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;     

Continuous citric acid fermentation by <Emphasis Type="Italic">Candida oleophila</Emphasis> under nitrogen limitation at constant C/N ratio

Summary The central aspect of this work was to investigate the influence of nitrogen feed rate at constant C/N ratio on continuous citric acid fermentation by Candida oleophila ATCC 20177. Medium ammonia nitrogen and glucose concentrations influenced growth and production. Space-time yield (STY) meaning volumetric productivity, biomass specific productivity (BSP), product concentration, product selectivity and citrate/isocitrate ratio increased with increasing residence time (RT). BSP increased in an exponential mode lowering nitrogen feed rates. Highest BSP for citric acid of 0.13 g/(g h) was achieved at lowest NH₄Cl concentration of 1.5 g/l and highest STY (1.2 g/l h) with 3 g NH₄Cl/l at a RT of 25 h. Citric acid 74.2 g/l were produced at 58 h RT and 6 g NH₄Cl/l. Glucose uptake rate seems to be strictly controlled by growth rate of the yeast cells. Optimum nitrogen concentration and adapted C/N ratio are essential for successful continuous citric acid fermentation. The biomass-specific nitrogen feed rate is the most important factor influencing continuous citric acid production by yeasts. Numerous chemostat experiments showed the feasibility of continuous citrate production by yeasts.     

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.     

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.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

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 <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

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.     

Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH₄NO₃) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.     

Optimization for producing cell-bound lipase from <Emphasis Type="Italic">Geotrichum</Emphasis> sp. and synthesis of methyl oleate in microaqueous solvent

An integrated optimization strategy involving a combination of different designs was employed to optimize producing conditions of cell-bound lipase (CBL) from Geotrichum sp. Firstly, it was obtained by a single factorial design that the most suitable carbon source was a mixture of olive oil and citric acid and the most suitable nitrogen source was a mixture of corn steep liquor and NH₄NO₃. Then, the Plackett–Burman design was used to evaluate the effects of 13 variables related to CBL production, and three statistically significant variables namely, temperature, olive oil concentration, and NH₄NO₃ concentration, were selected. Subsequently, the levels of the three variables for maximum CBL production were determined by response surface analysis as follows: 1.64% (v/v) olive oil, 1.49% (w/v) NH₄NO₃, and temperature 33.00°C. Such optimization resulted in a high yield of CBL at 23.15 U/ml, an enhanced 4.45-fold increase relative to the initial result (5.2 U/ml) in shake flasks. The dried CBL was used to synthesize methyl oleate in microaqueous hexane, resulting in 94% conversion after 24 h, and showed reusability with 70% residual activity and 69% conversion after eight cycles of batch operation, which indicating that CBL, as a novel and natural form of immobilized enzyme, can be effectively applied in repeated synthesis of methyl oleate in a microaqueous solvent.     

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.     

Oxytetracycline production by Streptomyces rimosus in solid-state fermentation of corncob

Corn-cob was used as a substrate in the production of oxytetracycline by Streptomyces rimosus TM-55 in a solidstate fermentation. Oxytetracycline was detected on day 4, and reached its maximum on day 8. Optimal conditions for oxytetracycline production were an initial pH of 5.2 to 6.3, an initial moisture content of 64% to 67%, supplementation with 20% (w/w) rice bran or 1.5% to 2.5% (w/w) (NH₄)₂SO₄ as sole N source, 1.0% (w/w) CaCO₃, 2% (w/w) MgSO₄.7H₂O, and 0.5% (w/w) KH₂PO₄, with incubation for 8 days at 25 to 30°C. Each g substrate produced 7 to 8 mg oxytetracycline.     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

Ram horn peptone as a source of citric acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis>, with a process

The present study deals with the production of citric acid from a ram horn peptone (RHP) by Aspergillus niger NRRL 330. A medium from RHP and a control medium (CM) were compared for citric acid production using A. niger in a batch culture. For this purpose, first, RHP was produced. Ram horns were hydrolyzed by treatment with acids (6 N H₂SO₄, 6 N HCl) and neutralizing solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined. RHP was compared with peptones with a bacto-tryptone from casein and other peptones. The results from RHP were similar to those of standard peptones. The optimal concentration of RHP for the production of citric acid was found to be 4% (w/w). A medium prepared from 4% RHP was termed ram horn peptone medium (RHPM). In comparison with CM, the content of citric acid in RHPM broth (84 g/l) over 6 days was 35% higher than that in CM broth (62 g/l). These results show that citric acid can be produced efficiently by A. niger from ram horn.     

Production and optimization of polygalacturonase from mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) peel using <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in solid state fermentation

Mango peel is one of the major wastes from fruit processing industries, which poses considerable disposal problems and ultimately leads to environmental pollution. The objective of the current research was to determine the significant parameters on the production of polygalacturonase from mango peel which is a major industrial waste. Solid state culture conditions for polygalacturonase production by Fusarium moniliforme from dried mango peel powder were optimized by Taguchi’s L-18 orthogonal array experimental design methodology. Eight fungal metabolic influencing variables, viz. temperature, mango peel, inoculum, peptone, ammonium nitrate (NH₄NO₃₎, magnesium sulphate (MgSO₄), zinc sulphate (ZnSO₄) and potassium dihydrogen phosphate (KH₂PO₄) were selected to optimize polygalacturonase production. The optimized parameters composed of temperature (30°C), mango peel (6.5%, g, w/v), inoculum (8%, ml, v/v), peptone (1%, g, w/v), NH₄NO₃ (0.60%, g, w/v), MgSO₄ (0.05%, g, w/v), ZnSO₄ (0.06%, g, w/v) and KH₂PO₄ (0.4%, g, w/v). Based on the influence of interaction of fermentation components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. The temperature, inoculum level, mango peel substrate and KH₂PO₄ showed maximum production impact at optimized conditions. From the optimized conditions the polygalacturonase activity was maximized to 43.2 U g⁻¹.     

Citric and gluconic acid production from fig by Aspergillus niger using solid-state fermentation

The production of citric and gluconic acids from fig by Aspergillus niger ATCC 10577 in solid-state fermentation was investigated. The maximal citric and gluconic acids concentration (64 and 490 g/kg dry figs, respectively), citric acid yield (8%), and gluconic acid yield (63%) were obtained at a moisture level of 75%, initial pH 7.0, temperature 30°C, and fermentation time in 15 days. However, the highest biomass dry weight (40 g/kg wet substrate) and sugar utilization (90%) were obtained in cultures grown at 35°C. The addition of 6% (w/w) methanol into substrate increased the concentration of citric and gluconic acid from 64 and 490 to 96 and 685 g/kg dry fig, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 298–304. Received 15 April 2000/ Accepted in revised form 11 August 2000     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

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     

Citric acid production by Yarrowia lipolytica: Effect of nitrogen and biomass concentration on yield and productivity

Summary Citric acid yields of Yarrowia lipolytica (NRRL Y-1095) grown on glucose ranged from 0.38–0.77 g/g and were dependent on both biomass and nitrogen concentration (as NH₄Cl and yeast extract). Increasing the biomass concentration by 3% (w/v) increased fermentor productivities from 0.6 to 1.22 g citric acid/L h.