to 7.5% substrate by mineral supplementation of the medium. The productivity of biomass increased from 0.6 to 1.9 per liter per hr; the protein content of the biomass, from 43 to 54%; and the protein yield, from 60 to 93%, respectively, as the substrate concentration (with mineral supplementation of the medium) was increased from 1 to 7.5%. Spent medium could be refortified and recycled as often as five times. The amino acids in the substrate protein appeared to be utilized for growth and metabolism more or less uniformly. Analysis of the B. megaterium biomass indicated considerable enrichment of the essential amino acids and reduction of proline, glycine, and hydroxyproline as compared to the collagen-derived substrate. The Protein Efficiency Ratios obtained on the collagen-derived substrate (SP-100) and on the B. megaterium biomass, expressed as percentages of the casein reference protein, were 14 and 74%, respectively. Thus, considerable improvement in nutritional value was effected by bacterial conversion of the collagen-derived substrate into single-cell protein.     

Single-cell protein production by photosynthetic bacteria cultivation in agricultural by-products

The growth of the nonsulfur photosynthetic bacterium Rhodopseudomonas gelatinosa was investigated as a possible way to produce single-cell protein from agricultural by-products. Of the various raw materials examined as potential feedstocks, wheat bran infusion was selected as the substrate for mass culture and continuous cultivation studies. Harvested photosynthetic cells contained approximately 65.0% crude protein and 5.1% nucleic acid (RNA). The amino acid content of harvested photosynthetic proteins was comparable with conventional proteins of plant and animal origin.     

Biochemical production capabilities of Escherichia coli

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. (c) 1993 John Wiley & Sons, Inc.     

Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.