Improved production of citric acid by a diploid strain of Aspergillus niger

Aspergillus niger strain CGU 87 was treated with UV radiation and some auxotrophic mutants were obtained. These mutants were less productive than CGU 87, which produced an average of 7.4% citric acid. All possible crosses in pair wise combinations were carried out between these auxotrophs, and three heterokaryons were synthesised. Finally, one heterozygous diploid was isolated from each of them. These heterokaryons and diploids showed improved productivity when compared with their component parents, but except in one diploid D5, all others produced less citric acid than CGU 87. The yield of D5 exceeded that of CGU 87 by 1.2 times and it produced 9% citric acid. This is a significant improvement and the increased productivity seems to be the result of successful adaptation of D5 to its fermentation environment.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.     

The location and analysis of two heterokaryon incompatibility (het) loci in strains of Aspergillus nidulans

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

K undu , P.N. & D as , A. 1985. A note on crossing experiments with Aspergillus niger for the production of calcium gluconate. Journal of Applied Bacteriology 59 , 1–5.
A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N-nitro-N-nitrosoguanidne (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.     

HETEROKARYOSIS AND PARASEXUALITY IN THE FUNGUS ASCOCHYTA IMPERFECTA

The object of this investigation was to discover whether heterokaryosis and parasexuality occur in the imperfect fungus Ascochyta imperfecta. Both phenomena have been observed. The wild type of A. imperfecta grows on a minimal medium containing only salts plus a carbon source. Auxotrophic and morphological mutants have been isolated after treatment with ultraviolet light. When 2 different mutant auxotrophs are inoculated together onto minimal medium, colonies are consistently formed. These colonies might be due, a priori, to back-mutation, diploidy, syntrophism or heterokaryosis. Back-mutation and diploidy have been eliminated, since no back-mutant nuclei have been isolated from any heterokaryon, and since the frequency of diploid nuclei is very low. The combination is primarily syntrophic (only 2% heterokaryotic hyphal tips) when the nicotinamide mutant is one component. The combination is primarily heterokaryotic (over 50% heterokaryotic hyphal tips) when both components are auxotrophs for amino acids. From the heterokaryotic hyphal tips, the 2 unaltered nuclear components have been isolated. Heterozygous diploid nuclei (4.2 X 10^−-7 per haploid nucleus) can be isolated from heterokaryons by plating, onto minimal medium, the primarily uninucleate conidia from a heterokaryon of 2 auxotrophs. The resulting colonies are isolated as potential diploids. Three properties of these isolates establish their diploid nature: (1) the isolates are wild type for nutrition and morphology; (2) their conidial length is uniformly greater than that of the haploids (1.21 times); (3) the isolates produce segregants with nonparental combinations of the marker genes. The diploid isolates are much more stable than heterokaryons. The recombinants from the diploids are still diploid, since (1) their conidial length falls in the diploid range, and (2) one of the recombinants has segregated a second-order recombinant. Many of the expected classes of recombinants have not been detected.     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism" of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.     

产黄青霉ds RNA病毒通过原生质体融合的转移

将国内青霉素产生菌(Penicillium chrysogenum)的黄孢子系统及绿孢子(包括淡绿,灰绿)系统的十多个菌株,经过病毒提取、电镜观察、奥氏免疫双扩散、凝胶电泳及放射免疫测定,证明黄孢子系统的菌株含有不同滴定度的、直径40nm的球形病毒,而绿孢子系统中检查不出病毒。从营养要求、孢子颜色不同的带病毒和无病毒菌体中分离原生质体,进行不同组合的原生质体的融合杂交,获得营养互补融合的异核体。异核体1中,病毒通过胞质融合转移到原来无病毒的灰绿孢子菌株及细胞核融合后的杂合二倍体中。灰绿孢子的病毒量接近二倍体的1/3。二倍体菌落生长稳定,低温保存二年后经0.01—0.02M对氟苯丙氨酸(PFA)诱发和分离,产生亲本类型的分离子,分离子及二倍体仍然含有病毒。异核体2作亲本性分离,黄孢子仍有病毒,淡绿孢子及细胞核融合后产生的二倍体均无病毒,表明非感染性为显性。此种淡绿孢子的突变体中存在非感病菌系,它不支持病毒的复制。提取各杂交组二倍体内的病毒所特有的dsRNA时,可看出dsRNA的存在和病毒的存在一致。多数杂合二倍体的青霉素产量比亲本高。     

Characterization of the Heterokaryotic and Vegetative Diploid Phases of MAGNAPORTHE GRISEA

The heterokaryotic and vegetative diploid phases of Magnaporthe grisea, a fungal pathogen of grasses, have been characterized. Prototrophic heterokaryons form when complementary auxotrophs are paired on minimal medium. Hyphal tip cells and conidia (vegetative spores) taken from these heterokaryons are auxotrophs with phenotypes identical to one or the other of the parents. M. grisea heterokaryons thus resemble those of other fungi that have completely septate hyphae with a single nucleus per cell. Heterokaryons have been utilized for complementation and dominance testing of mutations that affect nutritional characteristics of the fungus. Heterokaryons growing on minimal medium spontaneously give rise to fast-growing sectors that have the genetic properties expected of unstable heterozygous diploids. In fast-growing sectors, most hyphal tip cells are unstable prototrophs. The conidia collected from fast-growing sectors include stable and unstable prototrophs, as well as auxotrophs that exhibit a wide range of phenotypes, including many recombinant classes. Genetic linkage in meiosis has been detected between two auxotrophic mutations that recombine in vegetatively growing unstable diploids. The appearance of recombinants suggests that homologous recombination occurs during vegetative growth of M. grisea. No interstrain barriers to heterokaryosis and diploid formation have been detected. The mating type of the strains that are paired does not influence the formation of heterokaryons or diploids.     

Heterokaryosis between Aspergillus oryzae cyclopiazonic acid-defective strains: method for estimating the risk of inducing toxin production among cyclopiazonic acid-defective industrial strains.

Aspergillus oryzae strains are used extensively in the food industry. Some of these strains excrete alpha-cyclopiazonic acid (CPA), a mycotoxin which may provoke toxicoses in rats. Physicochemical methods may reveal the presence of this toxin, but they are inadequate to screen CPA-nonproducing (CPA-) strains. CPA production is revealed by either bacterial growth inhibition or alkalinization of the culture medium. This first biological property was used to devise a time-saving screening method to isolate mutants affected in their ability to produce CPA. The second method was used as a further test. After N-methyl-N'-nitro-N-nitrosoguanidine treatment, we isolated CPA- mutants from CPA producer strains (CPA+) and CPA+ mutants from CPA- strains. The mutants unable to produce CPA may be used in the food industry to reduce or eliminate the risk of intoxication in humans. Heterokaryon formation between different mutant strains was carried out to evaluate the risks of obtaining CPA from a mixture of mutants modified in their ability to synthesize this toxin. Pairings between two CPA+ strains always gave rise to CPA+ heterokaryons. Pairings between CPA+ and CPA- strains led, most often, to CPA+ heterokaryons. This could be directly correlated to the more frequent genotype (CPA+) in the heterokaryon. CPA hypoproducer and hyperproducer heterokaryons were obtained. Pairings between CPA- strains always gave rise to CPA- heterokaryons. These results suggest that the risks of producing this toxin from two CPA- individuals are not high.     

Heterokaryosis between Aspergillus oryzae cyclopiazonic acid-defective strains: method for estimating the risk of inducing toxin production among cyclopiazonic acid-defective industrial strains

Aspergillus oryzae strains are used extensively in the food industry. Some of these strains excrete alpha-cyclopiazonic acid (CPA), a mycotoxin which may provoke toxicoses in rats. Physicochemical methods may reveal the presence of this toxin, but they are inadequate to screen CPA-nonproducing (CPA-) strains. CPA production is revealed by either bacterial growth inhibition or alkalinization of the culture medium. This first biological property was used to devise a time-saving screening method to isolate mutants affected in their ability to produce CPA. The second method was used as a further test. After N-methyl-N'-nitro-N-nitrosoguanidine treatment, we isolated CPA- mutants from CPA producer strains (CPA+) and CPA+ mutants from CPA- strains. The mutants unable to produce CPA may be used in the food industry to reduce or eliminate the risk of intoxication in humans. Heterokaryon formation between different mutant strains was carried out to evaluate the risks of obtaining CPA from a mixture of mutants modified in their ability to synthesize this toxin. Pairings between two CPA+ strains always gave rise to CPA+ heterokaryons. Pairings between CPA+ and CPA- strains led, most often, to CPA+ heterokaryons. This could be directly correlated to the more frequent genotype (CPA+) in the heterokaryon. CPA hypoproducer and hyperproducer heterokaryons were obtained. Pairings between CPA- strains always gave rise to CPA- heterokaryons. These results suggest that the risks of producing this toxin from two CPA- individuals are not high.     

Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger

Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 3.7.1.1) was disrupted in a glucoamylase-producing strain of A. niger and the resulting strain was incapable of producing oxalic acid. The strain with the disrupted gene was compared with the wild-type strain producing oxalic acid in batch cultivations. The specific growth rate of both strains was 0.20 h(-1). The citric acid yields were identical, but the glucoamylase yield was only 50% in the disruptant compared with the wild-type strain. Batch experiments with 13C-labeled glucose as substrate were carried out to determine the metabolic fluxes through the central metabolism. The two strains had almost identical metabolic fluxes, which suggested that it was possible to disrupt the oah gene without pleiotropic consequences. The flux through the pentose phosphate pathway was around 60% of the glucose uptake for both strains, which suggested that a sufficient supply of NADPH was available for biosynthesis.     

Citric acid production by Aspergillus niger on wet corn distillers grains

AIMS: To determine which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. METHODS AND RESULTS: Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC 9029 and ATCC 201122. CONCLUSIONS: It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger was ATCC 9142. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.     

Heterokaryosis and the role of cytoplasmic inheritance in dark resting structure formation in Verticillium spp

Summary Auxotrophic and morphological mutants of Verticillium albo-atrum (producing darkly pigmented resting mycelium) and V. dahliae (forming dark microsclerotia) were isolated after treatment of conidia (haploid and uninucleate) with ultraviolet light. Hyphal tip and conidial analysis revealed that complementation between pairs of auxotrophs on minimal medium was due to a mosaic of homokaryotic and heterokaryotic regions with some hyphal tips growing syntrophically. A degree of incompatibility was observed in a few intraspecific, but in most of the interspecific, heterokaryon tests. Heterozygous diploid conidia (6–11 in length compared with 3–6 for haploids) were recovered at a frequency of 1 in 8x10⁶ by plating spores at high density on MM. Young diploid colonies segregated to give haploid and diploid sectors, some of which were recombinant types (parasexual cycle). Heterokaryons between complementary auxotrophs which were wild-type for dark pigmentation (hyl⁺) resembled wild-type and only darkly pigmented colonies were recovered by conidial analysis. Heterokaryons between hyl⁺ and hyaline (hyl) auxotrophs again resembled hyl⁺ morphology and usually only hyl⁺ colonies of both auxotrophic genotypes were recovered. Conidia from heterokaryons formed by stable hyl auxotrophs produced only hyl colonies of both auxotrophic genotypes. The important role played by cytoplasmic factors in the inheritance of darkly-pigmented resting structures in Verticillium was strongly confirmed by the present work.     

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.     

Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger.

Aspergillus niger Mulder strain when grown on a synthetic medium containing urea as the sole source of nitrogen at pH 5.2, formed a mixture of citric and gluconic acids. On growing the organism at pH 2.0 the gluconic acid content was reduced but citric acid yield remained low. Addition of NH4NO3 to the medium lowered the gluconic acid yields to undetectable levels with a simultaneous increase in the citric acid content. Of the sugars used for the production of citric acid, sucrose in an unautoclaved medium was found to be the best carbon source. Sucrose medium if autoclaved at pH 2.0, or a mixture of glucose and fructose instead of sucrose gave lower yields of citric acid. Under optimum conditions only citric acid was produced and the yield was 66-68 per litre after a growth period of about 10 days.     

MUTATIONS AFFECTING HETEROKARYOSIS IN SCHIZOPHYLLUM COMMUNE

Mutations that affect the basic characteristics of heterokaryons of S. commune occur spontaneously and are preferentially selected in the common-A heterokaryon and in its homokaryotic mimics, strains carrying a mutated B factor or strains disomic for heteroallelic B factors. Nine independent mutations were compared: all segregate independently of A and B incompatibility factors, and at least 3 distinct loci, of which 2 are linked, are involved. None of the mutations is phenotypically expressed in the homokaryon or in the common-AB heterokaryon. All 9 mutations increase vegetative vigor of the common-A the effects of all the mutations are additive in both heteroallelic and homoallelic combinations. At least 1 type-II mutation also affects nuclear distribution in common-B heterokaryons. Type-II mutations appear to reduce common-A, common-B, and compatible heterokaryons to a single type unlike any of the normal heterokaryons. Pseudoclamping often persists for extended periods in modified homokaryons isolated from modified heterokaryons. Several cases of somatic recombination have been observed among components of modified heterokaryons.     

A shift in nuclear state as the result of natural interspecific hybridization between two North American taxa of the basidiomycete complex Heterobasidion

A natural first generation hybrid fungus shows interspecific heterozygosity. The nuclear condition of a rare natural hybrid between two taxa of the Heterobasidion complex is investigated. Heterobasidion species are known to be either homokaryotic (haploid) or heterokaryotic (n+n), but heterokaryons are made up of both homokaryotic and heterokaryotic sectors. The natural hybrid appears to be either a heterokaryon undergoing a primary homothallic phase or a diploid with limited ability to exchange nuclei when mated with homokaryons. The natural hybrid is stable and long lived, suggesting hybridization may play an important role in the evolutionary history of this fungal complex.     

Globin synthesis in fibroblasts fused with erythroblasts. II. Human fibroblasts.

Fusion of human (diploid) fibroblast monolayers with erythroblasts from 3-day chick embryos resulted in cultures containing on the average 14% heterokaryons and 8% fibroblast homokaryons. When these heterokaryon-containing cultures were labeled with radioactive amino acids during the first 24 h after fusion, the proportion of labeled proteins found in the globin region of analytical polyacrylamide gels showed a 40-fold increase compared with fibroblast homokaryons (0.08% vs. 4% of protein synthesized). Incorporation of radioactivity into globin decreased sharply during the second 24 h. Purified 35S-methionine-labeled globin from heterokaryon cultures gave rise to a tryptic fingerprint containing peptides characteristic of chick embryonic globins as late as 4 days after fusion. While fibroblasts in the fusion culture continue to go through the cell cycle normally, heterokaryons stop cycling almost completely soon after fusion.