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.     

cAMP concentration,morphological differentiation and citric acid production in Aspergillus niger

Summary The possibility that adenosine 3,5 monophosphate exerts an effect on citric acid production by Aspergillus niger by influencing pellet morphology has been investigated. The effect of pH and inoculum size on pellet formation, citric acid production, and intracellular and extracellular cAMP levels were studied. High levels of intracellular and extracellular cAMP in the later stages of the fermentation, the period of maximum citric acid formation, were associated with those treatments which gave pellets of intermediate size. The highest cAMP levels were associated with those treatments which gave the highest citric acid titre. It was concluded that high cAMP levels are principally associated with an optimum physiological state for citric acid production and that cAMP levels do not vary directly with pellet size.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

Extended production of citric acid using an exchange filtration technique

A filtration technique has been applied to deep culture citric acid fermentation in an attempt to prolong the active phase of citric acid production. The fermentation was extended to 30 days with a daily production rate of between 9 and 10g^–1d^–1. A yield of over 70% citric acid was achieved.     

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.     

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.     

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.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

Studies on aspergilli xix. Role of carbon and nitrogen on citric acid production

Summary The effect of various carbon and nitrogen compounds on the production of citric acid byAspergillus luchuensis was studied under controlled conditions. Unhydrolysed sucrose was better than the hydrolysed one. The other carbon sources, viz., maltose, lactose, Gur, Ral and cane molasses were unsuitable for citric acid fermentation. With all these carbohydrates low yields were obtained. Sodium nitrate was found to be the best nitrogen source for the maximum yield of citric acid. It was closely followed by potassium nitrate. Ammonium compounds and amino acids, in general, were unsuitable for the production of citric acid.     

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.     

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     

Glutamic acid and by-product synthesis by immobilized cells of the bacterium Corynebacterium glutamicum

Summary The time course for the synthesis of glutamic acid and by-products from glucose was investigated using immobilized cell reactor of the bacterium C.glutamicum. Lactic acid, succinic acid, alanine acid and aspartic acid were formed early in the fermentation and during the active growth phase, whereas gluconic acid, -ketoglutaric acid and proline were produced late and during the active phase of glutamic acid synthesis. Oxygen transfer rate in fermentation broth had a pronounced effect on the nature and quantities of fermentation products. In continuous fermentation and at OTR of 102.5 mMO₂/l.h., formation of by-products greatly decreased and up to 58.5 g/l of glutamic acid were produced with a conversion efficiency of 74.6% of the theoretical value and volumetric productivity of 6.2 g/l.h.     

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.     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

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     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

Studies of the mixing character and flow distribution in mycelial fermentation broths

The influence of two mixing systems on the principal parameters of mycelial fermentations of Aspergillus niger, Fusicoccum amygdali Del. and Fusarium moniliforme Sheld. as well as their metabolite citric acid, fusicoccin and gibberellic acid production was analyzed from the viewpoint of flow energy distribution in a bioreactor. The growth and metabolite synthesis during fermentation was compared under different mixing conditions in the fermenter FU-8 with a turbine mixing system (TMS) and a counterflow mixing system (CMS). It was found that the growth, productivity and respiration characteristics as well as the morphology of these cultures varied dependent on the mixing system and agitation regime used. The counterflow mixing system was more favourable for large agglomerates (F. amygdali) or soft pellets (A. niger) forming fungi, while the turbine mixing system was more effective for F. moniliforme growing in the form of small clumps and freely dispersed hyphae. Flow characteristics under different mixing conditions were analyzed in a model fermenter. The kinetic energy of flow fluctuations was measured in gassed and ungassed water and different fermentation broth systems by using a Stirring Intensity Measuring Device (SIMD-F1). The difference of the energy values at different points was better expressed in the fermenter with a turbine mixing system in comparison with that having a counterflow mixing system. High viscous F. amygdali and A. niger broth provided higher energy values compared to water and low viscous F. moniliforme broth. It was observed that the intensity of growth and the intensity of the synthesis decreased at very high energy values, which was obviously connected to the influence of the irreversible shear stress on the mycelial morphology.     

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     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.