End product inhibition in methane fermentations: Effects of carbon dioxide and methane on methanogenic bacteria utilizing acetate

Summary The effects of pCO₂ and pCH₄ in the interval 0–1 bar on rates of acetate degradation and methane formation by methanogens as well as methane yields were studied in enrichment cultures in batch and continuous fermentations.In batch fermentations the rate of acetate utilization by methanogens was 1,000–1,500 mg/l · d at low levels of pCO₂. CO₂ was inhibitory and degradation rates were around 350 mg/l · d in 1 bar CO₂. The degradation of acetate was almost linear. In continuous culture maximum rates of acetate utilization around 2,500 mg/l · d were obtained and the acetate concentration in the substrate was reduced by 98–99%.The yields of methane on acetate substrates were close to the theoretical value (1 mole CH₄ per mole HAc) in the interval pCO₂-0–0.5 bar. In 1 bar CO₂ yields decreased by 20–30%.CH₄ was found to be only slightly inhibitory; the inhibiting effects of 1 bar CH₄ on acetate degradation rates were comparable to the effects of 0.3 bar CO₂. Also gas sparging and rapid mixing had small effects compared with a non-sparged, slowly mixed culture.The redox potential was usually around –200 mV during fermentations and no connection was found between acetate degradation rate, E_h and pCO₂.Acetate and propionate degradation were the reactions most sensitive to pCO₂ and to obtain maximum rates as well as maximum methane yields pCO₂-levels around 0.2 bar were found to be optimal.     

CO2 gas exchange of the understory plantAsarum europaeum L. in November

The CO₂ gas exchange rates of the Central European perennial understory plantAsarum europaeum L. were measured in late autumn (October 30 to November 30) in its natural habitat day and night.During these measurements the temperature ranged from 0 to 15°C and the absolute air humidity from 3 to 10 mg H₂O·1^–1. Temperature and absolute air humidity over these ranges did not affect CO₂ net assimilation which was determined almost entirely by quantum flux density.CO₂ net assimilation was light saturated at about 100 M·m^–2·s^–1 quantum flux density. The uptake rate at this point was 4.3 mg·dm^–2·h^–1. The compensation point occurred at approximately 1 M·m^–2·s^–1.     

Carbon dioxide efflux and pCO2 in soils of threeQuercus ilex montane forests

Soil CO₂ efflux and pCO₂ in the soil atmosphere were measured during one year at three montane sites of Mediterranean sclerophyllous forests in NE Spain. Two sites were located in the upper and lower slopes of a small catchment in the Prades mountains (mean precipitation 550 mm year^–1), and a third site was located on a lower slope in the Montseny mountains (mean precipitation 900 mm year^–1). The three sites were similar in bedrock and vegetation, but differed in soil characteristics and water availability. Seasonal variation of CO₂ efflux and soil pCO₂ were affected by soil temperature and, to a lesser extent, by soil moisture. Annual mean soil CO₂ efflux (considered as soil respiration) was similar at Montseny and at the comparably located site at Prades (83 ± 18 S.E. vs. 75 ± 9 mg CO₂ m^–2 hour^–1 , respectively), and was highest at the Prades upper slope site (122 ± 22 mg C0₂ m^–2 hour^–1 ). Despite those relatively similar CO₂ effluxes, mean soil pCO₂ was much higher at both Prades sites than at Montseny. Soil pCO₂ always increased with depth at Prades while maxima pCO₂ at Montseny were often at 20–30 cm depth. A model based on gas diffusion theory was able to explain why soil pCO₂ was much higher at Prades than at Montseny, and to reproduce the shape of the vertical profile of pCO₂ at the Prades soils. Nevertheless, the model failed to simulate the soil pCO₂ maximum found at 20–30 cm depth at the Montseny site. Model simulations using a time-variable CO₂ production rate suggested that pCO₂ maxima at intermediate depth could be the result of a transient situation instead of an equilibrium one.     

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.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Summary Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both elymoclavine-inhibitionless and clavine alkaloid-decompositionless fermentations if a combination of fermentation and separation units in a closed loop was used.Nomenlacture k ₁ rate constant of agroclavine synthesis (mg Agro · mg Elymo/l·g DW·day for stage 1, mg Agro/g DW·day for stage 2) - k ₂ parameter describing inhibition of agroclavine formation rate by elymoclavine (mg Elymo/l) - k ₃ specific rate of agroclavine decay (l/g DW·day) - k ₄ maximal specific rate of elymoclavine synthesis (stage 1, 1/g DW·day, stage 2, mg Elymo/g DW·day) - k ₄ ^– maximal specific rate of elymoclavine synthesis in stage 1 (inhibition-activation mechanism) (mg Elymo/g DW·day) - k ₅ physiological constant describing the elymoclavine decay rate (l²/g DW·day·mg Elymo) - k ₅ ^– physiological constant describing the activation of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₆ physiological constant describing the repression of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₇ maximal specific growth rate (1/day) - k ₈ specific rate of biomass decay (l/g DW·day) - A agroclavine concentration (mg/l) - E elymoclavine concentration (mg/l) - r _A specific rate of agroclavine biosynthesis (mg Agro/g DW·day) - r _E specific rate of elymoclavine biosynthesis (mg Elymo/g DW·day) - r _i specific rate of alkaloid biosynthesis (mg alkaloid/g DW·day) - X dry biomass concentration (g/l) - specific growth rate (1/day) Abbreviations Agro agroclavine - Elymo elymoclavine - Chano chanoclavine - DW dry weight of biomass     

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     

Interactions between substrate,fermentation end-products,buffering systems and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro

Summary In animal nutrition, incubation of feed samples with CO₂/HCO₃-buffered rumen fluid is used to predict the nutritional values of the feed. During fermentation, volatile fatty acids (VFAs) are produced, which release CO₂ from the buffer through their H⁺ ions. This indirect gas production amounted to 20.8 ml gas per mmol VFA. By incubating glucose, rice starch and cellulose, the relationship between direct and indirect gas production in relation to fermentation kinetics was studied. The total amount of gas formed was found to be dependent on the composition of the fermentation end-products formed. This could be described by: ml gas = M_v·mmol HAc + 2M_v·mmol HB + 0.87M_v·mmol Tot. VFA where HAc = acetic acid; HB = butyric acid; and M_v = molar gas volume. No clear relationship was found between the rate of fermentation and total gas production. From rice starch more total gas was produced than from glucose and cellulose, which were fermented faster and slower, respectively. Correspondence to: S. F. Spoelstra     

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014–0.076 h^-1) and extracellular pH (6.11–6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentrationto response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07–0.11 h^-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04–0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11–0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determined that maintenance coefficient (0.04–0.06 g cellulose/g cells · h) and true growth yield (0.23–0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower _max on microcrystalline cellulose.     

A horizontal bioreactor for ethanol production by immobilized cells

Continuous ethanol production in a three stage horizontal tank bioreactor (HTR) by yeast cells entrapped in Ca-alginate was about 30% higher than in a vertical type of bioreactor and reached 31 kg/(m³ · h) at 95% glucose utilization. Maximum ethanol productivity obtained was 41.2 kg/(m³ · h), however, with 38% of the glucose fed to the HTR being wasted. The higher performance of the HTR had been mainly accounted for the reduction of the adverse CO₂ gas phase effect and the more pronounced plug-flow character. Glucose and ethanol profiles along the HTR revealed that 50–80% of the overall fermentation activity was present in the first stage. Within a test period of 23 d the HTR showed an excellent operational stability.Compared to other continuous ethanol production processes using entrapped yeast cells the HTR presented here belongs to the top ones.     

Effect of Yeast Extract on Growth and Metabolism of H2-Utilizing Acetogenic Bacteria from the Human Colon

The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon. Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1–2 g. L⁻¹ in the medium did not enhance utilization of H₂/CO₂. Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain. Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain. In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate. The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria. Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species. Received: 6 March 1998 / Accepted: 3 April 1998     

Effect of Altered pO(2) in the Aerial Part of Soybean on Symbiotic N(2) Fixation

Dry matter accumulation, nitrogen content and N₂ fixation rates of soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to a pO₂ of 5, 10, 21, or 30% and a pCO₂ of 300 μl CO₂/l or a pO₂ of 21% and a pCO₂ of 1200 μl CO₂/l during the complete growth cycle were measured. Total N₂[C₂H₂] fixed was increased by CO₂/O₂ ratios greater than those in air and was decreased by ratios smaller than those in air; the effects on N₂ fixation of decreased pO₂ or elevated pCO₂ were quantitatively similar during the period of vegetative growth. Decreased pO₂ produced a smaller increase then elevated pCO₂ during the reproductive period, presumably because of the decreased sink activity of the arrested reproductive growth under subambient pO₂. At a pO₂ of 5% and a pCO₂ of 300 μl CO₂/l total N₂ fixed was increased 125% and per cent nitrogen content in the vegetative parts was increased relative to air while that in the seed was decreased. Dry matter production was increased and reproductive growth was arrested as previously reported for plants receiving only fertilizer nitrogen. At a pO₂ of 30% and a pCO₂ of 300 μl CO₂/l total N₂ fixed was decreased 50% and per cent nitrogen content in the vegetative part was increased relative to air while that in the reproductive structures was unaffected. Dry matter production was similarly decreased in both vegetative and reproductive structures. These effects of altered pO₂ in the aerial part on N₂ fixation are consistent with the hypothesis that the amount of photosynthate available to the nodule may be the most significant primary factor limiting N₂ fixation while sink activity of the reproductive structures may be a secondary factor.     

On-line biomass estimation using a modified oxygen utilization rate

A procedure for estimating biomass during batch fermentation from on-line gas analysis is presented. First, the respiratory quotient was used to determine the fraction of the total oxygen utilization rate required for cell maintenance and growth versus product synthesis. The modified oxygen utilization rate was then used to estimate biomass on-line by integrating the oxygen balance for cell synthesis-maintenance. The method is illustrated for the case of L-lysine synthesis by Corynebacterium glutamicum.List of Symbols CER mmol CO₂/l · h carbon dioxide evolution rate - M _{O 2}/x mmol O₂/h · g cells maintenance coefficient - OUR mmol O₂/l · h oxygen utilization rate - OUR _X mmol O₂/l · h OUR fraction for cell maintenance and growth - RQ mmol CO₂/mmol O₂ respiratory quotient(CER/OUR) - X g cells/l biomass concentration - Y _X/O₂ yield coefficients     

Thermoanaerobium lactoethylicum spec. nov. a new anaerobic bacterium from a hot spring of Kamchatka

A new anaerobic thermophilic Gram-positive, nonsporeforming bacterium strain ZE-1 was isolated from a hot spring of Kamchatka (USSR). The cells are rod-shaped, (0.5–0.8 · 2.0–20 m), non-motile. The bacterium can grow between 42 and 75°C; the optimal temperature is 65°C. The growth is possible between pH values 5.0 and 8.5; optimal pH is 7.0. The cultures grow on the media containing peptone, yeast extract, or casein hydrolysate as nitrogen sources in the presence of glucose or some other sugars, mannitol or starch. The main fermentation products of glucose are ethanol, acetate, lactate, H₂, CO₂; byproducts are propionic, butyric and isovaleric acids. Glucose is metabolized via Embden-Meyerhoff-Parnas pathway. Molecular hydrogen does not inhibit growth. The bacterium does not reduce aceton to isopropanol, but is able to form H₂S from elemental sulfur. The bacterium contains a soluble hydrogenase. This enzyme catalyzes both evolution and uptake of H₂ and is active in the presence of methyl viologen. The DNA-base composition is 34.6 mol%; the genome size 2.08x10⁹ D. The name proposed for the isolated bacterium strain ZE-1 is Thermoanaerobium lactoethylicum spec. nov.     

Alcoholic fermentation of glucose and xylose by Pichia stipitis,Candida shehatae,Saccharomyces cerevisiae and Zymomonas mobilis: oxygen requirement as a key factor

Summary To investigate simultaneous alcoholic fermentation of glucose and xylose derived from lignocellulosic material by separate or co-culture processes, the effect of oxygen transfer rate (OTR) on the fermentation of 50 g/l xylose by Pichia stipitis NRRL Y 7124 and Candida shehatae ATCC 22984, and the fermentation of 50 g/l glucose by Saccharomyces cerevisiae CBS 1200 and Zymomonas mobilis ATCC 10988 was carried out in batch cultures. The kinetic parameters of the xylose-fermenting yeasts were greatly dependent on the OTR. The optimum OTR values were found to be 3.9 and 1.75 mmol·1^–1·h^–1 for C. shehatae and P. stipitis, respectively. By contrast the fermentative parameters of S. cerevisiae were poorly affected by the OTR range tested (0.0–3.5 mmol·l^–1·h^–1) Under these conditions the ethanol yields ranged from 0.41 g·g^–1 to 0.45 g·g^–1 and the specific ethanol productivity was around 0.70 g·g^–1·h^–1. Z. mobilis gave the highest fermentative performance under strictly anaerobic conditions (medium continually flushed with nitrogen): under these conditions, the ethanol yield was 0.43 g·g^–1 and the average specific ethanol productivity was 2.3 g·g^–1·h^–1. Process considerations in relation to the effect of OTR on the fermentative performance of the tested strains are discussed. Offprint requests to: J. P. Delgenes     

Development of dependence on aerial respiration in Polypterus senegalus (Cuvier)

The respiratory behaviour and partitioning of O₂ uptake between air and water were investigated in Polypterus genegalus using continuous-flow and two-phase respirometers and lung gas replacement techniques P. senegalus rarely resorts to aerial respiration under normal conditions. Partitioning of O₂ consumption depends on the activity and age of fish and the availability of aquatic oxygen. Immature fish (12–22 g) cannot utilize aerial O₂ but older fish exhibit age-dependent reliance on aerial respiration in hypoxic and hypercarbic waters. Pulmonary respiration accounts for 50% of the total requirement at aquatic O₂ concentrations of about 3.5 mg · l^–1 (or CO₂ of about 5%) and fish rely exclusively on aerial respiration at O₂ concentrations of less than 2.5 mg · l^–1. Branchial respiration is initially stimulated by hypercarbia (CO₂: 0.5–0.8%) but increased hypercarbia (CO₂ – 1%) greatly depresses (by over 90%) brancial respiration and initiates (CO₂: 0.5%) and sustains pulmonary respiration.     

Does the threshold of transcutaneous partial pressure of carbon dioxide represent the respiratory compensation point or anaerobic threshold?

On reaching the respiratory compensation point (RCP) during rapidly increasing incremental exercise, the ratio of minute ventilation (V_E) to CO₂ output (VCO₂) rises, which coincides with changes of arterial partial pressure of carbon dioxide (P _aCO₂). Since P _aCO₂ changes can be monitored by transcutaneous partial pressure of carbon dioxide (PCO_2,tc) RCP may be estimated by PCO_2,tc measurement. Few available studies, however, have dealt with comparisons between PCO_2,tc threshold (T _AT) and lactic, ventilatory or gas exchange threshold (V _AT), and the results have been conflicting. This study was designed to examine whether this threshold represents RCP rather than V _AT. A group of 11 male athletes performed incremental excercise (25 W · min^–1) on a cycle ergometer. The PCO_2,tc at (44°C) was continuously measured. Gas exchange was computed breath-by-breath, and hyperaemized capillary blood for lactate concentration ([la^–]_b) and P _aCO₂ measurements was sampled each 2 min. The T _AT was determined at the deflection point of PCO_2,tc curve where PCO_2,tc began to decrease continuously. The V _AT and RCP were evaluated with VCO₂ compared with oxygen uptake (VO₂) and V_E compared with the VCO₂ method, respectively. The PCO_2,tc correlated with P _aCO₂ and end-tidal PCO₂. At T _AT, power output [P, 294 (SD 40) W], VO₂ [4.18 (SD 0.57)l · min^–1] and [la^–] [4.40 (SD 0.64) mmol · l^–1] were significantly higher than those at V _AT[P 242 (SD 26) W, VO₂ 3.56 (SD 0.53) l · min^–1 and [la^–]_b 3.52 (SD 0.75), mmol · l^–1 respectively], but close to those at RCP [P 289 (SD 37) W; VO₂ 3.97 (SD 0.43) l · min^– and [la^–]_b 4.19 (SD 0.62) mmol · l^–1, respectively]. Accordingly, linear correlation and regression analyses showed that P, VO₂ and [la^–]_b at T _AT were closer to those at RCP than at V _AT. In conclusion, the T _AT reflected the RCP rather than V _AT during rapidly increasing incremental exercise.     

Plant regeneration from protoplasts isolated from embryogenic suspension cultured cells of Cinnamomum camphora L.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1±2.1×10⁶/g FW) and viability (91.8±3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl₂·2H₂O, and 0.011% (w/v) NaH₂PO₄·2H₂O. First divisions occurred 7–10 days following culture initiation. The highest division frequency (24.6±2.9%) and plating efficiency (6.88±0.8%) were obtained in liquid medium (MS) supplemented with 30 g l^–1 sucrose, 0.7M glucose, 0.1 mg l^–1 NAA, 1.0 mg l^–1 BA, and 1.0 mg l^–1 GA₃. After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l^–1 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.     

Oxygen and carbon dioxide transport by the haemocyanin of an amphibious crab,Holthuisana transversa

Summary The oxygen and carbon dioxide transporting properties of the haemolymph from an amphibious Australian crab,Holthuisana transversa were investigated. Within the temperature range 15 to 35°C increasing temperature markedly decreased oxygen affinity (H=–54 kJ·mol^–1). The Bohr effect was small at all temperatures with a mean value of –0.13. Over the temperature range 15–35°C there was a significant increase in the cooperativity of oxygen binding. Changing the concentration of Ca,l-lactate or haemocyanin in the haemolymph could elicit no significant change in either O₂ affinity or cooperativity of O₂ binding. There was no evidence in support of a specific effect of CO₂ on oxygen affinity of either non-dialysed or dialysed haemolymph.The amount of CO₂ that could be carried byH. transversa haemolymph was significantly reduced by increased temperature (approx. 14 to 12.5 mmol·l^–1 CO₂). Comparisons of oxygenated and deoxygenated haemolymph at a fixed pH were unable to demonstrate the presence of a significant Haldane effect. Combining data from oxygenated and deoxygenated haemolymph the buffer value was calculated to be in the range –6.2 to –8.5 mmol·l^–1 HCO ₃ ^– ·pH unit^–1.The insensitivity ofH. transversa haemocyanin function to all modulating influences except temperature is discussed with respect to the ecology of this crab.     

Effect of pH,aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O₂L^–1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L^–1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attainingS. cerevisiae cells suitable for invertase production were: temperature=30°C; pH=5.0; DO=3.3 mg O₂L^–1; (S)=0.5 g L^–1 and sucrose added into the fermenter according to the equations: (V–V_o)=t²/16 or (V–V_o)=(V_f–V_o)·(e^0.6t–1)/10.This work was supported by FAPESP