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     

Mycotoxin Production by Fusarium proliferatum isolates from rice with Fusarium sheath rot disease

Twenty samples of unpolished (rough) rice collected in Arkansas and Texas during the 1995 harvesting season from fields exhibiting Fusarium sheath rot disease or panicle blight were previously shown to include 8 samples positive for fumonisin B₁(FB₁) in the range 2.2–5.2 ppm, and moniliformin (MON), but no beauvericin (BEA), deoxynivalenol, its derivatives or zearalenone were detected. Fifteen cultures of F. proliferatum were established from the 20 rough rice samples. Single spore isolates of each culture were grown on rice and tested for the production of fumonisins (FB₁, FB₂, FB₃, etc.), MON and BEA. All 15 isolates produced FB₁, FB₂, MON and BEA in culture on rice. No deoxynivalenol, its derivatives orzearalenone were detected. Seven cultures produced FB₁ at >50ppm (range 80–230 ppm), with therest producing FB₁ in the range 14–43 ppm.FB₂ was produced in the range 5–47 ppm, and those cultures which produced the most FB₁ also produced the most FB₂. Of the 15 cultures producing MON, 11 produced it at >100 ppm in the range 188–6018 ppm, with the rest producing in the range 7–64 ppm. BEA was produced in the range 109–1350 ppm. Other derivatives of fumonisins, including FA₁, FA₂ and partially hydrolyzed FB₁, as well asseveral unknown metabolites including a compound with MW 414, were identified in culture extracts by continuous flow fast atom bombardment with ion spraymass spectrometry (CF/FAB/MS). Further study is needed to identify the factors that control production of FB₁, MON and BEA by F.proliferatu in culture and in field samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of fumonisins byFusarium species of Taiwan

Twenty-nineFusarium species isolated from various sources in different districts of Taiwan were tested for their ability to produce fumonisins in corn cultures. OnlyFusarium moniliforme produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). The finding that the other 28Fusarium species produced neither FB₁ nor FB₂ is preliminary because only one strain per species was studied. The detection of FB₁ and FB₂ in cultures ofF. moniliforme was demonstrated by TLC and HPLC, and FB₁ was further confirmed by mass spectrometry. In a separate experiment, in which 38 strains ofF. moniliforme were tested for fumonisins, approximately 66% (25/38) produced FB₁ and/or FB₂. Of the 25 strains, 14 produced only FB₁ and 11 produced both FB₁ and FB₂, and the amounts of FB₁ and FB₂ produced by different strains varied greatly. This is the first report that fumonisins are found in corn cultures experimentally infected withF. moniliforme strains from Taiwan. It is safe to assume that fumonisin producing strains ofF. moniliforme are widely distributed among the economic crops such as corn, rice, sugarcane, and sorghum throughout the Island.Abbreviations FB₁ Fumonisin B₁ - FB₂ Fumonisin B₂ - OPA o-phthalidialdehyde     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Effects of fumonisin B1 alone and combined with deoxynivalenol or zearalenone on porcine granulosa cell proliferation and steroid production

Fumonisin B₁ (FB₁) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB₁, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B₁ had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB₁. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB₁. Regarding steroid production, FB₁ increased progesterone production, and FB₁ had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB₁ had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB₁ produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB₁. Fumonisin B₁ was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB₁ on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB₁ increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.     

Determination of fumonisins in corn: Evaluation of two purification procedures

The performance of two solid phase extraction (SPE) purification procedures, used in the determination of fumonlsin B₁ (FB₁), B₂ (FB₂) and B₃ (FB₂) In corn, was evaluated using both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Fewer interferences were observed In extracts prepared using the strong anion exchange (SAX) media, in contrast to those purified on C₁₈ media, where on occasions, visual discernment of the TLC bands was hampered by the presence of interfering compounds. Precipitate formation, resulting In the blocking of SPE cartridges was also encountered when using the C₁₈ procedure. HPLC analyses of extracts prepared by both media indicated that they gave comparable fumonlsin recoveries from naturally contaminated corn samples. The results suggest that the C₁₈ procedure, originally developed for the TLC analyses of FB₁ in mixed feeds, may also be applied to the determination of FB₂ and FB₂. However, where TLC is used quantitatively for fumonlsin levels <1 μg/g, purification of sample extracts on SAX media is recommended.     

Evaluation of the extraction and purification procedures of the maleyl derivatization HPLC technique for the quantification of the fumonisin B mycotoxins in corn cultures

The extraction and purification methods used in the maleyl derivatization HPLC technique was evaluated with respect to the pH of the extraction mixture, the extraction solvent and the purification methods used in order to determine optimum conditions for quantification of fumonisins B₁, B₂, and B₃ in corn cultures. The highest recovery of the three compounds was obtained by extraction at pH 3.5 with CH₃OH–H₂0 (3∶1), whilst the subsequent solvent partitioning and reversedphase C₁₈ Sep-pak purification have been shown to be very important in the quantification of the fumonisins in the corn cultures. The percentage recovery of the improved technique, utilizing a gradient HPLC solvent system for the simultaneous determination of the fumonisins, was 93.4% for FB₁, 68.0% for FB₂, and 82.6% for FB₃. The study indicates that the polarity of the fumonisins and consequently their solubility during extraction as well as their behavior during the subsequent purification step play an important role in quantification of these mycotoxins in corn cultures.     

Comparative study of aflatoxins M1 and B1 production in solid-state and shaking liquid cultures

Production of aflatoxins M1 (AFM) and B1 (AFB) by Aspergillus flavus NRRL 3251 in solid-state and shaking liquid cultures using rice as the carbon source was compared. In general, solid-state cultures produced more aflatoxins than shaking liquid cultures on an equal rice weight basis. Solid-state cultures with continuous shaking yielded higher levels of toxins than those with intermittent shaking. However, intermittent shaking is a feasible replacement for the continuous shaking method for AFM production. A typical solid rice culture supplemented with yeast extract produced 30 and 2600 mg per kg rice of AFM and AFB, respectively, in 8 days at 29 degrees C. The optimal culture conditions for toxin production in a shaking liquid culture were also studied. Parameters under consideration included the amount of carbon (rice) and nitrogen source, initial medium pH, and aeration rate. At optimum conditions, a representative shaking liquid culture produced 18 and 1680 mg per kg rice of AFM and AFB, respectively, in 5 days at 29 degrees C. This shaking liquid culture appears feasible for scaling up and routine production of AFM and AFB for toxicological investigations.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

娄彻氏链霉菌ATCC10739产抗生素Borrelidin发酵条件优化及其分离纯化

对娄彻氏链霉菌ATCC10739固体发酵产生十八元大环内酯抗生素Borrelidin进行了发酵条件的优化。首先筛选得到了理想的发酵培养基;其次考察了发酵时间、起始pH值以及在ISP-2培养基中添加附加碳源、氮源对Borrelidin产量的影响。初步确定最适发酵条件为:ISP-2培养基中添加1%甘油,起始pH值为6.0,培养温度30°C,发酵时间为7 d,产量可达1.336 mg/L。采用有机溶剂萃取、硅胶层析和半制备型高效液相色谱(HPLC)等分离技术纯化得到Borrelidin。     

Production of fumonisins by endophytic strains of <Emphasis Type="Italic">Tolypocladium cylindrosporum</Emphasis> and its relation to fungal virus infection

Fumonisins were first discovered in Fusarium verticillioides, a fungus associated to disease and asymptomatic infections in maize. Afterwards, other fungal taxa have been found to produce fumonisins. The entomopathogenic ascomycete Tolypocladium cylindrosporum has been isolated from soil and also as an endophyte from leaves of grasses. The objectives of this work were to determine the in vitro production of fumonisin B (FB) mycotoxins and the immunosuppressive compound cyclosporine A (CyA) in several strains of T. cylindrosporum, and to examine the effect of fungal virus infection and temperature in FB production. FB₁ was detected in 30% of the strains, ranging from 0.16 to 5.52 μg cm^?2 in solid media, and FB₂ was detected in 78% of the strains, ranging from 0.764 to 40.92 μg cm^?2. CyA was not detected in any strain. The mean FB₂ concentration of the endophytic strain Tc37W was three times greater (p?<?0.05) than that of any other strain. Up to 34% more of FB₂ was detected in strains infected by the virus TcV3 than in the corresponding virus-free versions. The effect of temperature on FB₂ content was interactively significantly dependent on fungal strain and growth medium; in the YES medium, the FB₂ of virus-infected strains Tc37-1V and Tc37W increased by 67 and 16%, respectively, at 26 °C as compared to 20 °C. The FB concentration in some fungal strains was similar to that in fungi associated to food and feed intoxications.     

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju

Pleurotus sajor-caju, strain Pl-27, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP activity was detected in fungal cultures supplemented with both high (26 mM-N) and low (2.6 mM-N) nutrient nitrogen although higher specific activity values were recorded under the latter conditions. Conversely, laccase production was not influenced by nutrient nitrogen levels under the growth conditions adopted. Both the titre and time of appearence of MnP were also affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 15 ppm Mn. Two MnP and five laccase isoforms were identified by FPLC and gel electrophoresis.     

Aerobic Biodegradation of 2,2′-Dithiodibenzoic Acid Produced from Dibenzothiophene Metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2′-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2′-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2′-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B₁₂, and it degraded 2,2′-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2′-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2′-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2′-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Effect of culture medium composition on the activity of extracellular lectins of <Emphasis Type="Italic">Lentinus edodes</Emphasis>

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. The lectin activity in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C:N ratio, (9.5–12):1) on day 15–18 of culturing at pH 8.0–9.0.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 200–203.Original Russian Text Copyright © 2005 by Tsivileva, Nikitina, Garibova.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

Effect of Nitrogen Source on End Products of Naphthalene Degradation

Soil cultures, enrichment cultures, and pure culture isolates produced substantial quantities of salicylic acid from naphthalene in a mineral salts medium containing NH₄Cl as the nitrogen source. However, when KNO₃ was substituted for NH₄Cl, these same cultures failed to accumulate detectable quantities of salicylic acid but did turn the medium yellow. When an isolate identified as a Pseudomonas species was used, viable cell numbers were much greater in the medium containing KNO₃, but up to 94% of the naphthalene was utilized in both media. After 48 h of incubation in a 0.1% naphthalene-mineral salts medium, the cultures containing NH₄Cl showed irregular clumped cells, a pH of 4.7, 42 μg of salicylic acid per ml, and the production of 4.4 ml of CO₂. Under the same conditions, the cultures in the medium containing KNO₃ showed uniform cellular morphology, a pH of 7.3, no salicylic acid, the production of 29.7 ml of CO₂, and a distinct yellow coloration of the medium. The differences between nitrogen sources could not be accounted for by pH alone since results obtained using buffered media were similar. Growth with NH₄NO₃ displayed a pattern similar to that obtained when NH₄Cl was used. The yellow coloration in the medium containing KNO₃ was apparently due to more than one compound, none of which were 1,2-naphthoquinone or acidic in nature, as suggested by other investigators. Further attempts to identify the yellow compounds by high-pressure liquid chromatography, infrared analysis, and gas chromatography-mass spectrometry have been unsuccessful thus far.     

Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under the conditions of submerged culture was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and pH of culture medium. The activity of lectin in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C : N ratio, (9.5-12): 1)) on the day 15-18 of culturing at pH 8-9.     

Mycoflora and fumonisin-producing strains ofFusarium moniliforme in mixed poultry feeds and component raw material

In a survey of the mycoflora and mycotoxins in foods and feeds, 66 samples of mixed poultry feeds and some component raw materials were investigated. Fungal counts ranged from < 10² to 1.3 × 10⁶ CFU/g.Fusarium spp. counts ranged from 10² to 1.0 × 10⁶ CFU/g. TheFusarium spp. strains isolated were screened for their potential to produce fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in maize cultures. Samples and maize cultures were analysed for FB₁ and FB₂ using TLC and fluorescamine-derivative HPLC. No fumonisins were detected in the samples (<6 ppm).Fusarium moniliforme was isolated in 59.1% of samples, and 97.4% of the strains produced FB₁ and 79.4% of strains produced FB₂ in maize cultures. Some isolates produced higher FB₁ and FB₂ levels than the reference strainF. moniliforme MRC 826.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Boron,an essential micro-element forAzotobacter chroococcum

Summary 1. It was shown that boron is an essential micro-element forAzotobacter chroococcum.2. Multiplication, CO₂ production, N fixation and pigmentation are closely related to the boron content of the culture medium. Of these, pigmentation is most susceptible to slight boron deficiency; with an easily assimilable carbon source (mannite, glucose) nitrogen fixation seems to be more susceptible than multiplication to boron deficiency.3. With increasing gifts of boron, the CO₂ production curves in quartz sand with nutrients as well as in liquid cultures follow each other at regular sequences.4. The optimum boron content for normal development ofAzotobacter amounts toca 2 ppm in liquid cultures, to 5 ppm in quartz sand cultures, whereas in soils sometimes 8 ppm B are tolerated, without noticeable loss of activity.