Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

Summary Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g^–1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g^–1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l^–1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O₂ l^–1 h^–1 and 0.95 mmol CO₂ l^–1 h^–1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g^–1 dw) by day 15.     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Nitrite uptake by Chlamydomonas reinhardtii cells immobilized in calcium alginate

When an initial cell loading of about 30–40 µg chlorophyll (Chl)·g^–1 gel and alginate suspension of 3% (w/v) were used for immobilization of Chlamydomonas reinhardtii, the resulting cell beads showed optimum nitrite uptake rate, at 30° C and pH 7.5, of 14 µmol NO _inf2 ^sup– ·mg^–1 Chl·h^–1, the photosynthetic and respiratory activities being about 120 µmol O₂ produced·mg^–1 Chl·h^–1, and 40 µmol O₂ consumed ·mg^–1 Chl·h^–1, respectively. The nitrite uptake activity required CO₂ in the culture and persisted after 8 days of cells immobilization, or in the presence of 0.2 mm ammonium in the medium. Our data indicate that alginate-entrapped C, reinhardtii cells may provide a stable and functional system for removing nitrogenous contaminants from waste-waters.Correspondence to: C. Vílchez     

Characterisation of kinetic parameters and metabolic transition of Corynebacterium glutamicum on l-lysine production in continuous culture

Kinetic parameters and physiological states of Corynebacterium glutamicum at the growing and l-lysine-overproducing phase were characterised in continuous culture on threonine-limited complex and minimal media. High l-lysine productivity occurred at dilution rates ranging from 0.1 h^–1 to 0.3 h^–1 on threonine-limited complex medium, and at dilution rates ranging from 0.1 h^–1 to 0.15 h^–1 in minimal medium. l-Lysine yields of 0.25 g/g (0.31 g/g as l-lysine hydrochloride) in complex medium, and of 0.17 g/g (0.21 g/g as l-lysine hydrochloride) in minimal medium, corresponding respectively to intrinsic yields of 0.533 g/g and 0.572 g/g were obtained. These intrinsic yield factors are closed to the theoretical ones (0.608 g/g, 0.75 mol/mol). Intrinsic biomass yields were calculated as 0.658 g/g in complex medium and 0.283 g/g in minimal medium. CO₂ production has been clearly related to l-lysine production. According to our results on specific uptake rates and specific productivities in complex medium, metabolic rearrangement should occur during the transition from the growing phase to the l-lysine-overproducing phase. This phenomenon was further investigated.     

Emission of N2O, N2, CH4, and CO2 from constructed wetlands for wastewater treatment and from riparian buffer zones

We measured nitrous oxide (N₂O), dinitrogen (N₂), methane (CH₄), and carbon dioxide (CO₂) fluxes in horizontal and vertical flow constructed wetlands (CW) and in a riparian alder stand in southern Estonia using the closed chamber method in the period from October 2001 to November 2003. The replicates’ average values of N₂O, N₂, CH₄ and CO₂ fluxes from the riparian gray alder stand varied from −0.4 to 58 μg N₂O-N m⁻² h⁻¹, 0.02–17.4 mg N₂-N m⁻² h⁻¹, 0.1–265 μg CH₄-C m⁻² h⁻¹ and 55–61 mg CO₂-C m⁻² h⁻¹, respectively. In horizontal subsurface flow (HSSF) beds of CWs, the average N₂ emission varied from 0.17 to 130 and from 0.33 to 119 mg N₂-N m⁻² h⁻¹ in the vertical subsurface flow (VSSF) beds. The average N₂O-N emission from the microsites above the inflow pipes of the HSSF CWs was 6.4–31 μg N₂O-N m⁻² h⁻¹, whereas the outflow microsites emitted 2.4–8 μg N₂O-N m⁻² h⁻¹. In VSSF beds, the same value was 35.6–44.7 μg N₂O-N m⁻² h⁻¹. The average CH₄ emission from the inflow and outflow microsites in the HSSF CWs differed significantly, ranging from 640 to 9715 and from 30 to 770 μg CH₄-C m⁻² h⁻¹, respectively. The average CO₂ emission was somewhat higher in VSSF beds (140–291 mg CO₂-C m⁻² h⁻¹) and at the inflow microsites of HSSF beds (61–140 mg CO₂-C m⁻² h⁻¹). The global warming potential (GWP) from N₂O and CH₄ was comparatively high in both types of CWs (4.8 ± 9.8 and 6.8 ± 16.2 t CO₂ eq ha⁻¹ a⁻¹ in the HSSF CW 6.5 ± 13.0 and 5.3 ± 24.7 t CO₂ eq ha⁻¹ a⁻¹ in the hybrid CW, respectively). The GWP of the riparian alder forest from both N₂O and CH₄ was relatively low (0.4 ± 1.0 and 0.1 ± 0.30 t CO₂ eq ha⁻¹ a⁻¹, respectively), whereas the CO₂-C flux was remarkable (3.5 ± 3.7 t ha⁻¹ a⁻¹). The global influence of CWs is not significant. Even if all global domestic wastewater were treated by wetlands, their share of the trace gas emission budget would be less than 1%.     

Fluxes of methane and carbon dioxide from a small productive lake to the atmosphere

The fluxes of CH₄ and CO₂ to the atmosphere, and the relative contributions of ebullition and molecular diffusion, were determined for a small hypertrophic freshwater lake (Priest Pot, UK) over the period May to October 1997. The average total flux of CH₄ and CO₂ (estimated from 7 sites on the lake) was approximately 52 mmol m^–2 d^–1 and was apportioned 12 and 40 mmol m^–2 d^–1 toCH₄ and CO₂ respectively. Diffusion across the air-water interface accounted for the loss of 0.4and 40 mmol m^–2 d^–1 of CH₄ and CO₂ respectively whilst the corresponding figures for ebullition losses were 12.0 (CH₄) and 0.23 (CO₂) mmol m^–2 d^–1. Most CH₄ (96%) was lost by ebullition, and most CO₂ (99%) by diffusive processes. The ebullition of gas, measured at weekly intervals along a transect of the lake, showed high spatial and temporal variation. The CH₄ content of the trapped gas varied between 44 and 88% (by volume) and was highest at the deepest points. Pulses of gas ebullition were detected during periods of rapidly falling barometric pressure. Therelevance of the measurements to global estimates ofcarbon emission from freshwaters are discussed.     

Carbon turnover in peatland mesocosms exposed to different water table levels

Changes of water table position influence carbon cycling in peatlands, but effects on the sources and sinks of carbon are difficult to isolate and quantify in field investigations due to seasonal dynamics and covariance of variables. We thus investigated carbon fluxes and dissolved carbon production in peatland mesocosms from two acidic and oligotrophic peatlands under steady state conditions at two different water table positions. Exchange rates and CO₂, CH₄ and DOC production rates were simultaneously determined in the peat from diffusive-advective mass-balances of dissolved CO₂, CH₄ and DOC in the pore water. Incubation experiments were used to quantify potential CO₂, CH₄, and DOC production rates. The carbon turnover in the saturated peat was dominated by the production of DOC (10–15 mmol m^–2 d^–1) with lower rates of DIC (6.1–8.5 mmol m^–2 d^–1) and CH₄ (2.2–4.2 mmol m^–2 d^–1) production. All production rates strongly decreased with depth indicating the importance of fresh plant tissue for dissolved C release. A lower water table decreased area based rates of photosynthesis (24–42%), CH₄ production (factor 2.5–3.5) and emission, increased rates of soil respiration and microbial biomass C, and did not change DOC release. Due to the changes in process rates the C net balance of the mesocosms shifted by 36 mmol m^–2 d^–1. According to our estimates the change in C mineralization contributed most to this change. Anaerobic rates of CO₂ production rates deeper in the peat increased significantly by a factor of 2–3.5 (DOC), 2.9–3.9 (CO₂), and 3–14 (CH₄) when the water table was lowered by 30 cm. This phenomenon might have been caused by easing an inhibiting effect by the accumulation of CO₂ and CH₄ when the water table was at the moss surface.     

Regulation of photosynthetic rates of submerged rooted macrophytes

Summary Fourteen temperate, submerged macrophytes were cultivated in the laboratory at high DIC levels (3.3–3.8 mM), 10.4–14.4 mol photons (PAR) m^-2 d^-1 and 15°C. Photosynthesis at photosaturation ranged between 0.59 and 17.98 mg O₂ g^-1 DW h^-1 at ambient pH (8.3) and were markedly higher between 1.76 and 47.11 mg O₂ g^-1 DW h^-1 at pH 6.5 under elevated CO₂ concentrations. Photosynthetic rates were significantly related to both the relative surface area and the chlorophyll content of the leaves. Consequently, the photosynthetic rate was much less variable among the species when expressed per surface area and chlorophyll content instead of dry mass. The chlorophyll content was probably a main predictor of photosynthesis of submerged leaves because of the direct relationship of chlorophyll to the light harvesting capacity and/or a coupling to the capacity for photosynthetic electron transport and carboxylation. A comparison with terrestrial leaves characterized the submerged leaves by their low chlorophyll concentrations and low photosynthetic rates per surface area due to the thin leaves. Photosynthetic rates per chlorophyll content in submerged leaves at CO₂ saturation, however, were at the same level as photosynthesis in terrestrial leaves measured at ambient CO₂ when appropriate corrections were made for differences in incubation temperature.     

Anaerobic fermentation of Salmonella typhimurium with and without pyruvate carboxylase

A plasmid that expressed pyruvate carboxylase (PYC) from Rhizobium etli was introduced into Salmonella typhimurium LT2. Anaerobic fermentations of S. typhimurium with and without PYC were compared with glucose as a carbon source. The presence of PYC increased the succinate yield from glucose from 0.044 g g^–1 to 0.22 g g^–1, while the lactate yield decreased from 0.31 g g^–1 to 0.16 g g^–1. Metabolic flux calculations during the early growth phase indicate that under these growth conditions in the presence of PYC more carbon flows to oxaloacetate via pyruvate carboxylase than via phosphoenolpyruvate carboxylase. Also, under these growth and induction conditions, the presence of PYC diminished the cell growth rate from 0.34 h^–1 to 0.28 h^–1, the specific rate of ATP formation from 45 mmol l^–1 h^–1 to 27 mmol l^–1 h^–1, and the specific rate of glucose consumption from 17 mmol l^–1 h^–1 to 10 mmol l^–1 h^–1.     

A new chemoheterotrophic bacterium catalyzing water-gas shift reaction

A new bacterium, Citrobacter sp. Y19, catalyzing the water-gas shift reaction was isolated from an anaerobic wastewater sludge digester. It grew aerobically with a high specific growth rate of 0.7 h^–1 in a mineral salt medium supplemented with yeast extract and Bacto-tryptone, and produced H₂ under anaerobic conditions after the cells were transferred to tryptone-deleted medium. The maximum H₂ production rate was 33 mmol H₂ g^–1 cell h, which was maintained for about 200 h. This is the first report on a chemoheterotrophic bacterium which utilizes CO with the production of H₂ and CO₂.     

Effect of benzoic acid on the ammonium transport system of Zygosaccharomyces bailii

Summary A study of the ammonium transport system of Zygosaccharomyces bailii was carried out using methylammonium as a non-metabolizable analogue. Benzoic acid in the growth medium decreased the capacity of the transport system from 1.46 ± 0.11 mmol.g^–1.h^–1 to 0.41±0.04 mmol.g^–1.h^–1, while the affinity for ammonium was not significatively affected. Although ammonium uptake was inhibited by benzoic acid, the ammonium transport system was still active at preservative concentrations which fully inhibited growth suggesting that inhibition of growth was not governed by the uptake of this nutrient.     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Deficiency of potassium but not phosphorus enhances root respiration

Root respiration of kohlrabi (Brassica oleraceavar. gongylodes) was measured non-destructivelyin vivo by infrared gas analysis of completeroot systems, using potted plants in sand culture andnutrient solutions, for six weeks under (a) nutrientsufficiency, (b) deficiency of all mineral nutrients,(c) potassium deficiency or (d) phosphorus deficiency.This was to study the adaptation to nutrient stress interms of changes in root growth, root respiration,assimilate allocation and energy requirement fornutrient uptake. Both deficiencies of phosphorus andpotassium increased the root:shoot-ratio. This wasattributed to the plants transferring a largerrelative proportion of assimilates to the roots thanto the shoots relative to nutrient-sufficient plants.Roots of nutrient-sufficient kohlrabi respired 1.7 or7.7 mg CO₂ h^–1 per g fresh or dry matter, respectively. However, potassiumdeficiency enhanced root respiration to 2.4 mgCO₂ h^–1 or 12.2 mg CO₂ h^–1 on a per g fresh or dry weight basis respectively. This originated from an additional2.6 mg glucose g^–1 dry matter h^–1 allocated to the roots and provided 50 Joule additional energy(150 versus 100 Joule g^–1 dry matter h^–1)which may become available for the proposedK⁺:H⁺ symporter for potassium uptake.     

Field energetics of a large carnivorous lizard,Varanus rosenbergi

Summary Field metabolic rates (FMRs; CO₂ production) and water influx rates of Varanus rosenbergi were measured seasonally by means of doubly-labelled water (³H₂ ¹⁸O). Metabolic rates and water influexes were highest in summer (0.18 ml CO₂ g^–1 h^–1 and 17.9 ml H₂O kg^–1 day^–1) and lowest in winter (0.04 ml CO₂ g^–1 h^–1 and 6.5 ml H₂O kg^–1 day^–1). FMRs and water fluxes were intermediate in spring. It is estimated that a 1-kg V. rosenbergi would need to consume 4.7 kg of prey each year to remain in energy balance and that the energy commitment to egg production requires a 40% increase in the food acquisition of a gravid female in late spring and early summer. It is calculated that water influxes in summer are totally provided from food but in other seasons, particularly winter, pulmocutaneous water exchange and/or drinking are significant avenues of water intake.     

Effects of growth temperature on maximal specific growth rate,yield, maintenance,and death rate in glucose-limited continuous culture of the thermophilic Bacillus caldotenax

Summary The influence of temperature on the growth of the theromophilic Bacillus caldotenax was investigated using chemostat techniques and a chemically defined minimal medium. All determined growth constants, that is maximal specific growth rate, yield and maintenance, were temperature dependent. It was striking that the very large maintenance requirement was about 10 times higher than for mesophilic cells under equivalent conditions. A death rate, which was very substantial at optimal and supraoptimal growth temperatures, was estimated by comparing the maintenance for substrate and oxygen. There was no indication for a thermoadaptation as postulated by Haberstich and Zuber (1974).Symbols D Dilution rate (h^–1) - D_c=_max Critical dilution rate (h^–1) - E Temperature characteristic (J mol^–1) - k Organism constant - k_d Death rate coefficient (h^–1) - k_m Maintenance substrate coefficient estimated from M_O (h^–1) - M_O Maintenance respiration, mmol O₂ per g dry biomass and h (mmol g^–1h^–1) - M_O Maintenance respiration, taking k_d into account - m_S Maintenance substrate coefficient, g glucose per g dry biomass and h (h^–1) - OD Optical density at 546 nm - QO₂ Specific O₂-uptake rate (mmol g^–1h^–1) - Q _O2 ^V Specific O₂-uptake rate for viable portion of biomass (mmol g^–1 h^–1) - Q_S Specific glucose uptake rate (h^–1) - Q _S ^V Specific glucose uptake rate for viable portion of biomass (h^–1) - R Gas constant 8.28 J mol^–1K^–1 - S Substrate concentration in reactor (g l^–1) - S_O Influent substrate concentration (g l^–1) - T_max Maximal growth temperature (°C) - T_min Minimal growth temperature (°C) - X Dry biomass (g l^–1) - X_tOt=X Dry biomass containing dead and viable cells - X_v Viable portion of biomass - Y _O ^m Potential yield for O₂ corrected for maintenance respiration (g mol^–1) - Y _S ^m Potential yield for substrate corrected for maintenance requirement, g biomass per g glucose (–) - Specific growth rate (h^–1) - _max Maximal specific growth rate (h^–1)     

Establishment and characterization of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia

A procedure is described for the rapid establishment of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia. Photoautotrophic growth was induced by lowering the glucose concentration to 2.5 g.l^–1 in the protoplast culture medium and by omitting glucose from the subsequent dilution medium. Four week-old highly viable suspensions were plated on an agar-medium without glucose in unsealed Petri dishes and kept in illuminated chambers flushed with 0.05 % or 2 % CO₂. Air-grown calli had net photosynthesis rates of 1.8 and 17 moles CO₂.g^–1 fresh wt.h^–1 in air at 0.034 % CO₂ and in air enriched with 1 % CO₂, respectively. Calli grown in 2 % CO₂ exhibited lower rates of net photosynthesis at the two CO₂ concentrations tested (0 and 7.5 moles CO₂.g^–1 fresh wt.h^–1, respectively). The contribution of photosynthesis to growth was estimated to be 80 % in air-grown calli and more than 90 % in calli grown in 2 % CO₂. The suitability of this photoautotrophic culture procedure is discussed with regard to the screening of photosynthetic mutants or transformants from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAA indoleacetic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

The influence of H2, CO2 and dilution rate on the continuous fermentation of acetone-butanol

Summary The effect of product gases, H₂ and CO₂, on solvent production was studied using a continuous culture of alginate-immobilized Clostridium acetobutylicum. Initially, in order to find the optimum dilution rate for aceton--butanol production in this system, fermentations were carried out at various dilution rates. With 10% H₂ and 10% CO₂ in the sparging gas, a dilution rate of 0.07 h^–1 was found to maximize volumetric productivity (0.58 g·l^–1·h^–1), while the maximum specific productivity of 0.27 g^–·h^–1 occured at 0.12 h^–1. Continuous cultures with vigorous sparging of N₂ produced only acids. It was concluded that in the case of continuous fermentation H₂ is essential for good solvent production, although good solvent production is possible in an H₂-absent environment in the case of batch fermentations. When the fermentation was carried out at atmospheric pressure under H₂-enriched conditions, the presence of CO₂ in the sparging gas did not slow down glucose metabolism; rather it changed the direction of the phosphoroclastic reaction and as a result increased the butanol/acetone ratio.     

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O₂ flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O₂ h^–1 dm^–3 for both culture modes, was slightly higher (0.425 g g^–1) for immobilized cultures than for their free counterparts (0.39 g g^–1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.     

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4--d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca²⁺, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4--d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h^–1, biomass yield coefficient of 0.5 g g^–1 and feed substrate concentration of 200 g L^–1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L^–1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g^–1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.Nomenclature C _ox Dissolved oxygen concentration (mg L^–1) - CPR Carbon dioxide production rate (mmol h^–1) - C _s0 Glucose concentration in the feed (g L^–1) - C _s Substrate concentration in the fermenter (g L^–1) - C _s.crit Critical substrate concentration (g L^–1) - E Ethanol concentration (g L^–1) - F _s Substrate flow rate (g h^–1) - i Sample number (–) - K _e Constant in Equation 6 (g L^–1) - K _o Constant in Equation 7 (mg L^–1) - K _s Constant in Equation 5 (g L^–1) - m Specific maintenance term (h^–1) - OUR Oxygen up-take rate (mmol h^–1) - q _ox Specific oxygen up-take rate (h^–1) - q _ox.max Maximum specific oxygen up-take rate (h^–1) - q _p Specific product formation rate (h^–1) - q _s Specific substrate up-take rate (g g^–1 h^–1) - q _s.max Maximum specific substrate up-take rate (g g^–1 h^–1) - RQ Respiratory quotient (–) - S Total substrate in the fermenter at timet (g) - S ₀ Substrate mass fraction in the feed (g g^–1) - t Fermentation time (h) - V Instantaneous volume of the broth in the fermenter (L) - V ₀ Starting volume in the fermenter (L) - V _si Volume of samplei (L) - x Biomass concentration in the fermenter (g L^–1) - X ₀ Total amount of initial biomass (g) - X _t Total amount of biomass at timet (g) - Y _p/s Product yield coefficient on substrate (–) - Y _x/e Biomass yield coefficient on ethanol (–) - Y _x/s Biomass yield coefficient on substrate (–) Greek letters Moles of carbon per mole of yeast (–) - Moles of hydrogen atom per mole of yeast (–) - Moles of oxygen atom per mole of yeast (–) - Moles of nitrogen atom per mole of yeast (–) - Specific growth rate (h^–1) - _crit Critical specific growth rate (h^–1) - _E Specific ethanol up-take rate (h^–1) - _max.E Maximum specific ethanol up-take rate (h^–1)