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.     

Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up.     

Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group

Aerobic metabolism and response to oxidative stress and starvation were studied in 11 Lactobacillus plantarum, L. paraplantarum and L. pentosus strains in order to assess the impact of aerobic metabolism on the growth and on the stress response. The strains were grown in aerobiosis without supplementation (AE), with hemin (AEH) or with hemin and menaquinone (AEHM) supplementation and in anaerobiosis (AN) in a complex buffered substrate. Growth rate, biomass yield, glucose and O₂ consumption, production of lactic acid and H₂O₂, catalase activity, oxidative and starvation stress tolerance were evaluated. Aerobic growth increased biomass yield in late stationary phase. Further increase in yield was obtained with both hemin (H) and menaquinone (M) addition. With few exceptions, the increase in biomass correlated with the decrease of lactic acid which, however, decreased in anaerobic conditions as well in some strains. Addition of H or H + M increased growth rate for some strains but reduced the duration of the lag phase. H₂O₂ production was found only for aerobic growth with no supplementation due to catalase production when hemin was supplemented. To our knowledge this is the first study in which the advantages of aerobic growth with H or H + M in improving tolerance of oxidative stress and long-term survival is demonstrated on several strains of the L. plantarum group. The results may have significant technological consequences for both starter and probiotic production.     

Metabolic,physiological and kinetic aspects of the alcoholic fermentation of whey permeate by Kluyveromyces fragilis NRRL 665 and Kluyveromyces lactis NCYC 571

Optimum growth conditions for the fermentation of non-concentrated whey permeate by Kluyveromyces fragilis NRRL 665 have been defined. Use of 3.75 g yeast extract l^?1, a growth temperature of 38°C and a pH of 4.0 allowed a maximum productivity of 5.23 g ethanol l^?1 h^?1 in continuous culture with a yield 91% of theoretical. Complete batch fermentation of permeate with 100 g lactose l^?1 was possible with a maximum specific growth rate of 0.276 h^?1 without any change in ethanol yield. Fermentation of concentrated permeate resulted, however, in a general decrease of specific substrate consumption rate, demonstrated by the inability to completely convert an initial 90 or 150 g lactose l^?1 in continuous culture, even at dilution rates as low as 0.05 and 0.08 h^?1, respectively. The decrease could be related to substrate inhibition, to an increase in osmotic pressure caused by lactose and salts, and to ethanol inhibition of both alcohol and biomass yield. The decrease in specific productivity could be counterbalanced by use of high cell density cultures, obtained by cell recycle of K. fragilis. Fermentation of a non-concentrated permeáte at a dilution rate of 1 h^?1 resulted in a productivity of 22 g l^?1 h^?1 at 22 g ethanol l^?1. Cell recycle using flocculating Kluyveromyces lactis NCYC 571 was also tested. With this strain a productivity of 9.3 g l^?1 h^?1 at 45 g product l^?1 was attained at a dilution rate of 0.2 h^?1, with an initial lactose concentration of 95 g l^?1.     

Effects of different nitrogen species on sensitivity and photosynthetic stress of three common freshwater diatoms

Different sources of nitrogen pose diverse effects to algal community, but the mechanism of inhibitory effects of nitrogen sources on freshwater diatoms is not fully understood. The purpose of this study was to compare biomass, photosynthetic activity, and morphological structure of three common freshwater diatoms (Cyclotella meneghiniana, Nitzschia sp., and Gomphonema parvulum) under different nitrogen sources (NO₃ ^? or NH₄ ⁺). The sorption characteristic of each diatom was investigated, and chlorophyll a (Chl-a) content and oxygen evolution rate were analyzed to investigate stress of different nitrogen sources on each diatom in the batch experiments. Ammonium lowered the growth rate of C. meneghiniana and Nitzschia sp. when it was supplied in addition to growth-saturating nitrate concentrations, suggesting a combined effect of inhibition of nitrate uptake and direct ammonium stress. Oxygen evolution rate of Nitzschia sp. showed that the direct ammonium stress on the photosynthetic activity can be alleviated by coexistence of nitrate in the nitrogen enriched treatment, but not for C. meneghiniana and G. parvulum, which may be caused by a different nitrate transporter system within algal cells. Transmission electron microscopy was used to assess the toxicity of ammonium on ultrastructural chloroplast of each diatom. Ultrastructural changes in chloroplasts showed undefined electron-dense granules and lipid droplets, but the membrane integrity of cell was maintained, suggesting an adaptation to adjustment to ammonia stress. Results showed that Cyclotella meneghiniana and Nitzschia sp. were more sensitive to ammonium stress than Gomphonema parvulum on growth, but the mechanism remains unclear.     

Physiological Consequences of Multiple-Target Regulation by the Small RNA SgrS in Escherichia coli

Cells use complex mechanisms to regulate glucose transport and metabolism to achieve optimal energy and biomass production while avoiding accumulation of toxic metabolites. Glucose transport and glycolytic metabolism carry the risk of the buildup of phosphosugars, which can inhibit growth at high concentrations. Many enteric bacteria cope with phosphosugar accumulation and associated stress (i.e., sugar-phosphate stress) by producing a small RNA (sRNA) regulator, SgrS, which decreases phosphosugar accumulation in part by repressing translation of sugar transporter mRNAs (ptsG and manXYZ) and enhancing translation of a sugar phosphatase mRNA (yigL). Despite a molecular understanding of individual target regulation by SgrS, previously little was known about how coordinated regulation of these multiple targets contributes to the rescue of cell growth during sugar-phosphate stress. This study examines how SgrS regulation of different targets impacts growth under different nutritional conditions when sugar-phosphate stress is induced. The severity of stress-associated growth inhibition depended on nutrient availability. Stress in nutrient-rich media necessitated SgrS regulation of only sugar transporter mRNAs (ptsG or manXYZ). However, repression of transporter mRNAs was insufficient for growth rescue during stress in nutrient-poor media; here SgrS regulation of the phosphatase (yigL) and as-yet-undefined targets also contributed to growth rescue. The results of this study imply that regulation of only a subset of an sRNA''s targets may be important in a given environment. Further, the results suggest that SgrS and perhaps other sRNAs are flexible regulators that modulate expression of multigene regulons to allow cells to adapt to an array of stress conditions.     

Co-metabolism of citrate and lactose by Leuconostoc mesenteroides subsp. cremoris

Citrate stimulated growth rate, increased the specific lactose consumption rate and enhanced the molar growth yield of Leuconostoc mesenteroides subsp. cremoris growing on lactose at pH 5.2 or 6.2 and at 22 or 30°C. As soon as citrate utilization began, diacetyl and acetoin were produced: 2,3-butylene glycol appeared later while acetoin decreased.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

Fermentative production of superoxide dismutase with <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg⁻¹) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h⁻¹), aeration pressure (0.3 vs 1.2 bar) and H₂O₂ (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H₂O₂ resulted in an increase in CuZn–SOD up to 475 U mg⁻¹. When a regulation of oxygen saturation was applied during batch cultures, CuZn–SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg⁻¹, respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg⁻¹, respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

Roles of extracellular lactose hydrolysis in cellulase production by Trichoderma reesei Rut C30 using lactose as inducing substrate

Lactose, an inexpensive, soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei. The fungus does not uptake lactose directly. Lactose is hydrolyzed to extracellular glucose and galactose for subsequent ingestion. The roles of this extracellular hydrolysis step were investigated in this study. Batch and continuous cultures were grown on the following substrates: lactose, lactose–glycerol mixtures, glucose, galactose, and glucose–galactose mixtures. Cell growth, substrate consumption, lactose hydrolysis, and lactase and cellulase production were followed and modeled. Cells grew much faster on glucose than on galactose, but with comparable cell yields. Glucose (at >0.3 g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D < ∼0.065 h⁻¹ where a basal level lactase production was observed. For cellulase production the optimal D was 0.055–0.065 h⁻¹ where the enzyme activity and productivity were both near maxima. The model suggested that lactase synthesis was subject to weak galactose repression. As the galactose concentration increased at high D (>0.1 h⁻¹), lactase synthesis became repressed. The insufficient lactase synthesis limited the lactose hydrolysis rate. Extracellular lactose hydrolysis was concluded to be the rate-limiting step for growth of T. reesei Rut C30 on lactose.     

Kinetic study of Lactococcus lactis strains (SLT6 and SLT10) growth on papain-hydrolysed whey

The effect of controlled whey hydrolysis by papain on growth of two lactic acid bacteria isolated from artisanal Leben: Lactococcus lactis var. diacetylactis (SLT6 and SLT10) was investigated. The higher biomass and maximum specific growth rate (μ _max) were obtained after 30 min of hydrolysis. HPLC analysis of peptides showed that whey hydrolysis reduced the amount of peptides of MW > 400 Da and increased those peptides of MW < 400 Da. The two studied strains exhibited different peptide requirements. The pH-controlled batch cultures in 30 min hydrolysed whey followed the Monod kinetic for growth and for lactate production. The values of the key kinetic constants were: maximum specific growth rate (μ _max), 1.08 and 0.56 h^?1; yield biomass on lactose (Y _x/s), 0.20 and 0.18 g g^?1 and saturation constant K _s, 4.2 and 2.8 g L^?1 for SLT6 and SLT10, respectively. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles.     

Kinetics of lactose transport inKluyveromyces fragilis grown in a chemostat on diluted whey permeate

Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK _m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V _max) are dependent on the dilution rate (D). At D=0.12 h^–1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h^–1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Evolution of a regulated operon in the laboratory

The evolution of new metabolic functions is being studied in the laboratory using the EBG system of E. coli as a model system. It is demonstrated that the evolution of lactose utilization by lacZ deletion strains requires a series of structural and regulatory gene mutations. Two structural gene mutations act to increase the activity of ebg enzyme toward lactose, and to permit ebg enzyme to convert lactose into allolactose, an inducer of the lac operon. A regulatory mutation increases the sensitivity of the ebg repressor to lactose, and permits sufficient ebg enzyme activity for growth. The resulting fully evolved ebg operon regulates its own expression, and also regulates the synthesis of the lactose permease.     

Genotypic variability in physiological,biomass and yield response to drought stress?in pigeonpea

Three pigeonpea (Cajanus cajan L. Millsp.) genotypes- GT-1, AKP-1 and PRG-158 with varying crop duration, growth habit and flowering pattern were evaluated for variability in their response for drought stress. Drought stress was imposed at initiation of flowering and the observations on biomass and seed yield parameters were recorded at harvest. The magnitude of response of individual component to drought stress was found to be genotype specific. Drought stress significantly decreased photosynthetic rate (P_N), transpiration rate (Tr) and relative water content (RWC) in all the genotypes, however the magnitude of reduction differed with genotype. With drought stress, the reduction of P_N was highest in GT-1 while reduction in Tr was highest in PRG-158. The genotype AKP-1, accumulated significantly higher concentrations of osmotic solutes especially proline under water deficit stress, this facilitated it to maintain higher relative water content (RWC) and lower malondialdehyde (MDA) content as compared to other genotypes. Drought stress also impacted biomass production and their partitioning to vegetative and reproductive components at harvest. There was significant variability between the genotypes for seed yield under drought stress while it was non-significant under well-watered condition. Drought stress enhanced flower drop and decreased flower to pod conversion resulting in reduced pod number and seed number in PRG-158 and GT-1. The genotype AKP-1 recorded superior performance for seed yield under stress environment due to its ability in maintaining pod and seed number as well as improved test weight (100 seed weight). Under drought stress, significant positive association of seed yield with proline, seed number, pod number and test weight clearly indicating their role in drought tolerance.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Single cell protein production from rayon pulp will waste by Paecilomyces variotii

Prehydrolysate liquor obtained from a rayon pulp mill was used as a substrate to produce single cell protein (SCP). Ten different strains of Paecilomyces variotii were locally isolated and screened for the production of SCP from following optimization creteria: rates and yield of biomass production, rates of substrate utilization, maximum specific growth rates and total biomass formation. The optimal SCP producer was found to be P. variotii TCRDC-M5 which gave a μmax of 0.11/h and a biomass yield of 96% of the theoretical, with 95% substrate utilization and 25 g dry wt. biomass/l in the Lab. fermentor. This resulted in a 70% reduction in the BOD of the prehydrolysate.