Photosynthetic inorganic carbon utilization and growth of Porphyra linearis (Rhodophyta)

Photosynthetic (oxygen evolution) and growth (biomass increase) responses to ambient pH and inorganic carbon (Ci) supply were determined for Porphyralinearis grown in 0.5 L glass cylinders in the laboratory, or in 40 L fibreglass outdoor tanks with running seawater. While net photosynthetic rates were uniform at pH 6.0–8.0, dropping only at pH 8.7, growth rates were significantly affected by pH levels other than that of seawater (c. pH 8.3). In glass cylinders, weekly growth rates averaged 76% at external pH 8.0, 13% at pH 8.7 and 26% at pH 7.0. Photosynthetic O₂ evolution on a daily basis(i.e. total O₂ evolved during day time less total O₂ consumed during night time) was similar to the growth responses at all experimental pH levels, apparently due to high dark respiration rates measured at acidic pH. Weekly growth rates averaged 53% in algae grown in fibreglass tanks aerated with regular air (360 mg L_-1 CO₂) and 28% in algae grown in tanks aerated with CO₂-enriched air (750 mg L^-1 CO₂). The pH of the seawater medium in which P. linear is was grown increased slightly during the day and only rarely reached 9.0. The pH at the boundary layer of algae submerged in seawater increased in response to light reaching, about pH 8.9 within minutes, or remained unchanged for algae submerged in a CO₂-free artificial sea water medium. Photosynthesis of P. linearissaturated at Ci concentrations of seawater (K_0.5560 μM at pH 8.2) and showed low photosynthetic affinity for CO₂(K_0.5 61 μM) at pH 6.0. It is therefore concluded that P. linearisuses primarily CO₂ with HCO₃ ^- being an alternative source of Ci for photosynthesis. Its fast growth could be related to the enzyme carbonic anhydrase whose activity was detected intra- and extracellularly. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of Environmental Factors in Aerobiosis and Anaerobiosis on the Growth and Activity of Propionic Acid Bacteria Evaluated by Pressure Measurement

The effects of air, N₂ and CO₂ atmospheres on the growth and metabolism of P. shermanii CIP 10 30 27 under “optimum conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and “stressful conditions” (pH 5.3, NaCl 2.1 %, temperature 20 °C) simulating cheese ripening conditions were investigated using a pressure measurement technique. Under both conditions, the inhibition of growth was at its maximum when a CO₂ atmosphere with an increase in the lag phase (L_P) from 3 to 80 h and a reduction in the maximum rate of pressure variation (V_max,P) from 2.5 to 0.4 kPa/h was used. The production of metabolites was not affected under “optimum conditions”, but under “stressful conditions”, propionate production increased and higher ratios of propionate to acetate of up to 2.45 were observed. The effects of air and N₂ atmospheres on both growth and metabolite production were also examined.     

Negative Effects of P-Buffering and pH on Photosynthetic Activity of Planktonic Desmid Species

The photosynthetic activities of three planktonic desmid species (Staurastrum brachiatum, Staurodesmus cuspidatus var. curvatus, and Staurastrum chaetoceras) were compared after adaptation to medium enriched with either a 20 mM Na⁺-phosphate (P) or HEPES buffer. Incubations up to 2 d were carried out at pH 6 or 8 under normal air or air enriched with 5 % CO₂. Gross maximum photosynthetic rate (P _max) and growth rate were decreased in both S. brachiatum and Std. cuspidatus at higher pH when using the HEPES buffer and this effect was independent of CO₂ concentration, indicating that pH had an inhibitory effect on photosynthesis and growth in these species. The P-buffer at pH 8 caused a large decrease in P _max and quantum yield for charge separation in photosystem 2 (PS2), compared to HEPES-buffered algae. This effect was very large in both S. brachiatum and Std. cuspidatus, two species characteristic of soft water lakes, but also significant in S. chaetoceras, a species dominant in eutrophic, hard water lakes. The decreased P _max in P-buffer could not be related to a significant increase in cellular P content known to be responsible for inhibition in isolated chloroplasts. Experiments at pH 6 and 8 showed that two conditions, high pH and high Na⁺ concentration, both contributed to the decreased P _max and quantum yield in the desmids. Effects of a P-buffer were less pronounced by using K⁺-P buffer. The use of P-buffer at pH 8 possibly resulted in high irradiance stress in all species, indicated by damage in the PS2 core complex. In the soft water species pH 8 resulted in increased non-photochemical quenching together with a high de-epoxidation state of the xanthophyll cycle pigments.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

THE RELEASE OF GLYCOLATE BY A FRESHWATER DIATOM1

Navicula pelliculosa (Breb) Hilse grown on 2% CO₂ in air released glycolate when incubated in light in buffer pH 8.0 containing 10 mM bicarbonate. Excretion ceased about 90 min after transfer to air and no excretion was detected with air-grown cells. These results indicate that glycolate release in this alga, as in species of other algal phyla, is an artifact of growth on high concentrations of CO₂.     

Oxygen effect on batch cultures of Leuconostoc mesenteroides: relationship between oxygen uptake,growth and end-products

Growth and lactose metabolism of a Leuconostoc mesenteroides strain were studied in batch cultures at pH 6.5 and 30° C in 101 modified MRS medium sparged with different gases: nitrogen, air and pure oxygen. In all cases, growth occurred, but in aerobiosis there was oxygen consumption, leading to an improvement of growth yield Y _x/s and specific growth rate compared to anaerobiosis. Whatever the extent of aerobic growth, oxygen uptake and biomass production increased with the oxygen transfer rate so that the oxygen growth yield, Y _x/o2, remained at a constant value of 11 g dry weight of biomass/mol oxygen consumed. Pure oxygen had a positive effect on Leuconostoc growth. Oxygen transfer was limiting under air, but pure oxygen provided bacteria with sufficient dissolved oxygen and leuconostocs were able to consume large amounts of oxygen. Acetate production increased progressively with oxygen consumption so that the total molar concentration of acetate plus ethanol remained constant. Maximal Y _x/s was obtained with a 120 l/h flow rate of pure oxygen: the switch from ethanol to acetate was almost complete. In this case, a 46.8 g/mol Y _x/s and a 0.69 h^–1 maximal growth rate could be reached.     

Characterization of Growth and Metabolism of Commercial Strains of Propionic Acid Bacteria by Pressure Measurement

Dairy propionibacteria are essential starters for Emmental cheese manufacture. The behavior of three commercial strains of Propionibacterium freudenreichii subsp. shermanii (P.f. 1, P.f. 2 and P.f 3) were studied in a liquid medium under air and N₂ atmosphere using an on‐line pressure measurement technique. Growth kinetics and metabolite production were characterized under conditions usually reported as “optimal conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and also evaluated under “stressful conditions” (pH 5.2, NaCl 2 %, temperature 20 °C) simulating the cheese ripening conditions. In both cases, the effects of oxygen on growth were strain‐dependent. Under “stressful conditions”, two of the three strains were inhibited by oxygen under conditions of air atmosphere, while all three strains grew under conditions of N₂ atmosphere. In the latter case, the duration of the lag phase and the maximum rate of pressure variation were significantly different, however, no significant differences were found between the strains with regard to the total fermentation time. Under “optimal conditions” metabolite production was strain‐dependent. In an air atmosphere, all strains produced more acetate and CO₂ and less propionate than in a nitrogen atmosphere.     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

Growth and lactic acid production ofPediococcus pentosaceus at different gas environments,temperatures, pH values and nitrite concentrations

Summary In order to obtain a better understanding of the behaviour ofPediococcus pentosaceus in food products as well to facilitate the designing of industrial production processes for the organism, the growth and lactic acid production ofPediococcus pentosaceus in a complex glucose medium was followed in batch cultures at different gas environments (CO₂, air, N₂ and static cultures without gasflow), temperatures (10–50°C), pH (4.3–7.3) and nitrite concentrations (0–700 ppm). Optimal growth was obtained in CO₂ at 40°C and pH 6.3 and resulted in a maximum specific growth rate ( _max) of 1.27 h^–1. In static culture at 40°C and pH 6.3 the _max was 1.21 h^–1. The _max was, compared with static culture, reduced in air (12%) and nitrogen (26%). At 10°C the _max was reduced by 99% and at 50°C by 88%. The reduction at pH 4.3 and 7.3 was 65% and 57%, respectively. Nitrite did not affect the _max at any pH but increased the lag phase at pH 4.3 by a factor of 12. The lactic acid production was linked to the growth. The total amount of lactic acid produced was the same in all the tested gases and nitrite concentrations and also within the wide temperature range (15–45°C) and pH range (5.3–7.3). Mainly L(+)-lactic acid was produced during the exponential growth phase, but after this growth declined about 30% of the L(+)-lactic acid was converted to D(–)-lactic acid. The lactic acid product yield and the cellmass yied were both affected by the temperature but not by the pH.     

Sialic acid and biomass production by Streptococcus agalactiae under different growth conditions

A fermentation process to increase type capsular polysaccharide production by different serotypes of Streptococcus agalactiae (group B Streptococcus) was established. As sialic acid is an integral component of the polysaccharide, its synthesis was used to monitor polysaccharide, its synthesis was used to monitor polysaccharide production. Culture conditions, examined both on laboratory and pilot-plant scales, allowed optimal bacterial growth and high polysaccharide production in a medium composed of ultrafiltered Todd Hewitt broth supplemented with 2% (w/v) glucose and 1.5% (w/v) Na₂HPO₄, at a constant pH of 7.2. Studies using different gas atmospheres (air, CO₂ or their mixture) showed that air greatly enhanced polysaccharide production. Correspondence to: C. von Hunolstein     

Accumulation of organic acids in cultivations of Neisseria meningitidis C

Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min⁻¹. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O₂) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O₂ control, varying glucose concentration from 1.0 to 3.0 g l⁻¹, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O₂ control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O₂ control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.     

Submersed macrophyte growth at low pH

Summary Vallisneria americana was grown for six weeks in a greenhouse on relatively fertile sediment to test for factors other than nutrient limitation which may slow growth of this submersed macrophyte at pH 5. On the basis of dry mass accumulated, (1) low pH significantly depressed Vallisneria growth at constant free CO₂ levels; (2) free CO₂ enrichment, however, greatly stimulated Vallisneria growth at pH 5, by 2.8-fold and 10-fold at 3.2 times and 10 times air-equilibrated CO₂ levels, respectively; and (3) growth was greater by far at pH 5 than at higher pH with constant total dissolved inorganic carbon (DIC). Free CO₂ availability was thus an important controller of growth at low pH by Vallisneria americana on fertile sediment, and low pH was not directly deleterious. Field surveys of acidic lakes in the Adirondack Mountains of New York state revealed that DIC levels in low pH lakes were often well above equilibrium values and could potentially support vigorous macrophyte growth. Aluminum and/or iron toxicity did not appear to impair growth at low pH, and aluminum concentrations in Vallisneria shoots significantly decreased with increasing free CO₂ concentrations at pH 5.0, perhaps due to growth dilution. Rosette production (a measure of asexual reproduction), maximum leaf length, and extent of flowering within treatments were positively correlated with plant biomass, rather than with pH or free CO₂ levels per se.     

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Mass-spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ were made to compare the rates of light-dependent O₂ evolution and uptake by Chlamydomonas reinhardtii Dang. grown in air (0.035% CO₂; low-C_i cells) or CO₂-enriched air (5% CO₂; high-C_i cells) at pH 5.5 and 8.0. While at pH 5.5, no differences were observed in the isotopic O₂-gas exchange of high- and low-C_i cells, at pH 8.0 the rates of true O₂ evolution and uptake were considerably higher in low-C_i than in high-C_i cells. The enhanced rates of O₂ uptake and evolution by low-C_i cells were completely inducible within 6 h after transferring high-C_i cells to ambient air. At pH 8.0, O₂ uptake in the light was inhibited by 2 M 3-(3,4-dichlorophenyl)-1,1 dimethylurea in both types of alga, but this effect was more pronounced in low-C_i than in high-C_i cells.When the cells were grown at pH 5.5 the activities of the superoxide-radical-degrading enzymes, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were similar regardless of the CO₂ concentration provided during growth. At pH 8.0, however, the activities of these enzymes were 4 to 20 times higher in low-C_i than in high-C_i cells. When high-C_i cells were allowed to acclimate to ambient air for 6 h at pH 8.0, the activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate dehydrogenase increased to more than 50% of the level observed with low-C_i cells. These results are consistent with an enhanced operation of O₂ photoreduction which could provide energy to the inorganic-carbon-concentrating mechanism via pseudo-cyclic photophosphorylation.     

Optical device for parallel online measurement of dissolved oxygen and pH in shake flask cultures

We describe a new device with parallel optical measurement of dissolved oxygen (DO) and pH in up to nine shake flasks applicable in any conventional shaking incubator. Measurement ranges are 0–500% of air saturation for oxygen and 5.5–8.5 for pH. It was used to characterize growth profiles of different l-lysine producing strains of Corynebacterium glutamicum, of Saccharomyces cerevisiae and of Escherichia coli. Cultures in unbaffled flasks were highly reproducible. Oxygen limitation was indicated online which is particularly important when cultivating fast growing cells as E. coli. C. glutamicum strains showed distinct characteristic patterns of DO and pH indicating biological events. During the cultivation of S. cerevisiae on glucose, fructose and galactose, oxygen uptake rate was determined using the predetermined value of k _L a. pH measurement was used to determine the minimum buffer requirement for a culture of C. glutamicum.     

Effects of solution pH and bicarbonate on the growth and nodulation of a range of grain legume species

Defining plant adaptation to soil conditions is critical for the successful introduction of grain legume species into farming systems. This nutrient solution study examined the effects of pH (4, 5, 6, 7 and 8) and bicarbonate (5 mM KHCO₃) on the growth and nodulation of 14 grain legume species supplied with N or reliant on N₂-fixation. Species includedPisum sativum L.,Cicer arietinum L.,Lens culinaris Med., and a range ofLupinus, Vicia andLathyrus species. Species differed greatly in response to solution pH. For both N-fertilized and N₂-fixing plants, shoot growth ofL. culinaris was very sensitive to low pH (pH < 7), whereas shoot growth ofLupinus angustifolius L. andLupinus albus L. was sensitive to higher pH (pH ≥ 6). Other species had a broader optimal pH range for growth when supplied with N, but were generally sensitive to low pH (pH < 7 forC. arietinum andVicia sativa L., pH < 6 forP. sativum, Vicia faba L.,Lathyrus sativus L. andLathyrus cicera L., and pH < 5 forVicia benghalensis L. andVicia narbonensis L.) when reliant on N₂-fixation. For these other species, symbiotic N₂-fixation appeared to be more sensitive than host plant growth to low pH. This finding was supported by lower nodule numbers and mass, and lower N concentrations in shoots of sensitive species at low pH relative to higher pH. ForL. culinaris, nodule numbers and mass were relatively unaffected by pH 5–8, N concentrations in shoots were high at low pH and plants developed symptoms relating to H⁺ toxicity at pH as high as 7. These results indicate that host plant growth ofL. culinaris is more sensitive to low pH than theRhizobium symbiosis. ForL. albus andL. angustifolius, both host plant growth and symbiotic N₂-fixation appeared to be equally sensitive to pH ≥ 6.Lupinus pilosus Murr. was more tolerant of high pH than the otherLupinus species. At pH 4, two genotypes ofC. arietinum had better early nodulation than other species.Vicia ervilia L. nodulated poorly at all levels of solution pH, indicating that the commercial Group E inoculum (Rhizobium leguminosurum bv.viceae SU303) may not be effective for this species in solution culture. Addition of bicarbonate decreased shoot growth, nodulation and N concentrations in shoots of most species. Early nodulation (nodule number) ofLathyrus ochrus (L.) DC was not affected by the bicarbonate treatment.     

Factors affecting the growth of Fusarium proliferatum and the production of fumonisin B1: oxygen and pH

Fumonisins are mycotoxins produced primarily by Fusarium moniliforme and Fusarium proliferatum in corn. In liquid culture, production of fumonisin B₁ (FB₁), the most common moiety of the family of fumonisins, can be obtained using a defined medium that is nitrogen-limited. Under nitrogen-limited conditions both growth and the production of FB₁ were greatly influenced by pH and aeration. At pH above 5.0, F. proliferatum grew normally but produced little FB₁ (<100 μg m⁻¹). At pH below 5.0, there was less growth but substantially more FB₁. Below a pH of 2.5, both growth and metabolism were slower with very little FB₁ produced. When the optimal pH range of between 3.0 and 4.0 under well-aerated conditions was used, both growth and FB₁ production were high. However, under oxygen-limited conditions, less growth occurred, glucose consumption was increased, and no FB₁ was produced. Received 16 May 1997/ Accepted in revised form 03 September 1997     

Control of nutrient solutions for studies at high pH

Little is known about factors effecting plant growth at high pH, with research often limited by the inability to separate nutritional deficiencies and HCO ₃ ⁻ toxicity from the direct limitations imposed under high pH conditions. Various methods of controlling dilute nutrient solutions for studies at high pH were investigated. For short-term studies, it was found that a solution without Cu, Fe, Mn and Zn and aerated with CO₂ depleted air, greatly reduced nutrient precipitation at high pH, thus eliminating nutritional differences between treatments. Manual pH adjustment and the use of ion exchange resins as pH buffers were unsuitable methods of pH control. However, pH control by automated titration had little effect on solution composition while maintaining constant pH. The system described is suitable for studies in which the pH of the bulk nutrient solution must be maintained. The system was used to examine OH⁻ toxicity in mungbeans (Vigna radiata (L.) Wilczek cv. Emerald), with root length reduced at a bulk solution pH of 8.5 and greater.     

The effect of pH onBrachionus calyciflorus Pallas (Rotifera)

Cohorts of the rotiferBrachionus calyciflorus were grown in media with pH of between 2.5 and 11.5 in increments of 1 unit. pH was shown to exert a major influence on the growth rate and reproductive capacity of theB. calyciflorus. At pH 2.5 theB. calyciflorus showed a negative capacity for population increase (r _c). Ther _c values of the cohorts reared at pH 3.5 and 4.5 were positive and these cohorts showed high net reproductive rates (R₀). Ther _c's of cohorts reared at pH of 5.5 and 6.5 were negative, while those of cohorts reared at pH 7.5 to 10.5 were positive with a peak in theR ₀ and cohort generation time (T _c) at pH 8.5 and 9.5. Total mortality occurred in less that 24 h at pH 11.5.Successive generations of the progeny from the cohort held at pH 3.5 were reared under the same conditions. Although ther _c of the F₁ and F₂ cohorts increased, the organism was unable to maintain the increased population growth rate, and the F₄ generation showed anr _c of almost 0.     

On the Relation Between the Effects of Auxin on Growth, pH and Potassium Transport

The relation between the effects of auxin on growth, pH and potassium transport in hypocotyl segments of Helianthus annuus was studied. In a solution containing 20 mM Na₂SO₄ auxin-induced growth was accompanied by an auxin-induced pH drop in the medium. (NH₄)₂SO₄, at the same concentration, brought about an almost complete abolishment of the effect of auxin on the pH. The magnitude of auxin-induced growth was, however, only slightly reduced. This result does not confirm the hypothesis according to which the action of auxin on growth is a result of its effect on the pH. In a solution containing 2 mM sodium phosphate buffer an inhibitory action of IAA on the release of potassium from the tissue was observed. Addition of 20 mM Na₂SO₄ to the medium brought about a complete abolishment of this effect. The magnitude of auxin induced pH drop was, however, similar in the two treatments. It was concluded that, although under suitable experimental conditions, a close relationship may exist between the effects of auxin on pH on K⁺ transport, the coupling between the two phenomena is not obligatory.     

Studies of the Growth in Culture of Excised Wheat Roots

Aeration of root culture medium with air containing 5 % or 10 % CO₂ enhances growth, particularly lateral initiation and growth, compared with aeration wilh air or CO₂-free air. This beneficial effect of enhanced CO₂ pressure on the growth of cultured roots of Atson Elite 56 wheat ean also be demonstrated in presence of a high (40 %) and inhibiting level of oxygen. ¹⁴C labelling from a carbonate–bicarbonate solution of pH 7.0 by cultured wheat roots is enhanced by light and this labelling is not inhibited by the presence of 1 % ethyl urethane.