Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production

Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.     

Does nitrogen nutrition restrict the CO2 response of fertile grassland lacking legumes?

The extent of the response of plant growth to atmospheric CO₂ enrichment depends on the availability of resources other than CO₂. An important growth-limiting resource under field conditions is nitrogen (N). N may, therefore, influence the CO₂ response of plants. The effect of elevated CO₂ (60 Pa) partial pressure (pCO₂) on the N nutrition of field-grown Lolium perenne swards, cultivated alone or in association with Trifolium repens, was investigated using free air carbon dioxide enrichment (FACE) technology over 3 years. The established grassland ecosystems were treated with two N fertilization levels and were defoliated at two frequencies. Under elevated pCO₂, the above-ground plant material of the L. perenne monoculture showed a consistent and significant decline in N concentration which, in general, led to a lower total annual N yield. Despite the decline in the critical N concentration (minimum N concentration required for non-N-limited biomass production) under elevated pCO₂, the index of N nutrition (ratio of actual N concentration and critical N concentration) was lower under elevated pCO₂ than under ambient pCO₂ in frequently defoliated L. perenne monocultures. Thus, we suggest that reduced N yield under elevated pCO₂ was evoked indirectly by a reduction of plant-available N. For L. perenne grown in association with T. repens and exposed to elevated pCO₂, there was an increase in the contribution of symbiotically fixed N to the total N yield of the grass. This can be explained by an increased apparent transfer of N from the associated N₂-fixing legume species to the non-fixing grass. The total annual N yield of the mixed grass/legume swards increased under elevated pCO₂. All the additional N yielded was due to symbiotically fixed N. Through the presence of an N₂-fixing plant species more symbiotically fixed N was introduced into the system and consequently helped to overcome N limitation under elevated pCO₂. Received: 11 November 1996 / Accepted: 20 May 1997     

Elevated CO2 and Phosphate Limitation Favor Micromonas pusilla through Stimulated Growth and Reduced Viral Impact

Growth and viral infection of the marine picoeukaryote Micromonas pusilla was studied under a future-ocean scenario of elevated partial CO₂ (pCO₂; 750 μatm versus the present-day 370 μatm) and simultaneous limitation of phosphorus (P). Independent of the pCO₂ level, the ratios of M. pusilla cellular carbon (C) to nitrogen (N), C:P and N:P, increased with increasing P stress. Furthermore, in the P-limited chemostats at growth rates of 0.32 and 0.97 of the maximum growth rate (μ_max), the supply of elevated pCO₂ led to an additional rise in cellular C:N and C:P ratios, as well as a 1.4-fold increase in M. pusilla abundance. Viral lysis was not affected by pCO₂, but P limitation led to a 150% prolongation of the latent period (6 to 12 h) and an 80% reduction in viral burst sizes (63 viruses per cell) compared to P-replete conditions (4 to 8 h latent period and burst size of 320). Growth at 0.32 μ_max further prolonged the latent period by another 150% (12 to 18 h). Thus, enhanced P stress due to climate change-induced strengthened vertical stratification can be expected to lead to reduced and delayed virus production in picoeukaryotes. This effect is tempered, but likely not counteracted, by the increase in cell abundance under elevated pCO₂. Although the influence of potential P-limitation-relieving factors, such as the uptake of organic P and P utilization during infection, is unclear, our current results suggest that when P limitation prevails in future oceans, picoeukaryotes and grazing will be favored over larger-sized phytoplankton and viral lysis, with increased matter and nutrient flow to higher trophic levels.     

Effect of carbon dioxide on growth of Zymomonas mobilis in continuous culture

Summary Biomass production of Zymomonas mobilis in continuous cultivation on synthetic or complex glucose media was increased up to 100% when CO₂ partial pressure was reduced from 1,460 to 95 mbar at high dilution rates. Biomass yield increased and specific glucose uptake and ethanol production rates decreased approximately 6% when pCO₂ was lowered from 1,160 to 95 mbar. Product yield was not affected by pCO₂. Long time adaptation superimposing these effects was considered.     

Yield response of Lolium perenne swards to free air CO2 enrichment increased over six years in a high N input system on fertile soil

After a step increase in the atmospheric partial pressure of CO₂ (pCO₂), the availability of mineral N may be insufficient to meet the plant's increased demand for N. Over time, however, the ecosystem may adapt to the new conditions, and a new equilibrium may be established in the fluxes of C and N. This would result in a higher dry mass (DM) yield response of the plants to elevated pCO₂. The effect of elevated atmospheric pCO₂ (60 Pa pCO₂) was studied in Lolium perenne L. swards with two N fertilization treatments (14 and 56 g m^?2 y^?1) in a six‐year FACE (Free Air Carbon dioxide Enrichment) experiment. In the high N treatment, the input of N with fertilizer considerably exceeded the export of N with the harvested plant material in both CO₂ treatments leading to an apparent net input of N into the ecosystem. Accordingly, the proportion of harvested N derived from ¹⁵N labelled fertilizer N, applied throughout the experiment (< 6 years), increased over the years. Under these high N conditions, the annual DM yield response of the Lolium perenne sward to elevated pCO₂ increased (from 7% in 1993 to 25% in 1998). In parallel, the response of N yield to elevated pCO₂ increased, and the initially negative effect of elevated pCO₂ on specific leaf area (SLA) disappeared. The high N input system seemed to overcome in part an initially limiting effect of N on the yield response to elevated pCO₂ within a few years. In contrast, there was no apparent net input of N into the ecosystem in the low N treatment, because N fertilization just compensated the export of N with the harvested plant material. Accordingly, the proportion of harvested N yield, derived from fertilizer N, which was applied throughout the experiment, remained low. At low N, the availability of mineral N strongly limited plant growth and yield production in both CO₂ treatments; the low yields of DM and N, the low concentration of N in the plant material, and the low SLA reflected this. Although the plants grew under the same environmental conditions and the same management treatment as plants in the high N treatment, the response of DM yields to elevated pCO₂ in the low N treatment remained weak throughout the experiment (5% in 1993 and 9% in 1998). The results are discussed in the context of the sizes of the different N pools in the soil, the allocation of N within the plant and the possible effects on temporal immobilization, and the availability of mineral N for yield production as affected by elevated pCO₂ and N fertilization.     

Iron Limitation Modulates Ocean Acidification Effects on Southern Ocean Phytoplankton Communities

The potential interactive effects of iron (Fe) limitation and Ocean Acidification in the Southern Ocean (SO) are largely unknown. Here we present results of a long-term incubation experiment investigating the combined effects of CO₂ and Fe availability on natural phytoplankton assemblages from the Weddell Sea, Antarctica. Active Chl a fluorescence measurements revealed that we successfully cultured phytoplankton under both Fe-depleted and Fe-enriched conditions. Fe treatments had significant effects on photosynthetic efficiency (F_v/F_m; 0.3 for Fe-depleted and 0.5 for Fe-enriched conditions), non-photochemical quenching (NPQ), and relative electron transport rates (rETR). pCO₂ treatments significantly affected NPQ and rETR, but had no effect on F_v/F_m. Under Fe limitation, increased pCO₂ had no influence on C fixation whereas under Fe enrichment, primary production increased with increasing pCO₂ levels. These CO₂-dependent changes in productivity under Fe-enriched conditions were accompanied by a pronounced taxonomic shift from weakly to heavily silicified diatoms (i.e. from Pseudo-nitzschia sp. to Fragilariopsis sp.). Under Fe-depleted conditions, this functional shift was absent and thinly silicified species dominated all pCO₂ treatments (Pseudo-nitzschia sp. and Synedropsis sp. for low and high pCO₂, respectively). Our results suggest that Ocean Acidification could increase primary productivity and the abundance of heavily silicified, fast sinking diatoms in Fe-enriched areas, both potentially leading to a stimulation of the biological pump. Over much of the SO, however, Fe limitation could restrict this possible CO₂ fertilization effect.     

Direct evidence that symbiotic N2 fixation in fertile grassland is an important trait for a strong response of plants to elevated atmospheric CO2

Although legumes showed a clearly superior yield response to elevated atmospheric pCO₂ compared to nonlegumes in a variety of field experiments, the extent to which this is due to symbiotic N₂ fixation per se has yet to be determined. Thus, effectively and ineffectively nodulating lucerne (Medicago sativa L.) plants with a very similar genetic background were grown in competition with each other on fertile soil in the Swiss FACE experiment in order to monitor their CO₂ response. Under elevated atmospheric pCO₂, effectively nodulating lucerne, thus capable of symbiotically fixing N₂, strongly increased the harvestable biomass and the N yield, independent of N fertilization. In contrast, the harvestable biomass and N yield of ineffectively nodulating plants were affected negatively by elevated atmospheric pCO₂ when N fertilization was low. Large amounts of N fertilizer enabled the plants to respond more favourably to elevated atmospheric pCO₂, although not as strongly as effectively nodulating plants. The CO₂‐induced increase in N yield of the effectively nodulating plants was attributed solely to an increase in symbiotic N₂ fixation of 50–175%, depending on the N fertilization treatment. N yield derived from the uptake of mineral N from the soil was, however, not affected by elevated pCO₂. This result demonstrates that, in fertile soil and under temperate climatic conditions, symbiotic N₂ fixation per se is responsible for the considerably greater amount of above‐ground biomass and the higher N yield under elevated atmospheric pCO₂. This supports the assumption that symbiotic N₂ fixation plays a key role in maintaining the C/N balance in terrestrial ecosystems in a CO₂‐rich world.     

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O₂ limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O₂ supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O₂ supply.Correspondence to: T. Yoshida     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Summary Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation.With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:Nomenclature D dilution rate (1/h) - DOT dissolved oxygen tension (%) - m_o2 maintenance coefficient (mmol O₂/g cell mass h) - q_o2 specific oxygen uptake rate (mmol O₂/g cell mass h) - q_s specific xylose uptake rate (g xylose/g cell mass h) or (mmol xylose/g cell mass h) - q_x specific xylitol production rate (g xylitol/ g cell mass h) or (mmol xylitol/ g cell mass h) - q_e specific ethanol production rate (g ethanol/ g cell mass h) or (mmol ethanol/ g cell mass h) - q_CO2 specific carbon dioxide production rate (mmol CO₂/g cell mass h) - S xylose concentration (g/1) - Y_cm/s cell mass yield coefficient, (g cell mass/mmol xylose) or (g cell mass/ g xylose consumed) - Y_cm/O2 cell mass yield coefficient, (g cell mass/mmol O₂) - Y_X/S xylitol yield coefficient (g xylitol/g xylose consumed) - Y_x/O2 xylitol yield coefficient (g xylitol/mmol O₂) - Y_e/s ethanol yield coefficient (g ethanol/g xylose consumed) - OUR oxygen uptake rate (mmol O₂/1h) - specific growth rate (1/h)     

Effects of glycerol on the thermal dependence of the stability of human erythrocytes

Incubation of human blood in saline solution of 0–36% (v/v) ethanol for 30 min produces lysis or stabilization of erythrocytes depending on the ethanol concentration. Under less elevated concentrations of ethanol, erythrocytes are present in expanded shapes (R state) that present lower stability and suffer lysis with increase in the ethanol concentration. Under more elevated concentrations of ethanol, erythrocytes are present in contracted shapes (T state) that have higher stability and suffer lysis at even more elevated ethanol concentrations. This work evaluated the effects of glycerol (0 to 2.0 M) and temperature (7 to 47°C) on the stability of the R erythrocytes, characterized by the ethanol concentration at the mid-transition point (D _50R ) of the hemolysis curve (D _50R ). D _50R declined sigmoidally with increase in the glycerol concentration or temperature, due to transition of the R to the T state erythrocytes. In 1.5 M glycerol, the erythrocytes stability decreased below 32 but increased above 37°C. The combination of temperature, glycerol and ethanol actions generates a critical value of osmotic pressure below which the R state predominates and above which the T state predominates. At 7°C 1.5 M glycerol decreased the erythrocytes stability against ethanol but increased the erythrocytes stability against hypotonic shock. Those conditions favor the R state, which has a lower stability against ethanol; however, in the absence of ethanol, glycerol determines less water entrance in the erythrocytes, making more difficult its lysis by hypotonicity.     

Ten years of free-air CO2 enrichment altered the mobilization of N from soil in Lolium perenne L. swards

Effects of free‐air carbon dioxide enrichment (FACE, 60 Pa pCO₂) on plant growth as compared with ambient pCO₂ (36 Pa) were studied in swards of Lolium perenne L. (perennial ryegrass) at two levels of N fertilization (14 and 56 g m^?2 a^?1) from 1993 to 2002. The objectives were to determine how plant growth responded to the availability of C and N in the long term and how the supply of N to the plant from the two sources of N in the soil, soil organic matter (SOM) and mineral fertilizer, varied over time. In three field experiments, ¹⁵N‐labelled fertilizer was used to distinguish the sources of available N. In 1993, harvestable biomass under elevated pCO₂ was 7% higher than under ambient pCO₂. This relative pCO₂ response increased to 32% in 2002 at high N, but remained low at low N. Between 1993 and 2002, the proportions and amounts of N in harvestable biomass derived from SOM (excluding remobilized fertilizer) were, at high N, increasingly higher at elevated pCO₂ than at ambient pCO₂. Two factorial experiments confirmed that at high N, but not at low N, a higher proportion of N in harvestable biomass was derived from soil (including remobilized fertilizer) following 7 and 9 years of elevated pCO₂, when compared with ambient pCO₂. It is suggested that N availability in the soil initially limited the pCO₂ response of harvestable biomass. At high N, the limitation of plant growth decreased over time as a result of the stimulated mobilization of N from soil, especially from SOM. Consequently, harvestable biomass increasingly responded to elevated pCO₂. The underlying mechanisms which contributed to the increased mobilization of N from SOM under elevated pCO₂ are discussed. This study demonstrated that there are feedback mechanisms in the soil which are only revealed during long‐term field experiments. Such investigations are thus, a prerequisite for understanding the responses of ecosystems to elevated pCO₂ and N supply.     

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study

The effect of individual environmental conditions (pH, pO₂, temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol⁻¹) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y _ATP and Q _ATP values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO₂) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y _ATP) and specific growth rate, however the specific ATP production rate (Q _ATP) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l⁻¹), propanol (>2 g l⁻¹) or NaCl (>15 g l⁻¹) lead at first to the decrease in the specific growth rate and Q _ATP, while Y _ATP was affected only at higher concentrations. The observed decrease in Q _ATP was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h⁻¹ and Y _ATP from 12.5 to 18.5 mol ATP g dwt⁻¹. This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.     

High CO2 partial pressure depresses productivity and bioenergetic growth yield of Chlorella pyrenoidosa culture

When low-CO₂ grown Chlorella pyrenoidosa (YSK strain) cells were exposed to high CO₂ partial pressures (pCO₂), the specific growth rate (μ) declined exponentially reaching a steady state value in about 20 h. A short interruption (up to 1 h) by temporarily lowering the pCO₂ did not prevent the continuous decline in μ when high pCO₂ was restored. In chemostate studies, light-limited cultures were supplied with 2 to 90 kPa of CO₂. The steady state biomass production rate and bioenergetic growth yield were related inversely to pCO₂ and the average energy uptake rate. The maintenance energy coefficient was, however, independent of dissolved pCO₂ in the pCO₂ range studied.     

Does a Low Nitrogen Supply Necessarily Lead to Acclimation of Photosynthesis to Elevated CO2?

Long-term exposure of plants to elevated partial pressures of CO₂ (pCO₂) often depresses photosynthetic capacity. The mechanistic basis for this photosynthetic acclimation may involve accumulation of carbohydrate and may be promoted by nutrient limitation. However, our current knowledge is inadequate for making reliable predictions concerning the onset and extent of acclimation. Many studies have sought to investigate the effects of N supply but the methodologies used generally do not allow separation of the direct effects of limited N availability from those caused by a N dilution effect due to accelerated growth at elevated pCO₂. To dissociate these interactions, wheat (Triticum aestivum L.) was grown hydroponically and N was added in direct proportion to plant growth. Photosynthesis did not acclimate to elevated pCO₂ even when growth was restricted by a low-N relative addition rate. Ribulose-1, 5-bisphosphate carboxylase/oxygenase activity and quantity were maintained, there was no evidence for triose phosphate limitation of photosynthesis, and tissue N content remained within the range recorded for healthy wheat plants. In contrast, wheat grown in sand culture with N supplied at a fixed concentration suffered photosynthetic acclimation at elevated pCO₂ in a low-N treatment. This was accompanied by a significant reduction in the quantity of active ribulose-1, 5-bisphosphate carboxylase/oxygenase and leaf N content.     

By-product formation by a novel glycerol-producing yeast,Candida glycerinogenes,with different O2 supplies

Candida glycerinogenes is an aerobe which does not depend on sulphite for production of glycerol. With a sufficient O₂ supply, up to 130 g glycerol l^–1 was produced with 2.6 g acetic acid l^–1 as by-product. However, with an insufficient O₂ supply – with higher volumes of medium or at higher corn steep liquid concentrations – the glycerol concentration was lower because the by-products, ethanol, pyruvate and lactic acid, were produced in greater amounts, up to 45 g l^–1, 4.3 g l^–1, 1.6 g l^–1, respectively, whereas, less acetic acid (0.6 g l^–1) was produced. In addition, ethanol decreased to 0.4 g l^–1 and the glycerol yield improved from 34 to 50% (w/w) by adding 50 g sulphite l^–1, nevertheless, acetic acid increased to 7.8 g l^–1.     

Interactive Effects of CO2, Temperature,Irradiance, and Nutrient Limitation on the Growth and Physiology of the Marine Diatom Thalassiosira pseudonana (Coscinodiscophyceae)

The marine diatom Thalassiosira pseudonana was grown in continuous culture systems to study the interactive effects of temperature, irradiance, nutrient limitation, and the partial pressure of CO₂ (pCO₂) on its growth and physiological characteristics. The cells were able to grow at all combinations of low and high irradiance (50 and 300 μmol photons · m⁻² · s⁻¹, respectively, of visible light), low and high pCO₂ (400 and 1,000 μatm, respectively), nutrient limitation (nitrate-limited and nutrient-replete conditions), and temperatures of 10–32°C. Under nutrient-replete conditions, there was no adverse effect of high pCO₂ on growth rates at temperatures of 10–25°C. The response of the cells to high pCO₂ was similar at low and high irradiance. At supraoptimal temperatures of 30°C or higher, high pCO₂ depressed growth rates at both low and high irradiance. Under nitrate-limited conditions, cells were grown at 38 ± 2.4% of their nutrient-saturated rates at the same temperature, irradiance, and pCO₂. Dark respiration rates consistently removed a higher percentage of production under nitrate-limited versus nutrient-replete conditions. The percentages of production lost to dark respiration were positively correlated with temperature under nitrate-limited conditions, but there was no analogous correlation under nutrient-replete conditions. The results suggest that warmer temperatures and associated more intense thermal stratification of ocean surface waters could lower net photosynthetic rates if the stratification leads to a reduction in the relative growth rates of marine phytoplankton, and at truly supraoptimal temperatures there would likely be a synergistic interaction between the stresses from temperature and high pCO₂ (lower pH).     

利用克雷伯肺炎杆菌限制性底物发酵生产1，3-丙二醇

研究了克雷伯肺炎杆菌（Klebsiella pneumoniae）批式流加发酵生产1,3-丙二醇的发酵工艺,根据1,3-丙二醇的生产和菌体生长相关的特点,采用营养基质限制性流加的发酵工艺,通过控制氮源氯化铵以保持细胞稳定生长。结果表明：过低的氮源浓度,细胞生长受到限制,影响产物1,3-PD的合成;过高的氮源浓度,细胞比生长速率增加,但1,3-PD关于消耗甘油的得率降低,用于生长和维持代谢所消耗的甘油量增加。以0.41 g/(L·h)的氮源流加速率,残余氯化铵浓度在0.1 g/L时,转化率和生产强度最高。发酵25 h～28 h后,1,3-丙二醇最终浓度达到52.03 g/L,生产强度为2.04 g/(L·h),相对于甘油的摩尔转化率为0.66,分别比氮源限制前提高了28.0 ％、35.1 ％及29.4 ％。通过限制性流加氯化铵,控制细胞的比生长速率,使底物甘油有效转变为发酵的目标产物1,3-PD,有效实现产物1,3-PD的高生产强度以及对甘油的高转化率。     

Metabolic selectivity and growth of<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis> in continuous culture under elevated hydrostatic pressure

The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure. A commercial limitation of ethanol production by C. thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate). At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10² relative to atmospheric pressure. Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure. A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures. Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures. The results are partially attributed to the increasing concentration of dissolved product gases (H₂, CO₂) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.