Fate and role of ammonium ions during fermentation of citric acid by Aspergillus niger

Stoichiometric modeling of the early stages of the citric acid fermentation process by Aspergillus niger revealed that ammonium ions combine with a carbon-containing metabolite inside the cell, in a ratio 1:1, to form a nitrogen compound which is then excreted by the mycelium. High-performance liquid chromatography analysis identified glucosamine as the product of the relationship between glucose and ammonium during the early stages of the citric acid fermentation process. Slightly acidic internal pHs, extremely low ammonium ion concentrations inside the cell, and glucosamine synthesis come into direct contradiction with the earlier theory of the ammonium pool inside the cell, regarded as responsible for inhibition of the enzyme phosphofructokinase. At later fermentation stages, when the mycelium is involved in a process of fragmentation and regrowth, the addition of ammonium sulfate leads to a series of events: the formation and secretion of glucosamine in elevated amounts, the short inhibition of citrate synthesis, growth enhancement, the utilization of glucosamine, and finally, the enhancement of citric acid production rates. Obviously, the enzymatic processes underlining the phenomena need to be reexamined. As a by-product of the citric acid fermentation, glucosamine is reported for the first time here. Suitable process manipulations of the system described in this work could lead to successful glucosamine recovery at the point of its highest yield before degradation by the fungus occurs.     

Growth ofMethylomonas methanolica: Factors Influencing growth yield

Summary The activities of ACH¹, NAD-ICDH, NADP-ICDH and CS were determined in cell extracts of high and low citric acid-producing strains of A. niger, cultivated on molasses medium by the surface or submerged method. A high differentiation in the activities of the enzymes studied was found to occur at various accumulation stages of citric acid and during its decomposition by moulds. During intensive citric acid synthesis, the activity of ACH and that of both dehydrogenases decreased significantly (though they did not disappear completely) compared to their initial activities during the growth period of the mycelium. CS activity, however, was maintained at almost the same level over the whole fermentation period or increased slightly, particularly in the case of low citric acid-producing strains. The relationships between the activities of these enzymes were reversed during citric acid decomposition by moulds. Moreover, it was shown that in the period of mycelium growth the maximal activities of the enzymes were much higher in submerged culture than in surface culture.     

Citric Acid Fermentation by Aspergillus niger on Low Sugar Concentrations and Cotton Waste

The possible use of cotton waste as a carbohydrate source of citric acid production by Aspergillus niger was examined. No citric acid was produced when A. niger was grown on cotton waste as a sole carbon source. In two-stage fermentations, however, mycelium obtained from surface cultures in cotton waste medium yielded more citric acid when transferred to sucrose-containing media than when directly inoculated to sucrose-containing media. It is concluded that cotton waste can be used for saving sucrose and for increasing yields of citric acid fermentation by A. niger.     

Kinetic studies on citric acid production by Aspergillus niger. II. The two-stage process.

A two-stage process of submerged citric acid fermentation with replacement of growth medium by fermentation medium has been worked out. The optimum composition of mineral nutrients and pH of the fermentation medium of the second stage of the process were determined. An addition of 0.5 g/l of NH4NO3 as nitrogen source and 0.1 g/l of MgSO4-7H2O as magnesium source ensured effective conversion of sucrose to citric acid. An addition to KH2PO4, on the other hand, was definitely unfavourable as it considerably reduced the product yield. The medium for the second stage of fermentation should be acidified to about pH 2.2, while the water used for washing the mycelium from the remains of the growth medium should have a pH of 2.5--3.5. Under these conditions, with an initial sucrose concentration of 100 g/l, after 132 hr fermentation at 26 degrees up to 90 g/l of citric acid was obtained, which corresponds to a productivity of over 16 g/l. day. The highest activity for citric acid formation was found in three- or four-day-old mycelium.     

过表达氨基葡萄糖脱氨酶对大肠杆菌氨基葡萄糖合成及中心碳代谢的影响

考察过表达氨基葡萄糖脱氨酶对氨基葡萄糖合成及大肠杆菌（Escherichia coli）中心碳代谢的影响。实验结果表明：过表达氨基葡萄糖脱氨酶使得在36 g/L葡萄糖,pH为9.0的发酵条件下,发酵24 h后,重组菌发酵液中氨基葡萄糖、丙酮酸和乙酸的量分别是对照菌Rosetta的2.1、1.48和1.74倍;而乳酸的量为2.53 g/L,对照菌Rosetta发酵液中的乳酸含量未检测到,重组菌发酵液中柠檬酸及α-酮戊二酸的含量分别是Rosetta的2.99和2.73倍。     

Production of citric acid using its extraction wastewater treated by anaerobic digestion and ion exchange in an integrated citric acid-methane fermentation process

In order to solve the problem of extraction wastewater pollution in citric acid industry, an integrated citric acid-methane fermentation process is proposed in this study. Extraction wastewater was treated by mesophilic anaerobic digestion and then used to make mash for the next batch of citric acid fermentation. The recycling process was done for seven batches. Citric acid production (82.4 g/L on average) decreased by 34.1?% in the recycling batches (2nd–7th) compared with the first batch. And the residual reducing sugar exceeded 40 g/L on average in the recycling batches. Pigment substances, acetic acid, ammonium, and metal ions in anaerobic digestion effluent (ADE) were considered to be the inhibitors, and their effects on the fermentation were studied. Results indicated that ammonium, Na⁺ and K⁺ in the ADE significantly inhibited citric acid fermentation. Therefore, the ADE was treated by acidic cation exchange resin prior to reuse to make mash for citric acid fermentation. The recycling process was performed for ten batches, and citric acid productions in the recycling batches were 126.6 g/L on average, increasing by 1.7 % compared with the first batch. This process could eliminate extraction wastewater discharge and reduce water resource consumption.     

The parasitism of the mouldPenicillium purpurogenum onAspergillus niger

The course of the parasitic action of the mouldPenicillium purpurogenum on the mycelium of a plant strain ofAspergillus niger is described in plant and laboratory conditions of surface citric acid fermentation. During the infection, a successive destruction of the mycelium ofAspergillus niger sets in, as well as a decrease in citric acid production, in this case also act the specific inhibitory thermostabile materials, secreted by the parasitic mould into the medium. The main factor is the cell lysis, effected by a specific enzymolytic system of the constitutive type. This system is evolved by the parasitic mould in the nutritions medium, resp. into the mycelium of the infected mouldAspergillus niger and causes the lysis both of entire hyphae, and of isolated cell walls. By this lytic system, up to 85% of the dry weight of the cell walls is transferred into the solution as sugars, mainly as glucose, so that the chief component undergoing the lysis is a polymer carbohydrate. The optimum temperature of the action of the lytic system lies within 30–41°C, optimum pH is 4.2–5.5. In case of several other representatives of the genusPenicillium, no lytic systems have been established, at the same time their inability for the parasitic form of life on the mycelium ofAspergillus niger was proved.     

The role of glucosamine-6-phosphate deaminase at the early stages of <Emphasis Type="Italic">Aspergillus niger</Emphasis> growth in a high-citric-acid-yielding medium

Glucosamine-6-phosphate (GlcN6P) deaminase seems to be the main enzyme in Aspergillus niger cells responsible for rapid glucosamine accumulation during the early stages of growth in a high-citric-acid-yielding medium. By determining basic kinetic parameters on the isolated enzyme, a high affinity toward fructose-6-phosphate (Fru6P) was measured, while in the reverse direction the K _m value for glucosamine-6-phosphate was lower than deaminases from other organisms measured so far. The enzyme characteristics of GlcN6P deaminase suggest it must compete with 6-phosphofructo-1-kinase (PFK1) for the common substrate—Fru6P in A. niger cells. Glucosamine accumulation seems therefore to remove an intermediate from the glycolytic flux, a situation which is reflected in slower citric acid accumulation and a specific growth rate after the germination of spores. When ammonium ions are depleted from the medium, one of the substrates for GlcN6P deaminase becomes limiting and Fru6P can be catabolised by PFK1 which enhances glycolytic flux. Other enzymatic features of GlcN6P deaminase such as pH optima for both aminating and deaminating reactions might play a significant role in rapid glucosamine accumulation during the early phase of fermentation and a slow consumption of aminosugar during the citric-acid-producing phase.     

The parasitism of the mouldPenicillium purpurogenum onAspergillus niger

The causes of the formerly described effect of the limitation of infection of surface citric acid fermentation byPenicillium purpurogenum by the action of an increased air supply have been ascertained. The influence of the increased air supply on the composition ofAspergillus niger mycelium and cell walls was followed, as well as their assailability by the lytic enzyme ofPenicillium purpurogenum. Further the effect of temperature, humidity, oxygen supply and presence of carbon dioxide on the growth of the parasitic mould was evaluated. The main cause of the mentioned effect seems to be the drainage of humidity by increased air supply.     

Bioconversion of palm oil mill effluent for citric acid production: statistical optimization of fermentation media and time by central composite design

A laboratory-scale study was conducted to evaluate the feasibility of using palm oil mill effluent (POME) as a major substrate and other nutrients for maximum production of citric acid using the potential fungal strain Aspergillus niger (A103). Statistical optimization of medium composition (substrate–POME, co-substrates–wheat flour and glucose, and nitrogen source–ammonium nitrate) and fermentation time was carried out by central composite design (CCD) to develop a polynomial regression model through the effects of linear, quadratic, and interaction of the factors. The statistical analysis of the results showed that, in the range studied, ammonium nitrate had no significant effect whereas substrate, co-substrates and fermentation time had significant effects on citric acid production. The optimized medium containing 2% (w/w) of substrate concentration (POME), 4% (w/w) of wheat flour concentration, 4% (w/w) of glucose concentration, 0% (w/v) of ammonium nitrate and 5 days fermentation time gave the maximum predicted citric acid of 5.37 g/l which was found to be 1.5 g/l in the experimental run. The determination of coefficient (R ²) from the analysis observed was 0.964, indicating a satisfactory adjustment of the model with the response. The analysis showed that the major substrate POME (P < 0.05), glucose (P < 0.01), nutrient (P < 0.05), and fermentation time (P < 0.01) was more significant for citric acid production. The bioconversion of POME for citric acid production using optimal conditions showed the higher removal of chemical oxygen demand (82%) with the production of citric acid (5.2 g/l) on the final day of fermentation process (7 days). The pH and biosolids accumulation were observed during the bioconversion process.     

Developmental cycle and pharmaceutically relevant compounds of Salinispora actinobacteria isolated from Great Barrier Reef marine sponges

The developmental cycle of the obligate marine antibiotic producer actinobacterium Salinispora arenicola isolated from a Great Barrier Reef marine sponge was investigated in relation to mycelium and spore ultrastructure, synthesis of rifamycin antibiotic compounds, and expression of genes correlated with spore formation and with rifamycin precursor synthesis. The developmental cycle of S. arenicola M413 on solid agar medium was characterized by substrate mycelium growth, change of colony color, and spore formation; spore formation occurred quite early in colony growth but development of black colonies occurred only at late stages, correlated with a change in spore maturity in relation to cell wall layers. Rifamycins were detected throughout the growth cycle, but changed in relative quantity at particular phases in the cycle, with a marked increase after 32 days. Expression of the spore division gene ssgA and the rifK gene for 3-amino-5-hydroxybenzoate synthase responsible for rifamycin precursor synthesis was seen even at early stages of the growth cycle. ssgA expression significantly increased between days 26 and 31, but rifK expression effectively remained constant throughout the growth cycle, consistent with the early synthesis of rifamycin. Factors other than precursor synthesis may be responsible for an observed late increase in rifamycin production. A useful approach for measuring and exploring the regulation of antibiotic synthesis and gene expression in the marine natural product producer S. arenicola has been established.     

Synergism of ammonium and palmitic acid in uncoupling of electron transfer and ATP synthesis in chloroplasts

Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 μM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H⁺). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H⁺ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.     

Optimizing medium constituents and fermentation conditions for citric acid production from palmyra jaggery using response surface method

The quantitative effects of pH, temperature, time of fermentation, sugar concentration, nitrogen concentration and potassium ferrocyanide on citric acid production were investigated using a statistical experimental design. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for increasing the yield of citric acid using Aspergillus niger MTCC 281 by submerged fermentation. Regression equations were used to model the fermentation in order to determine optimum fermentation conditions. Higher yields were obtained after optimizing media components and conditions of fermentation. Maximum citric acid production was obtained at pH 5.35, 29.76 °C, 5.7 days of fermentation with 221.66 g of substrate/l, 0.479 g of ammonium nitrate/l and 2.33 g of potassium ferrocyanide/l.     

Effect of propionic acid on citric acid fermentation in an integrated citric acid–methane fermentation process

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.     

A recombinant <Emphasis Type="Italic">E. coli</Emphasis> bioprocess for hyaluronan synthesis

An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent metabolic engineering efforts.     

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.     

Application of carbon balance to submerged citric acid production by Aspergillus niger

Summary In an air-lift fermenter, the following production was obtained from 125 g sucrose in mineral medium at pH 2.5 : 15.76 g mycelium dry wt, 107.2 g citric acid anhydrous and 0.594 mol CO₂ within 138 h (run I) and 13.72 g mycelium dry wt, 114.28 g citric acid and 0.516 mol CO₂ within 144 h (run II). Initially, the carbon content of consumed sugar and products did not balance. At the end of fermentation, the carbon content of the products was 0.9%–5.5% higher than that of the consumed sugar. For the purpose of the calculations the carbon content in mycelium was accepted as 0.462.The work was a part of Project No 04.11 CPBP, topic No 2.24     

Inhibition of palatal epithelial cell death by altered protein synthesis.

Programmed cell death occurs in a defined region of the secondary palatal epithelium (medial edge) during its development in vivo or in vitro. The purpose of this study was to examine the effects of various inhibitors of macromolecular synthesis, particularly glycoprotein synthesis, on the death of palatal medial-edge epithelial cells in vitro. Rat palatal shelves explanted on gestational Day 15 and cultured singly showed medial-edge cell degeneration after 48 hr, whereas in the presence of 6-diazo-5-oxo-l-norleucine (DON), 2-deoxyglucose (DOG), or cycloheximide, cell death was prevented. This was in contrast to the lack of inhibition of cell death in the presence of actionomycin D or cytosine arabinoside. Selectively adding metabolites which bypass the effect of DON on various synthetic pathways demonstrated that the inhibition of cell death by DON was most likely due to a block in glucosamine formation. The synthesis and activity of various lysosomal enzymes were not affected by DON, whereas a marked decrease was observed with both cycloheximide and DOG. These results suggest that palatal epithelial cell death is not a passive event, but an active process requiring the programmed synthesis of specific proteins.     

Sudden substrate dilution induces a higher rate of citric acid production by Aspergillus niger.

On the basis of the present knowledge of Aspergillus niger metabolism during citric acid fermentation, an idea on how to improve the process was formed. Initially, a higher sucrose concentration was used for the germination of spores, which caused a higher intracellular level of the osmoregulator, glycerol, to be present. When citric acid started to be excreted into the medium, the substrate was suddenly diluted. Optimization of this procedure resulted in a nearly tripled volumetric rate (grams per liter per hour) of acid production, while the overall fermentation time was halved compared with the usual batch process. Yet, a characteristic delay was observed at the start of the acid excretion after the dilution. Hypo-osmotic shock caused a prominent elevation of intracellular cyclic AMP levels. Simultaneously, the specific activity of 6-phosphofructo-1-kinase increased significantly, probably due to phosphorylation of the protein molecule by cyclic AMP-dependent protein kinase. Specific 6-phosphofructo-1-kinase activity was much higher in the treated than in the normally growing mycelium. The metabolic flow through glycolysis was expected to be higher, which should contribute to a higher volumetric rate of acid production.