Enhancement of lactic acid production with ram horn peptone by Lactobacillus casei

Ram horns are a waste material from the meat industry. The use of ram horn peptone (RHP) as a supplement for lactic acid production was investigated using Lactobacillus casei. For this purpose, first, RHP was produced. Ram horns were hydrolysed by treating with acids (3 M H₂SO₄ and 6 M HCl) and neutralizing the solutions to yield ram horn hydrolysate (RHH). The RHH was evaporated to yield RHP. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined and compared with a bacto-tryptone from casein. When the concentrations (1–6% w/v) of the RHP were used in bacterial growth medium as a supplement, 2% RHP (ram horn peptone medium) had a maximum influence on the production of lactic acid by L. casei. The content of lactic acid in the culture broth containing 2% RHP (43 g l^–1) grown for 24 h was 30% higher than that of the control culture broth (33 g l^–1) and 10% higher than that of 2% bacto-tryptone (39 g l^–1). RHP was demonstrated to be a suitable supplement for production of lactic acid. This RHP may prove to be a valuable supplement in fermentation technology.     

A High Performance Fermentation Process for Citric Acid Production by Aspergillus niger NGGCB-101, Using Vermiculite as an Additive in a Stirred Bioreactor

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG^GCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levels of vermiculite were added as an additive into the fermentation medium. The best results were observed when 0.20 g/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citric acid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelial pellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at different vermiculite levels was monitored for Q _p , Q _s and q _p , there was a significant enhancement (P 0.05) in these variables over the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basic parameters for high performance citric acid fermentation.     

Utilization of ram horn peptone in the production of glucose oxidase by a local isolate Aspergillus niger OC-3

Glucose oxidase (GO) is an enzyme that is used in many fields. In this study, ram horn peptone (RHP) was utilized as the nitrogen source and compared with other nitrogen sources in the production of GO by Aspergillus niger. To obtain higher GO activity, 14 A. niger strains were isolated from soil samples around Erzurum, Turkey. Among these strains, the isolate that was named A. niger OC-3 achieved the highest GO production. The production of GO was carried out in 100 mL scaled batch culture. The fermentation conditions such as initial pH, temperature, agitation speed, and time were investigated in order to improve GO production. The results showed that the cultivation conditions would significantly affect the formation of GO, and the utilization of the RHP achieved the highest enzyme production (48.6 U/mL) if compared to other nitrogen sources. On the other hand, the maximum biomass was obtained by using the fish peptone (7.2 g/L), while RHP yielded 6.4 g/L. These results suggest that RHP from waste ram horns could effectively be used in the production of GO by A. niger OC-3.     

Coffee husk: an inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system

Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH₄)₂SO₄, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India;     

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca⁺⁺ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl₂ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl₂ at a level of 2.0 M, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NG^GCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

Redox potential in submerged citric acid fermentation

Summary The role and importance of the redox potential phenomena in submerged citric acid production are discussed. The redox potential of the fermentation broth is the result of oxydo-reduction processes where the metabolic activity of the microorganism Aspergillus niger plays the most significant role. The course of the redox curve for a good yielding citric acid production is presented and interpreted. The experiments of submerged citric acid production were carried out on beet molasses treated with potassium hexacyanoferrate and inoculated with A. niger spores.     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Effect of medium on the bacteriocin production byKlebsiella pneumoniae

The influence of growth media and media constituents on bacteriocin production byKlebsiella pneumoniae was studied. Among the standard laboratory media used trypticase soy broth (TSB) showed the maximum production and poor yields resulted from growth in peptone water and nutrient broth. A number of peptones differed in their bacteriocin production. Best yields were observed in tryptone and proteose peptone water. Addition of 1 % yeast extract to TSB further stimulated bacteriocin production. However, activity was low when glucose, glycine, sodium mercaptoacetate or bile salt mixture were added to the medium. Supression of synthesis by certain agents as well as inhibition of formed bacteriocin by the others appears to affect the bacteriocin yield. No proteinase activity was detected during the entire incubation period.     

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.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.     

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.     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.     

Evaluation of peptones from chicken waste as a nitrogen source for micro-organisms

In this study, chicken peptone was produced by hydrolysing inedible parts derived from chickens using endo-protease and exo-protease. The usefulness of chicken peptone as a nutrient source for bacteria was evaluated in comparison with other commercially produced peptones (animal, soy and casein-derived peptone). Escherichia coli and Bacillus subtilis were used as test strains to determine the effect of peptones from different sources on their growth ability. Both bacteria were successfully cultured in chicken peptone solution, which is similar to peptone solution containing commercial peptones apart from animal peptone. In chemical analysis, chicken peptone contained 12·0% nitrogen; this was similar to the nitrogen content from other commercial peptone sources, except for the 9·0% nitrogen found in soy peptones. The molecular weight of the peptone was determined by gel filtration chromatography, and those of all peptone, except animal-derived peptone, were found to be <5000 Da. In addition, when B. subtilis was cultured in a medium containing chicken peptone, it was shown that the protease activity was highest as compared with other commercial peptones. From these results, it is suggested that chicken peptone can be utilized for microbial culture, and this is an effective method to reuse chicken waste.     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Ultrasound effects on invertase from Aspergillus niger

Ultrasound effects on the release and activity of invertase from Aspergillus niger cultivated in a medium containing sucrose and peptone and in another with sugar-cane molasses and peptone were investigated. Irradiation was conducted for periods of 2–10 min. with waves of amplitude 20 and 40 using an ultrasound processor of 20 kHz. Product formation was determined as reducing equivalents formed by time units using 3,5-dinitrosalicylic acid. Total and specific activities of the culture supernatants were compared in the presence and absence of sonication. Both amplitudes promoted a significant increase of total invertase activity in the time periods investigated and the highest values were obtained with an amplitude of 20. Ultrasound irradiation caused cell disruption, thus releasing invertase and, after 4 min, activation of the enzyme also occurred. The best conditions for production, extraction and activation of invertase were in molasses medium containing peptone and irradiation with ultrasound waves at 20 for 8 min. This method showed high efficiency for the extraction and activation of invertase from A. niger as well as a great potential for use in industrial processes.     

Production of Bacillus sphaericus larvicide on industrial peptones

Summary A range of industrial peptones in combination with other carbon and nitrogen sources were investigated in order to define a medium for production of insecticide from Bacillus sphaericus, strain 2362. Industrial protein hydrolysates with an average peptide chain length above 30 were found to be poor substrates. Fermentation of B. sphaericus on protein-based media supplemented with glycerol resulted in production of 4–5x10³ units of larvicide per ml fermentation broth, and toxicity of the larvicide up to 10⁴ units per mg. Equally high larvicide production was obtained, when peptone was substituted with 30 mM l-glutamate and 2 mM proline. Addition of 2 mM l-arginine to this medium prevented sporulation and larvicide production.     

Submerged citric acid fermentation: rheological properties ofAspergillus niger broth in a stirred tank reactor

Summary A large number of submerged citric acid fermentations in a beet molasses substrate was studied. The development of Aspergillus niger from conidia to pellets was followed. Rheological characteristics of the fermentation broth including the pellets were determined. The results obtained confirm the fact that the non-Newtonian pseudoplastic behaviour of the fermentation broth was due to the presence of mycelial pellets. The most significant changes in rheological properties occurred during the period of maximal citric acid production and increase in biomass. Offprint requests to: M. Berovi