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THE task of testing and localizing enzymes in a protein mixture subjected to paper electrophoresis is a tedious one. This is particularly true in a fractionation study where the number of tests may be numerous. During the course of such an investigation of the various carbohydrases in an Aspergillus niger fermentation, it became necessary to employ a rapid qualitative test for the detection of these enzymes. Keilin and Hartree1 have reported that glucose oxidase (notatin) can be successfully used for the quantitative assay of various carbohydrases. Monod and Torriani2 employed notatin to determine glucose in their studies of amylomaltase. Recently, Whistler et al. 3 showed that D-glucose in corn syrups can also be quantitatively estimated by using notatin. The latter enzyme oxidizes glucose to yield two products—gluconic acid and hydrogen peroxide.

SUMMARY: A method for isolating and identifying high acid-yielding mutants of Aspergillus niger is described. This involves cultivation of the organisms on absorbent paper soaked in an indicator medium. The advantages of the technique and the criteria used for selecting biochemically interesting mutants are briefly described. The greater acid production of mutants selected by paper culture has been confirmed by comparing yields of citric acid in surface and submerged fermentations with those given by the wild type strain.

A laboratory procedure for the realistic screening of paper mill toxicants is described.This method is modified for the pulp substrate method that was already proposed by Appling et al in 1951. The technique consists of two parts, that is ;The technique I is using 1% of pulp solution for the test culture substrate, and Pseudomonas melogenes, Penicillium steckii, Aspergillus niger and Humicola grisea are inoculated on it for the test organisms.The technique II is employed as slime sample which was taken directly from an area of the mill system in stead of the test organisms. The dilution plate method is made for an evaluation of biosides mixing substrates in which slime forming organisms had been growing.This technique is available for the selection of slime control agents at the laboratory of paper mills.

Washed mycelium of Aspergillus niger , incubated in buffer solution, was found to incorporate suitable 14 C-labeled amino acids efficiently into the cyclic peptide, malformin, as well as into protein. Optimum conditions were developed for the assay of this process. Evidence for the specificity of the biosynthesis was that only component amino acids of the malformin molecule were utilized. The chemical identity of the isolated malformin fraction was confirmed by paper chromatography and by altered paper electrophoresis following oxidation with performic acid.

Introduction NIELSEN and HARTELIUS (2) succeeded in forming substance B by chemical means. Their first results were obtained by autoclaving the "Rhizopus-nutrientsolution" in the presence of filter paper and assaying by dry weight yields of Aspergillus niger. They also showed that the mineral salts played no part, while the important factors were the nitrogen source, the carbon source, and the filter paper. Various nitrogen sources were tried, and the results indicated that all organic ammonium salts were effective, that NH4Cl had no effect, and that NaNO3 produced an inhibiting factor. Tests with various carbon sources showed that all were more or less effective. Six different filter papers were tried, and Schleicher and Schull no. 597 proved to be the most effective. They also state that the ash of the filter paper gave results similar to those of the filter paper itself. This synthesized substance not only functioned similarly to substance B of the Rhizopus filtrate, but also-like the latter-it was soluble in water, insoluble in ether, and stable to oxidation by perhydrol. In a subsequent paper (3), these workers demonstrated the production of this active substance by autoclaving 2 per cent lactic acid and 2 per cent glucose, at

Yukioka , M. (University of Hawaii, Honolulu), and T. Winnick . Synthesis of malformin by an enzyme preparation from Aspergillus niger . J. Bacteriol. 91: 2237–2244. 1966.—An enzyme fraction derived from disrupted Aspergillus cells was able to utilize each of the component labeled amino acids of malformin for the synthesis of this cyclic pentapeptide. The process was stimulated by adenosine triphosphate, K + , and Mg ++ , and was optimal at approximately p H 8.5. It was not affected by inhibitors of protein synthesis (ribonuclease, chloramphenicol, puromycin). There is evidence that cysteine, rather than cystine, was incorporated into peptide linkage, so that the disulfide bridge of malformin was formed subsequently. Although only the d isomers of cysteine and leucine occur in the malformin molecule, the l , as well as the d form of these amino acids, was readily utilized by the enzyme preparation. As in the case of several other microbial peptide systems, it appears that the d enantiomorph can arise from the l isomer at an intermediate stage of polypeptide synthesis.

Aspergillus niger NRRL 330 was grown in submerged culture in media containing d -glucose- 1 - 14 C or d -mannose- 1 - 14 C . Glucoamylase I [α- d -(1→4)-glucan glucohydrolase, E. C. 3.2.1.3] and II were isolated from the culture filtrate in high purity by precipitation with ethanol followed by chromatography on DEAE-cellulose and Bio-Gel P-150. The carbohydrate residues were released from the purified enzymes by acid hydrolysis and were separated by paper chromatography. The hexoses were oxidized to aldonic acids, which were purified by chromatography on an ion-exchange resin. Following oxidation of the purified aldonic acids with sodium metaperiodate, radioactivity was measured in the carbon dioxide (C-1 of the hexose) and formaldehyde (C-6 of the hexose) by liquid-scintillation techniques. A large percentage of the radioactivity was retained in C-1 of the hexose isolated from the enzyme, indicating direct incorporation of the hexose into the glycoprotein without prior breakdown into smaller units followed by resynthesis. Involvement of a nucletide-hexose pathway in the incorporation of the carbohydrate residues into glucoamylase I and II is consistent with these results. UDP-Glucose pyrophosphorylase (UTP:α- d glucosyl phosphate uridylytransferase, E.C. 2.7.7.9) activity was observed in a cell-free extract obtained from mycelia of a A. niger.

With a view to examine the applicability of the method of colloid titration to the estimation of pectic enzyme activity, the decomposition processes of pectin and pectic acid by the crude extracts of Sclerolinia liberliana Fcl and Aspergillus niger were followed by means of the colloid titration. The results were compared with the values obtained by estimation of the decrease in viscosity as well as of the increase in amounts of the liberated reducing- sugars of the test solution. Paper chromatography of the end-products was also conducted at the same time. Consequently, it was confirmed that this method was specific to galacturogenic polygalacturonase and pectin methylesterase, but indifferent to depolymeric polygalacturonase.

Pectic glycosidases produced by a strain of Aspergillus niger comprise at least two kinds of polygalacturonases, each of which is formed in different ways depending on the culture conditions, especially on the kinds of C-sources. The one, depolymeric polygalacturonase (DPG), produced constitutively by the mold, attacks the polygalacturonase chain at random, reducing the molecular size rapidly, and leaving tri-, di- and D-galacturonic acids as end products in the final reaction mixture where more than 50% of the substrate is hardly hydrolyzed. This enzyme was effectively purified by adsorption onto pulpified filter paper, followed by the general method of precipitation, from the culture liquor which did not contain pectic substrate and other adaptive pectic enzymes. DPG is easily adsorbed in a salt-free state at a pH near 4 and eluted with a more neutral salt-solution. The reaction pH has the optimum value of 3.4-4.6, variable with the substrate and with the method of determination. DPG is most stable at pH 3.6, where the half activity is reduced by treatment at 50° for 1hour. This enzyme seems to hydrolyze the non methoxylated glycosidic chain. The another polygalacturonase, galacturonogenic polygalacturonase (GPG), the formation of which is induced by the pectic substrate in contrast to DPG, splits the terminal galacturonic acid from one end of the polygalacturonide chain, not reducing the molecular size of the substrate as rapidly as DPG does. Other properties of GPG are similar to those of DPG though there are some differences which might be due to the lesser purity.

The action pattern and kinetics of an Aspergillus niger extracellular endopolygalacturonase were studied with oligogalacturoic acids (digalacturonic acid through hexagalacturonic acid) and their derivatives in which the terminal aldehyde group was reduced. The rate of hydrolysis catalyzed by the enzyme decreases with the shortening of oligogalacturonide chain length; digalacturonic acid is not hydrolyzed. With tetragalacturonic acid one productive complex is formed resulting in (1 + 3) cleavage. Two and three modes of cleavage occur with pentagalacturonic acid and hexagalacturonic acid, respectively. The enzyme is competitively inhibited by trigalacturonic acid whereas digalacturonic acid does not affect the activity. Reduced pentagalacturonic acid forms one productive complex. Reducing trigalacturonic acid and nonreducing digalacturonic acid are the products of the reaction. Lower reduced oligogalacturonic acids are not degraded by the enzyme. The action pattern and k2 values obtained with the oligouronides suggest that the binding site of the endopolygalacturonase contains four subsites and that the catalytic groups of the enzyme are located in the proximity of the first bond, counting from the reducing end, of the substrate segment bound in the complex.

1. A highly dextrorotatory polysaccharide (alpha(D)+232 degrees in n-sodium hydroxide), previously isolated as a fragment of Aspergillus niger cell walls, was prepared from whole mycelium and subjected to partial acid hydrolysis. 2. Fractionation of the hydrolysate on a charcoal column with a linear gradient of ethanol yielded a series of oligosaccharides. The disaccharide member was shown to be nigerose (3-O-alpha-d-glucopyranosyl-alpha-d-glucopyranose), although a small proportion of the disaccharide peak (10%) was present as maltose despite the fact that all the nigeran had been removed from the starting material. 3. The oligosaccharides forming the main peaks from the column were shown to be members of a polymer-homologous series (nigerodextrins) by (a) the relationship between the logarithm of their chromatographic mobility and degree of polymerization, (b) obeying the Freudenberg relationship, and (c) partial acid hydrolysis.

1. An exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 290-fold purification and a recovery of 8.6%. 2. The enzyme displayed its full activity only in the presence of Hg(2+) ions; K(A) for mercuric chloride was about 6x10(-8)m. 3. The mercury-activated enzyme progressively removed the terminal galacturonic acid residues from alpha-(1-->4)-linked galacturonide chains and converted digalacturonic acid, trigalacturonic acid, tetragalacturonic acid and pectic acid into galacturonic acid.

1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.

Enzymes attached to insoluble supports offer some particularly attractive advantages for many purposes including their use in the food processing industry. These advantages include repeated reuse of the enzyme, ease of removal from the reaction mixture, good or improved enzyme stability and properties which permit use in columns and large continuous reactors. In order to be successful an application must meet these criteria; in addition the costs for the support, the enzyme, the process for attachment, the equipment, and the operating costs must all be low. Previously we have reported on the use of phenol-formaldehyde resins as supports for immobilizing enzymes (1). This paper presents some of the most recent findings with this method of immobilization from pilot scale runs using a β-galactosidase from Aspergillus niger as the model enzyme for the hydrolysis of lactose in milk, whey, and deproteinized whey. Some examples of the variations possible with this method of enzyme immobilization are also discussed.

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 63-α-glucosylmaltotriose, 62-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.

Publisher Summary Enzyme activity can be assayed either by measuring ammonia formed, colorimetrically or by measuring nicotinic acid formed or nicotinamide hydrolyzed. Nicotinamidase in Torula cremoms is a constitutive enzyme. This chapter focuses on the assay method, purification procedure, and properties of the enzyme nicotinamide deamidase from Torula cremoris . Nicotinamide deamidase activity is measured by following the formation of nicotinic acid 7- 14 C from nicotinamide 7- 14 C with a strip counter or liquid scintillation counter after separation of the reaction components by paper chromatography. Modest requirements of nicotinamidase for optimal activity and extremely high sensitivity of the assay method permits it application to crude extracts. Thus, the method is successfully applied for measuring nicotinamidase activity in dialyzed cell-free extracts of Bacillus stearothermophilus, Aspergillus niger, and Clostridium acetobutylicum . Torula cremoris is grown for 24 hours at 37°C with continuous vigorous aeration in batches of 10–12 liters of nutrient medium. The reaction is apparently irreversible. Enzyme preparations representing the steps of purification retain full activity for several weeks when stored at 4°C.

UNDER this title, Walker, Hall and Hopton1 have described the formation of pyruvic and α-keto-glutaric acids from a glucose–arsenite medium. Further evidence for the close relationship of the metabolism of Aspergillus niger to the tricarboxylic acid cycle is now reported with the simultaneous identification of oxalic, gluconic, citric, malic, glycollic, succinic, pyruvic and fumaric acids. These products were separated for analysis on filter paper as free acids2 or as the 2:4 dinitrophenylhydrazones3. Approximate quantitative determinations were made by comparing the unknown spots with a range of standard acids of known concentration. Citrate and oxalate were also examined by more accurate methods4,5. All metabolic solutions were inoculated with a spore suspension of A. niger, which rapidly formed an actively growing mycelium.

IT has previously been reported that sake and koji extract produced from rice by the application of Aspergillus oryzae contains two new disaccharides1, one of which has been confirmed to be 1 : 3-α-linked gluco-biose (kojibiose)3,4 by its conversion into glucose with acid, R F in paper chromatography, M G in ionophoresis in borate buffer, its resistance to almond β-glucosidase, lack of formation of osazone and the similarity of its stain (aniline hydrogen phthalate spray) to that of sophorose. Peat et al. 4 have also described the isolation of a gluco-biose (octaacetate; melting point 166°, [α]D + 153°), believed to contain the 1 : 2-α-linkage, from a mixture of disaccharides synthesized from glucose by Aspergillus niger. One of us (K. A.) has detected kojibiose in the acid hydrolysate (hydrol) of sweet potato starch which is known to have undergone marked reversion5. We now report the isolation of this sugar from hydrol.

Evidence obtained by paper chromatography indicates that corn root curvatures caused by culture filtrates of Aspergillus niger are caused by the same compound which causes curvatures and malformations on the stems and petioles of bean plants. The R f values obtained for this compound were substantially different from those of indoleacetic acid.

Exhaustive extraction of the cell walls of Aspergillus niger with 10% NaOH solution leaves an alkali-resistant residue containing chitin and glucan as the major components. The glucan in this residue comprises 58.7% of the total cell wall glucan and was characterized by permethylation, and identification of the resulting O- methyl- D -glucoses obtained after hydrolysis by gas-liquid chromagtography and mass spectrometry of the derived partially acetylated, partially methylated, [1- 2 H]alditols. The glucan was separated from the chitin by acetylation of the alkali-resistance material, a procedure which separates a large portion of the total glucan as a chloroformsoluble acetate, abd by treatment of the alkali-insoluble residue with nitrous acid, a procedure which was found to render the complex soluble in dimethylsulfoxide and amenable, therefore, to permethylation. The data collected suggests that the preparation is an essentially linear glucan containing 85–95% 1 → 3 linkages and 10–15% 1 → 4 linkages. An analysis of the glycosidic linkages using NMR spectroscopy indicate that both α and β linkages are present in the ratio of 4:1. An identical glucan appears to be present in the cell walls of Penicillium chrysogenum as well as the spore cell walls of both organisms, as evidenced by methylation studies.

Glucose oxidase from Aspergillus niger is shown to possess a glycoprotein structure. This enzyme is composed of most of the common amino acids but is relatively high in aspartic acid, glutamic acid, glycine, and alanine, and relatively low in cystine, methionine, and tryptophan. The carbohydrate residues comprise about 16% of the enzyme molecule and include mannose, galactose, and glucosamine, probably as the N-acetyl derivative. The possible types of linkages of the carbohydrate residues to the amino acids of the polypeptide chain of the glucose oxidase are considered.

Aspergillus niger produces two forms (isoenzymes) of glucoamylase that are separable by electrophoresis or by chromatography on DEAE-cellulose and that are designated glucoamylase I and II. The molecular weight of glucoamylase I is 99,000, and that of glucoamylase II is 112,000. Both forms of the glucoamylase contain covalently linked carbohydrate (containing d -mannose, d -glucose, and d -galactose residues) and are therefore glycoenzymes. The carbohydrate-protein linkage in the glycoenzyme is primarily glycosidic to the hydroxyl group of l -serine and l -threonine residues, but glycosylamine linkages to l -asparagine and l -glutamine may also be present. Glucoamylase I and II possess identical amino acid compositions and, presumably, identical amino acid sequences. However, the two glycoenzymes differ in carbohydrate content, glucoamylase II containing nearly twice as many carbohydrate residues per molecule as glucoamylase I. Accordingly, it is suggested that the two forms of glucoamylase are isoglycoenzymes. The difference in electrophoretic and chromatographic properties of the isoglycoenzymes is probably due to a difference in the number of amide groups or glycosylaminically linked carbohydrate units in the polypeptide chains.

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.

A MODIFICATION of the normal Pettenkoffer tube has been developed to save time and materials and at the same time ensure accuracy in the estimation of carbon dioxide evolved during soil respiration experiments. It has also proved of use in the study of the respiration of Aspergillus niger in work at this Station by Dr. D. J. D. Nicholas.

In einer normalen Ultrazentrifugenzelle wird ein Bodenbelag aus Filtrierpapier angebracht, in welchen das Enzym hineinsedimentiert. Aus dem Aktivitatsverlust oberhalb des Bodenbelags wird die Sedimentationskonstante berechnet. Durchfuhrung und Auswertung der Versuche werden zu einer Routinemethode ausgearbeitet. Die Methode wird dazu benutzt, die Sedimentationskonstanten der beiden Glukosedehydrogenasen aus Penicillium notatum bzw. Aspergillus niger zu bestimmen. Es ergibt sich 7,91 bzw. 8,81 SVEDBERG. Die nach dem fruher beschriebenen Aktivitatstest bestimmten Diffusionskonstanten sind 5,56 bzw. 4,47 · 10−7 cm2·sec−1. Daraus ergeben sich die Molekulargewichte 138 000 bzw. 192000 und die Reibungsverhaltnisse 1,11 bzw. 1,24. On the bottom of a normal ultracentrifuge cell was placed a layer of filter paper into which the enzyme sediments. The sedimentation constant was calculated from the loss of activity above the filter paper. The execution and evaluation of the experiments were developed to a routine method. The method was used to determine the sedimentation constants of glucosedehydrogenase from Penicillium notatum and from Aspergillus niger. These were found to be 7,91 and 8,81 SVEDBERG units respectively. When combined with the previously determined diffusion constants 5,56 and 4,47 · 10−7 cm2·sec−1, one obtains molecular weights of 138,000 and 192,000, and friction ratios of 1,11 and 1,24 respectively.

POSTLETHWAIT, S. N., AND RoY W. CURTJS. (Purdue U., Lafayette, Imid.) Histology of malformations produced on bean plants by culture filtrate of Aspergillus niger. Amer. Jour. Bot. 46(1): 31-35. Illus. 1959.-The stem and leaf petioles of bean seedlings treated with culture filtrates of A. niger exhibit severe curvatures, increased diameter, reduced length, and irregular protrusions. These malformations result from an apparent partial loss of polarity of the cells in the actively growing region of young internodes and petioles. Enlargement and multiplication of cells in the affected regions occur mostly at an angle to the longitudinal axis of the plant. The effect is localized and the portion of the plant produced from the apical meristem subsequent to treatment matures in a normal way. ONE OF THE writers has reported that culture filtrates of the fungus, Aspergillus niger 56-39, induce severe curvatures and grotesque malformations on bean plants (Curtis, 1958). The plants were treated with a drop of the culture filtrate on each side of the unexpanded bud between the simple leaves above the cotyledons. After 5-10 days, malformations commonly appeared on the first and second internodes above the simple leaves. The stems and petioles were short and curved, greatly enlarged in diameter, relatively flat in transverse section, and somewhat irregularly ridged longitudinally. It was found subsequently that ether extracts of the culture filtrate of A. niger caused pronounced curvatures on corn roots (Curtis, in press) . Evidence obtained through the use of paper chromatography indicated that the compound which caused malformations on the stems of bean plants was the same as the one which induced curvatures on corn roots. Although early, indirect evidence suggested that A. niger produces indoleacetic acid (BoysenJensen, 1932; Kogl and Kostermans, 1934), the work of Curtis (1958) indicated that the compound active on corn and bean plants was not IAA. The present paper is an attempt to describe in more detail morphological and histological changes in plants treated with the culture filtrate of A. niger. Special emphasis has been placed on the histology of the irregular stem enlargements. MATERIALS AND METHODS.-The methods used to obtain culture filtrates of A. niger have been described earlier (Curtis, 1957). The crude culture filtrates (pH 5) were extracted 3 times with ethyl ether and then evaporated to dryness; the residue was taken up in water containing 2 drops tween 80/100 ml. This preparation, referred to as an ether extract, was used without further purification for treating seedlings of bean plants (Phaseolus vulgaris var. 'Black Valentine') grown in vermiculite. Usually, the concentration of the active compound used in treating plants was twice that in the crude culture filtrate. The treatment was made 'Received for Publication June 19, 1958. 2Journal Paper No. 1297 of the Purdue Agricultural Experiment Station, Lafayette, Indiana. by placing a drop of the ether extract on both sides of the young apical bud when the laminae of the simple leaves above the cotyledons were approximately 4-5 cm. long. This was done on each of 2 alternate days. Samples for study were collected at various intervals up to 14 days after treatment from treated and untreated plants. Dissection and observation of the apex were made with the aid of a stereomicroscope. Transverse and longitudinal sections were studied from both free-hand sections and paraffin sections. Permanent sections were made from material killed and fixed in FAA, dehydrated in the ethyl alcohol series, embedded in paraffin, sectioned at 20 microns and stained with safranin and fast green. RESULTS.-Untreated bean stem.-The development of a young internode of the bean stem from a short internode between leaf primordia at the apex to a greatly elongated internode at maturity is very complex due to the initiation of the vascular system before primary enlargement has been completed (Esau, 1953). Three internodes are differentiated in the apex before any appreciable elongation occurs. When the simple leaves above the cotyledons are about 4-5 cm. in length (treatment was applied at this stage of ontogeny), the apex consists of three internodes and three trifoliolateleaf primordia. For convenience of reference in this paper, the first internode above the cotyledons will be referred to as internode 1 and internodes constituting the young apex will be numbered successively in an acropetal sequence as internode 2, internode 3, and internode 4. Internode 2, which is 3-5 mm. long and the most basal of the three internodes in the apex, elongates rapidly, reaching its maximum length in 7-10 days under average greenhouse conditions. Internode 3, which is about 1 mm. long, and internode 4, which is less than 1 mm. long, elongate progressively later. Internode elongation occurs as a result of both cell division and cell enlargement. This activity takes place throughout the length of the younger internodes and throughout most of the length of internode 2 except for the more rnature basal portion. During

The synthesis of a number of compounds likely to possess fungicidal activity has been described by the authors in the earlier papers of this series (Sen & Joshi, 1948, 1949, 1951a, b). This paper incorporates the results of an investigation of the fungicidal action of 55 of those compounds on the fungus Aspergillus niger employing the agar-growth technique. The results have been reviewed with the idea of correlating chemical constitution and fungicidal activity in the light of theories put forward by various authors from time to time. As expected, most of the compounds tested have been found to be considerably toxic to the fungus. Interesting results have been obtained with aryloxy-fatty acids, which stimulate growth at lower concentrations.

The coenzymes Q found in 18 molds were examined by paper chromatography, melting point, and ultraviolet absorption spectroscopy. In the six following organisms, CoQ 9 was the major component: Aspergillus niger, Mucor abundans, Penicillium brevi-compactum, Penicillium charlesii, Penicillium chrysogenum , and Penicillium notatum . Two molds contained CoQ 10 : Aspergillus fumigatus and Cladosporium fulvum . The following ten organisms contained dihydro CoQ 10 as the major component: Alternaria solani, Aspergillus flavus, Aspergillus flavus-oryzae, Aspergillus quadrilineatus, Aspergillus terreus, Cephalosporium sp., Chaetomium globosum, Curvularia lunata, Gibberella fujikuroi , and Neurospora crassa . In the latter molds, the presence of ten C 5 units was further confirmed by hydrogenation to the perhydroquinone structure, followed by paper chromatographic comparison with authentic perhydro CoQ 10 . In many cases, small amounts of other isoprenelogs accompanied the major CoQ component. In all of these molds, ergosterol was the major sterol component.

Summary Monovalent phosphorus applied as ammonium hypophosphite is not suited to serve as a P source for soil micro-organisms, but is not toxic. Phosphorus of the +3 degree of oxidation in the form of diammonium phosphite cannot be utilized as an ingredient of nutrient solutions by Aspergillus niger; it is, however, incorporated into the metabolism by microflora under soil conditions. Ammonium diphosphite displays its effect more slowly than diammonium hydrogen phosphate, but as an ingredient of nutrient solutions it gives nearly the same good growth values for Aspergillus niger. Likewise, the influence exercised on the microflora sets in slowly and is equal in its extent at least to that of orthophosphate. Low-valent phosphorus of the diphosphite is transformed into quinquevalent phosphorus by hydrolysis in an acid medium with active participation of Aspergillus niger. Only in the +5 degree of oxidation is phosphorus as nutrient suitable for microorganisms. Besides a method designed to determine the P(+1) content in addition to the total P content in glucose-containing nutrient solution the paper Chromatographic inventory of hydrolysis products with quinquevalent phosphorus is described.

In an investigation upon tam& acid fermentation reported in the previous paper, it was found that when cane sugar and tannic acid are offered simultaneously to either Aspergillus niger or Penicillium sp., the sugar is utilized as the source of carbon while the tannic acid is fermented, gallic acid resulting. Some of the results indicated that the rate of fermentation was influenced by the concentration of the sugar. It was deemed important, therefore, to determine if varying the relative amounts of tannic acid and sugar in the nutrient solution has an influence upon the amount of the enzyme tannase produced in the fungus thus grown. Since tannic acid is probably not commonly utilized in nature by Aspergitlus niger and Penitillium sp. as a source of carbon, experiments were also made to determine if the enzyme is produced when the fungus is cultivated on nutrient solution lacking tannic acid. The writer wishes to acknowledge his indebtedness to Prof. B. M. Duggar for assistance received during the course of the investigation.

The fungistatic activity of the methyl to n-amyl p-amino-benzoates and of the ortho, meta and para-aminobenzoic acids and their methyl esters has been determined in the inhibition of the growth rate of Aspergillus niger, Byssochlamys fulva and Penicillium roqueforti. An increased inhibition of P. roqueforti in the presence of nitrate nitrogen, pH being maintained at about 3.5, has been noted and provision made to avoid this effect, as well as the complications mentioned in an earlier paper. The data are treated (i) on the basis of the reciprocal of concentration required for 50% inhibition (I50); (ii) as related to an adsorption phenomenon. On the basis of I50 values there is a general increase in activity from the methyl to the amyl p-amino-benzoate, also the meta isomer is markedly less effective than the corresponding ortho- or para-compound. The adsorption approach has proved of little use with P. roqucforti, but with B. fulva it seems to permit a useful analysis of the activity of the esters, particularly in terms of increasing biological adsorbability. The esters tested against A. niger do not give the same clear cut picture. With both B. fulva and A. niger the low I50 values of the meta isomer may be explained on the basis of the adsorption approach.

It was shown in a previous communication [Stephenson and Whetham, 1922 (1)] that the Timothy Grass Bacillus can be cultivated on a synthetic medium containing potassium phosphate (KH 2 PO 4 : 0·4 per cent.), and magnesium sulphate (MgSO 4 . 7H 2 O : 0·07 per cent.), with ammonium phosphate ((NH 4 ) 2 . HPO 4 : 0·4 per cent.) as the sole source of nitrogen, and glucose (1 percent.) as the sole source of carbon. The cultivation was carried out in Roux bottles, and the hydrogen ion concentration was kept constant at P H = 8·0 by the presence of excess of calcium carbonate. Under these conditions, the glucose was completely used in about 21 days; no breakdown products other than carbon dioxide were found; the protein nitrogen and lipoid contents of the bacillus were estimated at intervals and found to attain a maximum immediately before the glucose was used up; the fat of the organism then rapidly fell off, though no decrease was detected in the protein as estimated by Kjeldahl’s method. It therefore appeared that while growing in a medium containing a sufficiency of glucose, the organism stored fat, which was oxidised when the glucose was exhausted—the organism living at the expense of its fat and saving its protein. More recently, somewhat similar work on Aspergillus niger has been reported by Terroine, Wurmser and Montané [1922 (2)], who deprived the fully grown fungus of both nitrogenous and carbon-containing foodstuffs, and found a considerable fall in the percentage content of nitrogen in the organism. Kosinski [1902 (3)] had observed that the respiratory quotient of A . nigher on a glucose-containing medium is about 1, but falls off to 0·8 during starvation. This result, taken in conjunction with their own, led Terroine, Wurmser, and Montané to the conclusion that A . niger uses protein as a reserve material rather than cellulose. No particular observations seem to have been made on the fat content of A . niger . The work is not exactly comparable with ours on the Timothy Grass Bacillus, as ammonia was always present in the medium in our experiments.