Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

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 H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), 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 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ 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).     

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.     

Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.     

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor

AIMS: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. METHODS AND RESULTS: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P相似文献   

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture

AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.     

Pretreatment of indian cane molasses for increased production of citric acid

Surface culture citric acid fermentation was carried out by Aspergillus niger T55, a strain isolated from its natural source, using cane molasses, either untreated or treated by various methods. Citric acid biosynthesis is seriously impaired by both organic and inorganic inhibitors. A combined treatment of molasses with tricalcium phosphate, hydrochloric acid, and Sephadex fractionation minimizes the level of inorganic and organic inhibitors in molasses and increases the production of citric acid (65% weight yield based on total reducing sugar). The optimum level of individual metal ions for citric acid production depends on the concentration of other metals in the medium.     

Stimulatory effect of alcohols (methanol and ethanol) on citric acid productivity by a 2-deoxy D-glucose resistant culture of aspergillus niger GCB-47

The present study describes citric acid fermentation by Aspergillus niger GCB-47 in a 15-1 stainless steel stirred fermentor. Among the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid (90.02 +/- 2.2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. The sugar consumed and % citric acid was 108 +/- 3.8 g/l and 80.39 +/- 4.5%, respectively. The desirable mycelial morphology was in the form of small round pellets having dry cell mass 14.5 +/- 0.8 g/l. Addition of ethanol, however, did not found to enhance citric acid production, significantly. The maximum value of Yp/x (i.e., 5.825 +/- 0.25 g/g) was observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after inoculation when the initial pH of fermentation medium was kept at 6.0.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Application of kaolin to improve citric acid production by a thermophilic Aspergillus niger

Citric acid production by a thermophilic strain of the filamentous fungus Aspergillus niger IIB-6 in a medium containing blackstrap cane molasses was improved by the addition of kaolin to the fermentation medium. The fermentation was run in a 7.5-l stirred bioreactor (60% working volume). The optimal sugar concentration was found to be 150 g/l. Kaolin (1.0 ml) was added to the fermentation medium to enhance volumetric production. The best results in terms of product formation were observed when 15 parts per million (ppm) kaolin was added 24 h after inoculation. With added kaolin, citric acid production was enhanced 2.34-fold, compared to a control fermentation without added kaolin. The length of incubation to attain this product yield was shortened from 168 to 96 h. The comparison of kinetic parameters showed improved citrate synthase activity of the culture (Y (p/x)=7.046 g/g). When the culture grown at various kaolin concentrations was monitored for Q (p), Q (s), and q (p), there was significant improvement in these variables over the control. Specific production by the culture (q (p)=0.073 g/g cells/h) was improved several fold. The addition of kaolin substantially improved the enthalpy (DeltaH (D)=74.5 kJ/mol) and entropy of activation (DeltaS=-174 J/mol/K) for citric acid production, free energies for transition state formation, and substrate binding for sucrose hydrolysis. The performance of fuzzy logic control of the bioreactor was found to be very promising for an improvement ( approximately 4.2-fold) in the production of citric acid (96.88 g/l), which is of value in commercial applications.     

Optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger NGd-280

The present investigation is concerned with the optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger NGd-280 in a 15 l stirred tank bioreactor. Nutrients, especially nitrogen source have a marked influence on citrate productivity because it is an essential constituent of basal cell proteins. Citric acid has been known to be produced when the nitrogen source was the limiting factor. Ammonium nitrate was employed as a nitrogen source in the present study and batch culture experiments were carried out under various concentrations of ammonium nitrate. Specific growth rate was decreased and the biosynthesis of citric acid was delayed at higher concentrations of ammonium nitrate. Specific citric acid production rate was the highest when intracellular ammonium ion concentration was between 2.0 and 3.0 mmol g(-1) cells. Citrate production was however, stopped when intracellular ammonium ion concentration decreased below 1.0 mmol g(-1) cell.     

Influence of calcium on fungal growth and citric acid production during fermentation of a sugarcane molasses-based medium by a strain of Aspergillus niger

The effect of CaCl₂ on the growth, morphology and citric acid production from sugarcane molasses by Aspergillus niger 419 was studied. The addition of 0.5g CaCl₂/l to the fermentation medium induced a loose pelleted form of growth, reduced the biomass concentration and increased the volumetric productivity (g citric acid/h) and the specific production (g citric acid/g biomass dw) from 0.02 and 0.37 to 0.13 and 3.72, respectively.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Citric acid fermentation by the mutant strain of theAspergillus niger resistant to manganese ions inhibition

Summary A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM^–3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP⁺ -isocitric dehydrogenase, one of the control points for citric acid accumulation.     

甘蔗渣和糖蜜混合发酵制备燃料丁醇

以抗逆突变株Clostridium beijerinckii IB4为研究对象,葡萄糖为C源,对其进行补料分批发酵过程的优化,同时将该优化工艺应用于甘蔗渣和糖蜜混合发酵制备燃料丁醇。结果表明:在5 L发酵罐中,先加入作为还原糖的甘蔗渣酸解糖液10 g/L,16 h后补加甘蔗糖蜜30 g/L,于35℃、100 r/min发酵50 h,丁醇和总溶剂产量分别达到11.1和15.3 g/L,丁醇比例高达72.5%。     

Mathematical modeling of citric acid production by repeated batch culture

A mathematical model has been created for the process of citric acid biosynthesis by yeast (mutant strain Yarrowia lipolytica) cultivated by the repeated batch (RB) method on ethanol under conditions of nitrogen limitation. The model accounts for cell growth as a function of nitrogen concentration in the culture liquid; nitrogen uptake by growing cells; citric acid production; pH control in the fermentor by means of NaOH addition; and changes in system volume. The model represents a system of five nonlinear differential equations. Experimental measurements of cell concentration, citric acid concentration, and cultivation broth volume were used with the least squares method to determine the values of eight model parameters. The parameter values obtained were consistent with literature data and general concepts of cell growth and citric acid biosynthesis. The model has been used to predict optimum RB culture conditions.     

Production of citric acid from starch-hydrolysate by Aspergillus niger

The present investigation explored the possible use of a rarely used agro-industrial by-product, maize starch-hydrolysate, for economic production of citric acid. To achieve this, seventeen strains of Aspergillus niger were screened for their capacity to produce citric acid using starch-hydrolysate as a substrate. The most efficient strain, ITCC-605 was selected for further improvement in citric acid content by mutation. Mutants developed by treatment with EMS and UV, singly and in combination, produced citric acid in the range of 0.51-64.7 g kg(-1) of glucose consumed. The mutant UE-1 produced the maximum citric acid which was about 130 times more than that produced by the parent strain, ITCC-605. For further increase in citric acid production from this substrate, the cultural conditions were optimized: concentration of starch-hydrolysate, 15% (glucose equivalent); ammonium nitrate, 0.25%; KH2PO4, 0.15%; nicotinic acid, 0.0001% and initial pH of 2.0. Under these conditions, the mutant strain UE-1 yielded 490 g citric acid kg(-1) of glucose consumed in 8 days of incubation at 30 degrees C. The productivity of 341 mgl(-1)h(-1) corresponded to 49% substrate conversion to citric acid.     

Formation of autodiploid strains in Aspergillus niger and their application to citric acid production from starch

Autodiploid strains were induced by colchicine treatment of Aspergillus niger WU-2223L, a citric acid-producing strain. In shaking culture, a representative autodiploid strain, L-d1, yielded higher citric acid than the parental strain, WU-2223L. When glucose was used as a carbon source, L-d1 and WU-2223L produced 67.2 g/l and 62.0 g/l of citric acid, respectively, from 120 g/l of glucose in 9 d-cultivation. Furthermore, the autodiploid strain L-d1 produced 49.6 g/l of citric acid, 1.4 times as much as that produced by WU-2223L from 120 g/l of soluble starch. During the whole period of cultivation with starch, the extracellular glucoamylase activity of L-d1 was on the same level as that of WU-2223L, but the extracellular acid-protease activity of L-d1 was much higher. The addition of pepstatin, an inhibitor of acid protease, to the culture broth at 2 d greatly increased the extracellular glucoamylase activity, and citric acid production by L-d1 reached a level of 59.0 g/l. During several subcultivations on both minimal and complete agar media, the autodiploid strains were genetically stable since they formed diploid conidia in their uniform colonies without producing sectors, and maintained citric acid productivity. However, when cultivated on minimal and complete agar media containing benomyl as a haploidizing reagent, the autodiploid strains readily formed sectors of haploid segregants. The properties of the haploid strains obtained by the benomyl treatment of the autodiploid strains were similar in morphology and citric acid productivity to those of the parental strain, WU-2223L. These results indicated that the enhanced production of citric acid from soluble starch by the autodiploid strains was due to autodiploid formation but not to gene mutation caused by the colchicine treatment.     

Economical succinic acid production from cane molasses by Actinobacillus succinogenes

In this work, production of succinic acid by Actinobacillus succinogenes CGMCC1593 using cane molasses as a low cost carbon source was developed. In anaerobic bottles fermentation, succinic acid concentration of 50.6+/-0.9 g l(-1) was attained at 60 h using an optimum medium containing molasses pretreated with sulfuric acid, resulting in a succinic acid yield of 79.5+/-1.1% and sugar utilization of 97.1+/-0.6%. When batch fermentation was carried out in a 5-l stirred bioreactor with pretreated molasses, 46.4 g l(-1) of succinic acid was attained at 48 h and faster cells growth was also observed. Fed batch fermentation was performed to minimize the substrate (sugar) inhibition effect, giving 55.2 g l(-1) of succinic acid and 1.15 g l(-1)h(-1) of productivity at 48 h. The present study suggests that the inexpensive cane molasses could be utilized for the economical and efficient production of succinic acid by A. succinogenes.     

Repeated batch production of citric acid from sugarcane molasses using recycled solid-state surface culture ofAspergillus niger

Summary The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.