Solid state fermentation of apple pomace for citric acid production

Summary A solid state fermentation process is described for the production of citric acid from apple pomace byAspergillus niger NRRL 567. The yields of citric acid varied with the pomace varieties, and were dependent on (1) the amount of methanol present in the pomace, and (2) the fermentation time and temperature. The process yielded as much as 90 g citric acid/kg apple pomace fermented in the presence of 3–4% methanol at 30°C in five days.

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Resumen Se describe un proceso de fermentación en estado sólido para la producción de ácido cítrico a partir de residuos de manzana, utilizandoAspergillus niger NRRL 567. La producción de ácido cítrico varió con el tipo de residuo utilizado y dependió de (1) la cantidad de metanol presente en los residuos y (2) del tiempo y la temperatura del proceso. La fermentación puede producir hasta 90 g de ácido cítrico/kg de residuo de manzana, fermentado en presencia de 3–4% de metanol a 30°C de temperatura durante 5 días.

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Résumé Le procédé de fermentation en milieu solide qui est décrit est déstiné à la production d'acide citrique à partir du marc de pommes parAspergillus niger NRRL 567. Les rendements en acide citrique varient suivant les variétés de pommes et dépendent: (1) de la quantité de méthanol présente dans le marc, et (2) du temps de fermentation et de la température. On peut obtenir jusqu'à 90 g d'acide citrique par kg de marc de pommes fermenté, en présence de 3 à 4% de méthanol, à 30°C et en 5 jours.

     

Apple pomace: A potential substrate for citric acid production by Aspergillus niger

Summary Apple pomace was used as a fsubstrate for citric acid production by five strains of Aspergillus niger. A. niger NRRL 567 produced the greatest amount of citric acid from apple pomace in the presence of 4% methanol. The yield was 88% based on the amount of sugar consumed.     

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.     

A fed batch surface culture process for the production of citric acid with reuse of Aspergillus niger mycelia

Citric acid production in surface culture process with reuse of mycelia for more than one batch of media has been reported in this work. A strategy for replacing media and thus increasing productivity in subsequent batches is discussed. An explanation for the decrease in citric acid accumulation beyond the second batch is suggested on the basis of oxygen limitation.     

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.     

Grape pomace: A novel substrate for microbial production of citric acid

Summary Grape pomace was used as substrate for microbial production of citric acid. Of the five cultures examined,Aspergillus niger NRRL 567 was found to produce the greatest amount of citric acid from grape pomace in the presence of methanol at a concentration of 3% (vol/wt). The yield was 60% based on the amount of fermentable sugar consumed.     

Mineral nutrition ofAspergillus niger for citric acid production

The mineral requirements of a strain ofAspergillus niger for the production of citric acid in a synthetic medium were studied. It was observed that K₂HPO₄ and MgSO₄.7 H₂O were required at concentrations of 0.1% and 0.02% respectively. The optimum level of each of the trace elements Fe, Mn and Zn was 1.0 μg/ml. NaCl and CaCl₂ at lower concentrations had no effect on citric acid production. Trace elements, Cu, Co and Mo, had an adverse effect on the production of citric acid while Ni and V were without effect.     

Selection of mutants of Aspergillus niger showing enhanced productivity of citric acid from starch in shaking culture

Mutants with enhanced citric acid production from soluble starch were induced from Aspergillus niger WU-2223L. After UV-irradiation of a conidial suspension of strain WU-2223L, mutants were selected on modified starch-methyl red agar plates on the basis of higher amylolytic activity and acid productivity. The 8 mutants selected showed enhanced citric acid production from soluble starch in shaking culture. Among them, a representative mutant strain, 2M-43, produced 48.0gg/l of citric acid from 120 g/l of soluble starch in 9 d of cultivation in shaking culture, whereas strain WU-2223L produced 35.1 g/l. Glucoamylase activities in the culture filtrates of strains 2M-43 and WU-2223L reached maximum levels of 3.62 U/ml and 2.11 U/ml, respectively, both at 3 d of cultivation, and thereafter decreased.     

Channelling of glucose by methanol for citric acid production from Aspergillus niger

Citric acid produced by Aspergillus niger was increased from 4.6g l^-1 to 7.8gl^-1 by supplementing basal medium with methanol (30mll^-1). While stimulating citric acid production, methanol did not improve membrane permeability of the fungus for citric acid. Methanol inhibited the germination of Aspergillus spores. An increase in glucose concentration from 50gl^-1 to 100gl^-1 in the presence of methanol (30mll^-1) improved citric acid production (1.6-fold) while at higher levels of glucose concentration methanol had no effect on citic acid production.     

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;     

L-lactic acid production from apple pomace by sequential hydrolysis and fermentation

The potential of apple pomace (a solid waste from cider and apple juice making factories) as a source of sugars and other compounds for fermentation was evaluated. The effect of the cellulase-to-solid ratio (CSR) and the liquor-to-solid ratio (LSR) on the kinetics of glucose and total monosaccharide generation was studied. Mathematical models suitable for reproducing and predicting the hydrolyzate composition were developed. When samples of apple pomace were subjected to enzymatic hydrolysis, the glucose and fructose present in the raw material as free monosaccharides were extracted at the beginning of the process. Using low cellulase and cellobiase charges (8.5 FPU/g-solid and 8.5 IU/g-solid, respectively), 79% of total glucan was saccharified after 12 h, leading to solutions containing up to 43.8 g monosaccharides/L (glucose, 22.8 g/L; fructose, 14.8 g/L; xylose+mannose+galactose, 2.5 g/L; arabinose+rhamnose, 2.8g/L). These results correspond to a monosaccharide/cellulase ratio of 0.06 g/FPU and to a volumetric productivity of 3.65 g of monosaccharides/L h. Liquors obtained under these conditions were used for fermentative lactic acid production with Lactobacillus rhamnosus CECT-288, leading to media containing up to 32.5 g/L of L-lactic acid after 6 h (volumetric productivity=5.41 g/L h, product yield=0.88 g/g).     

Improved xylanase production using apple pomace waste by Aspergillus niger in koji fermentation

Xylanase production by Aspergillus niger NRRL‐567 in solid‐state fermentation (koji fermentation) was optimized using 2⁴ factorial design and response surface methodology. The evaluated variables were the initial moisture level and concentration of inducers [veratryl alcohol (VA), copper sulphate (CS), and lactose (LAC)], leading to the response of xylanase production. Initial moisture level and LAC were found to be the most significant variable for xylanase production (p<0.05). The highest xylanase production was observed with 3578.8 ± 65.3 IU/gds (gram dry substrate) under optimal conditions using initial moisture of 85% (v/w), pH 5.0 and inducers VA (2 mM/kg), LAC 2% (w/w), and CS (1.5 mM/kg) after 48 h of incubation time. Higher xylanase activity of 3952 ± 78.3 IU/gds was attained during scale‐up of the process in solid‐state tray fermentation under optimum conditions after 72 h of incubation time. The present study demonstrates that A. niger NRRL‐567 can efficiently be used to achieve xylanase production with an economical and environmental benefit in solid‐state tray fermentation. The developed process can be used to develop an effective process for commercially feasible bioproduction of xylanases for speciality applications, such as conversion of lignocellulosic biomass to biofuels and other value‐added products.     

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H₂SO₄-pretreated black strap molasses (containing 150 g sugars/l) in Vogel's medium. On the basis of comparison of kinetic parameters, namely the 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 had the ability to overproduce citric acid.     

Galactose inhibition of citric acid production from glucose by Aspergillus niger

Summary Previous work in this laboratory has demonstrated that although Aspergillus niger can readily utilize galactose, no citric acid is produced from this carbon source (Hossain et al. 1984). Experiments were now conducted where galactose was added at various concentrations to synthetic growth medium containing glucose as carbon source, so that the effect of galactose on citric acid production from glucose could be observed. The results showed that the presence of galactose or a product of galactose metabolism caused inhibition of citric acid production, and also reduced the rate of glucose utilization. Enzyme analyses using mycelial cell-free extracts indicated that galactose interfered with the glucose-repression of the key enzyme 2-oxoglutarate dehydrogenase.     

Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

Improved reproducibility of citric acid production in Aspergillus niger

Summary We report the derivation of Aspergillus niger strains having a lower standard deviation in citric acid production by the use of ⁶⁰Co gamma radiation and by the parasexual cycle. On the basis of the results obtained by isolating diploids and segregants, diploidization seems to be sufficient for reducing variability. The implications of these results in terms of improvement of industrial strains are discussed.     

Enhanced solid‐state citric acid bio‐production using apple pomace waste through surface response methodology

Aims: To evaluate the potential of apple pomace (AP) supplemented with rice husk for hyper citric acid production through solid‐state fermentation by Aspergillus niger NRRL‐567. Optimization of two key parameters, such as moisture content and inducer (ethanol and methanol) concentration was carried out by response surface methodology. Methods and Results: In this study, the effect of two crucial process parameters for solid‐state citric acid fermentation by A. niger using AP waste supplemented with rice husk were thoroughly investigated in Erlenmeyer flasks through response surface methodology. Moisture and methanol had significant positive effect on citric acid production by A. niger grown on AP (P < 0·05). Higher values of citric acid on AP by A. niger (342·41 g kg^?1 and 248·42 g kg^?1 dry substrate) were obtained with 75% (v/w) moisture along with two inducers [3% (v/w) methanol and 3% (v/w) ethanol] with fermentation efficiency of 93·90% and 66·42%, respectively depending upon the total carbon utilized after 144 h of incubation period. With the same optimized parameters, conventional tray fermentation was conducted. The citric acid concentration of 187·96 g kg^?1 dry substrate with 3% (v/w) ethanol and 303·34 g kg^?1 dry substrate with 3% (v/w) methanol were achieved representing fermentation efficiency of 50·80% and 82·89% in tray fermentation depending upon carbon utilization after 120 h of incubation period. Conclusions: Apple pomace proved to be the promising substrate for the hyper production of citric acid through solid‐state tray fermentation, which is an economical technique and does not require any sophisticated instrumentation. Significance and Impact of the Study: The study established that the utilization of agro‐industrial wastes have positive repercussions on the economy and will help to meet the increasing demands of citric acid and moreover will help to alleviate the environmental problems resulting from the disposal of agro‐industrial wastes.