Mutant Strain Of Aspergillus Niger Research Articles

EFFECT OF GROUNDNUT CAKE AND SOYA BEANS ON ENHANCED CITRIC ACID PRODUCTION FROM PAWPAW AND ORANGE PEEL BY MUTANTS OF ASPERGILLUS NIGER

This present study was concerned with the biosynthesis of citric acid (CA) with mutant strain of Aspergillus niger using pawpaw and orange peel as substrates by solid state fermentation process. The A. niger strain isolated from spoilt orange was identified, screened for CA production on Czapek-Dox Agar and subjected to mutation by ethidium bromide. The effect of carbon sources, nitrogen sources and substrates were also determined. Among the mutant strains, A. niger PJ-02 A120 was found to be the best mutant that produced citric acid (65.00±0.58f) after 48 hours in Vogel’s medium. The effects of carbon sources (sucrose and glucose) on CA production from each substrate (orange and pawpaw peel) using mutant A. niger PJ-02 was determined and sucrose, the best carbon source was combined with two the nitrogen sources (groundnut cake and soyabeans) to determine the most suitable supplement for CA production. Groundnut cake enhances the production of citric acid while soyabeans was inhibitory. Citric acid was further produced in pawpaw peel and orange peel medium containing sucrose (5 %) groundnut cake (2 %), methanol (1.5 %) and the mutant strain. The orange peel substrates yielded 112.07g/kg of CA while 107.17g/kg was recorded for pawpaw peel when fermented for 5 days at 30°C. The Production of citric acid with mutant Aspergillus niger proved better with orange peel than pawpaw peel when optimized with alcohol.
 
 

Decolourization of Textile effluent using Mutant strains of Aspergillus niger and Penicillium spp

Decolourization of Textile effluent using Mutant strains of Aspergillus niger and Penicillium spp

Optimization of citric acid production using a mutant strain of Aspergillus niger on cassava peel substrate

Abstract In this study, mutation by UV-radiation and medium optimization were used to enhance production of citric acid through the screening of five local strains of Aspergillus niger using hydrolyzed cassava peel medium. Mutant strains were evaluated for citric acid production, and the physico-chemical parameters for citric acid production were further optimized using Central Composite Design (CCD) of Response Surface Methodology. Among the wild strains, a strain (FUO 2) gave highest citric acid yield of 1.93 g/l, while the mutant strain FUO 1 10 obtained after 10 min of exposure to UV-radiation yielded 9.4 g/l, an improvement of 4.87 fold. Process optimization using the mutant strain (FUO 1 10 ) on the input parameters of substrate concentration, process time, inoculum size and initial pH gave a process model with a coefficient of determination R 2 of 0.75 and a validated citric acid yield of 88.73 g/l, an improvement of 45.97 fold over the best wild strain. The response surface graphs showed significant interactive effects of input parameters on citric acid formation. This study established the usefulness of UV-mutation and medium optimization in improving the yield of citric acid. This study highlights the practicability of enhancing citric acid yield using mutant strains of A. niger on agro waste such as cassava peels, coupled with a suitable combination of optimal operational parameters. To the best of our knowledge, this work represents the first reference to the utilization of cassava peel-malted sorghum blend in submerged condition for the production of citric acid by A. niger .

Effect of Biomass and Sugar in Citric Acid Production by Aspergillus niger Using Molasses and Jackfruit as Substrates

Under surface culture condition, a number of cheap indigenous raw materials like cane molasses and jackfruits were selected for citric acid fermentation and gamma ray induced mutant strains of 14/20 and 79/20 of Aspergillus niger were used. The rate of citric acid production was also influenced by biomass production and residual sugar used. In both cases on the day 13 without presence of Prescott salt for the mutant strain of Aspergillus niger 14/20 the citric acid production was highest. The biomass production was found different in various fermentation medium. It was found to increase with the increase of fermentation period and maximum biomass was found on day 13. It is also found that biomass production was higher in mutant strain of A. niger 14/20 than 79/20. In addition to this, wet-weight biomass was higher than dry weight biomass. Without presence of Prescott salt, wet weight of mycelial body of the fungus Aspergillus niger 14/20 was found highest on day 13 in mixed substrates which was 160.15. Mixed substrate prepared with molasses and jackfruit media was proved to be the best and potential for biomass production. Sugar utilization rate was also different in various media during citric acid production by A. niger 14/20 and 79/20. Prescott salt was also found to have remarkable effect on sugar utilization for citric acid production except on days 0, 3 and 5. In presence of Prescott salt, sugar utilization was lower than without presence of Prescott salt in the molasses, jackfruit and mixed fermentation media during production of citric acid. With the increase of fermentation period, sugar concentration in the medium was reduced and maximum reduction was found on day 13. Without presence of Prescott salt and in mixed fermentation medium the highest production of citric acid was found by further mutated Aspergillus niger 14/20 which was about 16.16mg/ml.

Starch saccharification and fermentation of uncooked sweet potato roots for fuel ethanol production

An energy-saving ethanol fermentation technology was developed using uncooked fresh sweet potato as raw material. A mutant strain of Aspergillus niger isolated from mildewed sweet potato was used to produce abundant raw starch saccharification enzymes for treating uncooked sweet potato storage roots. The viscosity of the fermentation paste of uncooked sweet potato roots was lower than that of the cooked roots. The ethanol fermentation was carried out by Zymomonas mobilis, and 14.4g of ethanol (87.2% of the theoretical yield) was produced from 100g of fresh sweet potato storage roots. Based on this method, an energy-saving, high efficient and environment-friendly technology can be developed for large-scale production of fuel ethanol from sweet potato roots.

GAMMA RAYS MUTAGENESIS OF ASPERGILLUS NIGER FOR HYPERPRODUCTION OF MUTAROTASE

Aspergillus niger was subjected to gamma rays mutagenesis at different dose rates (40 to 120 k.Rads). The mutant strains of Aspergillus niger were isolated and selected by random screening method. The selected mutant derived strains were compared with parent type of Aspergillus niger for enhanced production potential of mutarotase. A mutant strain of Aspergillus niger with maximum production potential of mutarotase was finally selected.

Purification and characterization of an acid protease produced by a mutant strain of Aspergillus niger

Purification and characterization of an acid protease produced by a mutant strain of Aspergillus niger

PURIFICATION AND CHARACTERIZATION OF AN INTRACELLULAR α-GLUCOSIDASE WITH HIGH TRANSGLYCOSYLATION ACTIVITY FROM A. niger M-1

An intracellular α-glucosidase with high transglycosylation activity was purified from a mutant strain of Aspergillus niger M-1 by sequential chromatography using a DEAE-cellulose 52 column, a DEAE-Sepharose CL-6B column, and a Sephadex G-100 column. The molecular mass of the purified enzyme was determined to be 116 kD with no subunits and a pI of 5.23. Maximal α-glucosidase activity occurred at pH 6.0 and 50°C. The N-terminal amino acid sequences were identified as N-SVPGTEYVV-. The presence of Ca2+ enhanced the enzyme activity by 20%, while the α-glucosidase activity was strongly inhibited by p-chloromercuribenzoate, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, monochloroacetic acid, and 2-mercaptoethanol. In addition, Ag+, n-bromosuccinimide, and acetylacetone inhibited enzyme activity by 70%, 50%, and 22%, respectively. K m values of 4.32 m mol L−1 and V max of 3.10 × 10−2 mol L−1 min−1 were found for methyl-α-D-glucopyranoside (α-MG). Maltose was identified as the preferred substrate. The high-performance liquid chromatography (HPLC) analysis indicated that the oligosaccharide products contained 10.54% of isomaltose, 8.08% of panose, and 9.29% of isomaltotriose, and the amount of glucose, maltose, maltotriose, and maltotetrose was dropped from 22.21% to 15.80% using the purified enzyme in the solution of 25% maltose and 3% glucose. This intracellular α-glucosidase has potential applications in the synthesis of sugar derivatives and the investigation of associated mechanisms.

Enhanced Production of Extracellular Lipase by Novel Mutant Strain of Aspergillus Niger

ABSTRACTTwelve fungal strains belonging to 9 different species of genera Aspergillus, Pencilillium, Trichoderma and Mucor were screened for extracellular lipase production. The most active lipase producing strain A. niger was selected for strain improvement by induced mutagenesis with UV light and N-methyl-N-nitro-N-nitrosoguanidine (NMG). Chemical mutagenesis was found to be more effective in comparison to the physical one. By two stages mutagenesis with 200 μg cm−3 NMG for 4 h, lipase activity of parental strain was enhanced more than two times. The selected mutant A. niger NMG12/4 was stable and maximum lipase activity of 15.5 U cm−3 was reached at 96 h, which corresponded to the end of the exponential growth phase. The selected mutant is prospective for the development of industrial biotechnology for production of extracellular lipase.

Application of 2-factorial design on the enhanced production of calcium gluconate by a mutant strain of Aspergillus niger

Sixty-eight isolates of the filamentous fungus Aspergillus niger were examined for calcium gluconate production under submerged culture conditions in 500-ml Erlenmeyer flasks. The isolate Chem-15 was selected for improvement through ultraviolet (UV) light-induced mutagenesis. Among viable mutants, strain 32 exhibited the best gluconate productivity, and it was subjected to N-methyl N-nitro N-nitroso guanidine (NG) treatment. Mutant strain NG-7 gave the highest gluconate production (86.48 g/L) which varied significantly ( p ⩽ 0.05) from that of the wild type. The mutant was cultured overnight and plated on 5-fluorocytosine-PDA medium. Gluconate productivity was increased by 35% when the process parameters, incubation period (72 h), initial pH (6.5), glucose as carbon source (15%), inoculum size (1.875 × 10 6 CFU/ml) and corn steep liquor (CSL) as nitrogen source (0.5%) were optimized using a 2-factorial Plackett-Burman design. Maximal glucose oxidase activity (28 U/ml/min) was achieved at the optimal fermentation conditions with 26.5 g/L DCM. The model terms were highly significant thus suggesting the potential commercial utility of the mutant (HS, df = 3 ∼ 0.0182).

Removal of heavy metals from contaminated sewage sludge using Aspergillus niger fermented raw liquid from pineapple wastes

The environmental benefits derived from using citric acid in the removal of heavy metals from contaminated sewage sludge have made it promising as an extracting agent in the chemical extraction process. At present, citric acid is produced commercially by fermentation of sucrose using mutant strains of Aspergillus niger ( A. niger), and chemical synthesis. In recent years, various carbohydrates and wastes (such as pineapple wastes) have been considered experimentally, to produce citric acid by A. niger. This study investigated the potential of using A. niger fermented raw liquid from pineapple wastes as a source of citric acid, in extracting chromium (Cr), copper (Cu), lead (Pb), nickel (Ni) and zinc (Zn) from anaerobically digested sewage sludge. Results of the study revealed that metal removal efficiencies varied with pH, forms of metals in sludge and contact time. At pH approaching 4, and contact time of 11 days, A. niger fermented liquid seemed to remove all Cr and Zn while removing 94% of Ni. Moreover, chemical speciation studies revealed that metals which are predominantly in the exchangeable and oxidizable phases seemed to exhibit ease of leachability (e.g., Zn). The by-products of the process such as pineapple pulp and mycelium which are rich in protein, can still be used as animal feed. It can be said therefore that this novel process provides a sustainable way of managing contaminated sewage sludge.

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 ± 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro- N-nitroso-guanidine (MNNG). Out of 3,2-deoxy- d-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 ± 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H 2SO 4 pre-treated blackstrap molasses in Vogel’s medium. On the basis of kinetic parameters such as volumetric substrate uptake rate ( Q s), and specific substrate uptake rate ( q s), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 × 10 −5 M MgSO 4 · 5H 2O into the fermentation medium reduced the Fe 2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 ± 5 g/l).

Evidence that cleavage of the precursor enzyme by autocatalysis caused secretion of multiple amylases by Aspergillus niger

The observation that a mutant strain of Aspergillus niger isolated for protease overproduction accumulated Taka-amylase supported an earlier report that processing of the precursor amylase by protease resulted in the secretion of multiple amylases. Studies using a mutant strain revealed that such processing was not due to aspergillopepsin but to autocatalysis by an inherent protease activity of the precursor and glucoamylase. Alignment of protease sequences with glucoamylase showed regions of consensus with serine carboxypeptidase of A. niger. Thus point mutations in this region due to ultraviolet radiation apparently caused the mutant to evolve with enhanced protease activity that degraded the precursor and accumulated Taka-amylase.

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger.

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 degrees C and glucose, maltose and maltodextrins at 70 degrees C as primary products, suggested significant applications for the enzyme in starch-processing industries.

Digestion of residual β-cyclodextrin in treated egg using glucoamylase from a mutant strain of Aspergillus niger (CFTRI 1105)

Glucoamylase enzyme isolated from the cultures of a mutant strain of Aspergillus niger (CFTRI 1105) was found to digest β-cyclodextrin in standard 1% solutions as well as in β-cyclodextrin-treated egg yolk and whole egg samples. About 70–95% of β-cyclodextrin in samples was digested with both dilute (reducing sugar activity 870 units) and concentrated (34,800 units) enzyme solutions at 40°C and 70°C for incubation periods of 15–360 min.

Yam bean starch: a novel substrate for citric acid production by the protease-negative mutant strain of Aspergillus niger

Selection of protease-negative mutant strains of Aspergillus niger in semi-solid culture was carried out in order to enhance citric acid production from yam bean. The protease-negative mutants were obtained by UV-irradiation of the parental strain Yang no. 2. Using a halo-selection medium, a number of mutants with decreased extracellular protease activity were selected. Citric acid productivity by the selected mutant strains was tested on a modified starch–methyl red agar plate. The best mutant strain YW-112 was obtained and produced 106 g/l of citric acid, whereas the parental strain Yang no. 2 produced 58 g/l, from 140 g/l of soluble starch in semi-solid culture at 5 days of cultivation time. During the whole period of cultivation with soluble starch, the extracellular glucoamylase activity of YW-112 was higher than that of the parental strain Yang no. 2. In addition, when 17 g/plate of rod-shaped yam bean was used instead of soluble starch, the protease-negative mutant strain YW-112 produced 490 g/l of citric acid, which is approximately 1.5 times as much citric acid as Yang no. 2 produced and showed enhanced extracellular glucoamylase production.

Citric acid production from cellulose hydrolysate by a 2-deoxyglucose-resistant mutant strain of Aspergillus niger

Citric acid production from cellulose hydrolysate was done by a semi-solid culture of Aspergillus niger Yang no. 2, a parental strain, and C192, which is a 2-deoxy- d-glucose-resistant mutant strain. Cellulose powder was hydrolyzed to mainly glucose and a low concentration of cellobiose by a commercial cellulase in acetate or citrate buffer. The hydrolysate prepared in acetate buffer was concentrated to contain 150 g/l of reducing sugars and used as a medium for fermentation. Yang no. 2 and C192 produced 77·9 g/l and 98·7 g/l of citric acid in 9 days from the concentrated hydrolysate, respectively. Acetate buffer delayed the fermentation. When the hydrolysate was prepared in citrate buffer and used as a medium, Yang no. 2 and C192 produced 92·2 g/l and 102·3 g/l of citric acid within 3 days from the concentrated hydrolysate containing 150 g/l of reducing sugars, respectively. It is concluded that citric acid production from cellulose hydrolysate with high conversion yield, maximally 68·2% based on the reducing sugars supplied, was achieved first by C192.

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q s (g per L per h) and specific substrate consumption rate,q s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y p/x), volume productivity (Q p) and specific product yields (q p), the mutant strain is significantly (p≤0.05) improved over the parental strain.

Production of the homologous pectin lyase B protein in six genetically defined protease-deficient Aspergillus niger mutant strains.

An expression cassette has been transformed into six protease-deficient (prt) mutant strains of Aspergillus niger. Transformants were tested for improved production of the proteolytically susceptible PELB tester protein. In four complementation groups (prtA, B, D and F) distinct improvement of PELB yield was observed. These in vivo experiments in single prt mutants confirmed earlier in vitro PELB degradation data and demonstrated how the use of protease-deficient mutants can significantly improve protein production in A. niger. The strong effects of several prt alleles on the stability of the PELB tester protein have initiated a more detailed genetical and molecular characterization of the prt mutations. Mapping of the cloned protease genes pepA [I], B [II], C [IV], D [I], E [IV] and F [IV] indicated that none of the prt mutations represent alleles of the presently cloned protease (pep) genes from A. niger. Analysis of the expression of the pep genes in prt strains demonstrated that the strongly reduced protease activities observed in several prt mutants are not reflected by reduced transcription levels for a number of extracellular proteases. These results indicate that the mode of action of the prt genes constitute an interesting group of new genetic functions which severely affect protease production, and as such improve protein production, in A. niger.

Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger

Mutants having impaired protein synthesis, that is cycloheximide-sensitive mutants of a citric-acid-hyper-accumulating strain, were induced from Aspergillus niger WU-2223L. Selection was on the basis of a presumption that the mutants should be more sensitive to cycloheximide than WU-2223L. In shake culture without methanol as a promotor substance, seven mutants accumulated approximately 1.8–3.5 times as much citric acid as WU-2223L. The best mutant, CHM I-C3, accumulated 69.4 mg citric acid/ml from 120 mg glucose/ml in shake culture without methanol, this amount being 1.1 times the amount accumulated by WU-2223L with methanol. Furthermore, under the conditions without methanol the mutants appeared to be more efficient than WU-2223L in employing the consumed glucose for the accumulation of citric acid. It was also confirmed that CHM I-C3 exhibited a significantly increased level of intracellular NH 4 + accumulation. The addition of 2% (v/v) methanol or 20 μg cycloheximide/ml to the medium caused a remarkable increase of citric acid accumulation by WU-2223L: about 3.1 and 2.4 times respectively. However, the addition of these substances produced negative effects on citric acid accumulation by the mutants. With 2% (v/v) methanol, WU-2223L showed a remarkably decreased level of protein accumulation but a substantially increased level of intracellular NH 4 + accumulation. However, these phenomena were also observed in CHM I-C3 without methanol. These results indicate that the intracellular circumstances of the cycloheximide-sensitive mutants without methanol were similar to those of WU-2223L with methanol, and that the impairment of protein synthesis contributed to increased citric acid accumulation by the mutants in the absence of methanol.