Pineapple waste - a novel substrate for citric acid production by solid-state fermentation

Summary Citric acid production in solid-state fermentation by Aspergillus foetidus ACM3996 was better on pineapple waste than on apple pomace, wheat bran or rice bran. The highest citric acid content achieved on pineapple waste was 16.1 g per 100 g dried pineapple waste, with a moisture content of 70% and in the presence of 3% methanol. This represents a yield of 62.4% based on the sugar consumed.     

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.     

The effect of methanol on citric acid production from galactose by Aspergillus niger

Summary The effect of methanol on the ability of several strains of Aspergillus to produce citric acid from galactose has been investigated. In the absence of methanol, very little production (less than 1 g/l) was observed. In the presence of methanol (final concentration 1% v/v), however, citric acid production and yeilds were increased considerably. Strong relationships were observed between citric acid production and the activities of the enzymes 2-oxoglutarate dehydrogenase and pyruvate carboxylase in cell-free extracts. During citric acid production, in the presence of methanol, the activity of 2-oxoglutarate dehydrogenase was low and that of pyruvate carboxylase high. In the absence of methanol, where little citric acid was produced, the reverse was true. It is suggested that the presence of methanol may increase the permeability of the cell to citrate, and the cell responds to the diminished intracellular level by increasing production via repression of 2-oxoglutarate dehydrogenase.     

Optimization of citric acid production by Aspergillus niger using two downgraded Algerian date varieties

In the present work, the GHARS and the MECH DEGLA downgraded date varieties were used in a fermentation medium in order to produce citric acid by the Aspergillus niger. The biochemical characteristics of the dates were investigated, along with the chemical and physical characteristics of the solutions of both samples. The analyzed parameters included the moisture and sugar content, the ash residual, the pH values, and the electrical conductivity. The effect of the following fermentation parameters was studied: initial pH, temperature, incubation period, and methanol. For the GHARS and MECH DEGLA date varieties respectively, the ash residual measured at 1.90% and 2.47%. For each date variety, the moisture and total sugars were measured at 11.59% and 85%, for the GHARS, and 12.82% and 80.47% for the MECH DEGLA. Citric acid production using either of the two varieties of dates showed a high yield in a short time.The obtained results showed that the highest production of citric acid by both medium of dates was achieved at the initial pH value of 3.0, temperature 30 °C, and an incubation period of 8 days. Also, the maximum amount of citric acid was produced when both mediums contained 4% of methanol. Both varieties of dates showed a good yield for the citric acid and can be used as a culture medium since they are economic and ensure good growth for the Aspergillus niger.     

Application of statistical experimental designs for the optimization of medium constituents for the production of citric acid from pineapple waste

Statistical experimental designs were applied for the optimization of medium constituents for citric acid production by Yarrowia lipolytica NCIM 3589 in solid state fermentation (SSF) using pineapple waste as the sole substrate. Using Plackett-Burman design, yeast extract, moisture content of the substrate, KH(2)PO(4) and Na(2)HPO(4) were identified as significant variables which highly influenced citric acid production and these variables were subsequently optimized using a central composite design (CCD). The optimum conditions were found to be yeast extract 0.34 (%w/w), moisture content of the substrate 70.71 (%), KH(2)PO(4) 0.64 (%w/w) and Na(2)HPO(4) 0.69 (%w/w). Citric acid production at these optimum conditions was 202.35 g/kg ds (g citric acid produced/kg of dried pineapple waste as substrate).     

Response surface optimization of medium components for citric acid production by Aspergillus niger NRRL 567 grown in peat moss

Aspergillus niger NRRL 567 was grown in an inert support material for citric acid production. Optimization of the medium components, including ethanol, methanol, phytate, olive oil and surfactant was carried out using "one-factor-at-a-time" and central composite design (CCD) methods. Optimization using "one-factor-at-a-time" was performed and the supplement of ethanol and methanol between 15 and 30 g/kg dry peat moss (DPM) enhanced citric acid production while higher levels than 30 g/kg DPM had an inhibitory effect on citric acid production at 48 and 72 h of incubation. Based on the results of "one-factor-at-a-time" optimization, phytate, olive oil and methanol were the selected additives to test the effect on citric acid production using CCD. The three variables were identified to have significant effects on citric acid production and the maximum citric acid production of 354.8 g/kg DPM was resulted from the combination of 19 g phytate/kg DPM, 49 g olive oil/kg DPM and 37 g methanol/kg DPM at 120 h. Maximum citric acid production in optimized condition by CCD represented about a 2.7-fold increase compared to that obtained from control before optimization.     

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     

Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose

Citric acid production by Aspergillus niger NCIM 548 and Candida lipolytica NCIM 3472 has been studied in shake culture using glucose and molasses as carbon sources. Methanol addition (3% v/v) at 40 h of fermentation enhanced the production of citric acid by Aspergillus niger whereas a reduction in citric acid production by Candida lipolytica was observed with addition of methanol. Maximum citric acid concentration of 12 kg/m³ was obtained with Aspergillus niger using molasses in the presence of methanol, while maximum citric acid concentration of 8.4 kg/m³ was obtained with Candida lipolytica using glucose without methanol. It appears that product formation by Aspergillus niger is either non-growth associated or partially growth associated depending on the substrate. Methanol addition changes the nature of product formation in case of Candida lipolytica.     

Stimulatory effect of alcohols (methanol and ethanol) on citric acid productivity by a 2-deoxy D-glucose resistant culture of aspergillus niger GCB-47

The present study describes citric acid fermentation by Aspergillus niger GCB-47 in a 15-1 stainless steel stirred fermentor. Among the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid (90.02 +/- 2.2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. The sugar consumed and % citric acid was 108 +/- 3.8 g/l and 80.39 +/- 4.5%, respectively. The desirable mycelial morphology was in the form of small round pellets having dry cell mass 14.5 +/- 0.8 g/l. Addition of ethanol, however, did not found to enhance citric acid production, significantly. The maximum value of Yp/x (i.e., 5.825 +/- 0.25 g/g) was observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after inoculation when the initial pH of fermentation medium was kept at 6.0.     

Citric acid production by solid state fermentation using sugarcane bagasse

A solid state fermentation (SSF) method was used to produce citric acid by Aspergillus niger DS 1 using sugarcane bagasse as a carrier and sucrose or molasses based medium as a moistening agent. Initially bagasse and wheat bran were compared as carrier. Bagasse was the most suitable carrier, as it did not show agglomeration after moistening with medium, resulting in better heat and mass transfer during fermentation and higher product yield. Different parameters such as moisture content, particle size, sugar level and methanol concentration of the medium were optimised and 75% moisture level, 31.8 g sugar/100 g dry solid, 4% (v/w) methanol and particles of the size between 1.2 and 1.6 mm were found to be optimal. Sucrose and clarified and non-clarified molasses medium were also tested as moistening agents for SSF and under optimised conditions, 20.2, 19.8 and 17.9 g citric acid /100 g of dry solid with yield of 69.6, 64.5 and 62.4% (based on sugar consumed) was obtained in sucrose, clarified and non-clarified molasses medium respectively, after 9 days of fermentation.     

Simultaneous Removal of Lindane,Lead and Cadmium from Soils by Rhamnolipids Combined with Citric Acid

This study investigated the performance of rhamnolipids-citric acid mixed agents in simultaneous desorption of lindane and heavy metals from soils. The capacity of the mixed agents to solubilize lindane, lead and cadmium in aqueous solution was also explored. The results showed that the presence of citric acid greatly enhanced the solubilization of lindane and cadmium by rhamnolipids. A combined effect of the mixed agents on lindane and heavy metals removal from soils was observed. The maximum desorption ratios for lindane, cadmium and lead were 85.4%, 76.4% and 28.1%, respectively, for the mixed agents containing 1% rhamnolipidsand 0.1 mol/L citric acid. The results also suggest that the removal efficiencies of lead and cadmium were strongly related to their speciations in soils, and metals in the exchangeable and carbonate forms were easier to be removed. Our study suggests that the combining use of rhamnolipids and citric acid is a promising alternative to simultaneously remove organochlorine pesticides and heavy metals from soils.     

Effect of substrate moisture content on fungal production of citric acid in a solid state fermentation system

Summary Under solid state fermentation conditions, the production of citric acid byAspergillus niger varied considerably with the initial moisture content of apple pomace. The stimulating effect of methanol on fungal production of citric acid decreased markedly as the substrate moisture decreased.     

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.     

Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger

 Mutants having impaired protein synthesis, that is cycloheximide-sensitive mutants of a citric-acid-hyper-accumulating strain, were induced from Aspergillus niger WU-2223L. Selection was on the basis of a presumption that the mutants should be more sensitive to cycloheximide than WU-2223L. In shake culture without methanol as a promotor substance, seven mutants accumulated approximately 1.8–3.5 times as much citric acid as WU-2223L. The best mutant, CHM I-C3, accumulated 69.4 mg citric acid/ml from 120 mg glucose/ml in shake culture without methanol, this amount being 1.1 times the amount accumulated by WU-2223L with methanol. Furthermore, under the conditions without methanol the mutants appeared to be more efficient than WU-2223L in employing the consumed glucose for the accumulation of citric acid. It was also confirmed that CHM I-C3 exhibited a significantly increased level of intracellular NH⁺ ₄ accumulation. The addition of 2% (v/v) methanol or 20 μg cycloheximide/ml to the medium caused a remarkable increase of citric acid accumulation by WU-2223L: about 3.1 and 2.4 times respectively. However, the addition of these substances produced negative effects on citric acid accumulation by the mutants. With 2% (v/v) methanol, WU-2223L showed a remarkably decreased level of protein accumulation but a substantially increased level of intracellular NH⁺ ₄ accumulation. However, these phenomena were also observed in CHM I-C3 without methanol. These results indicate that the intracellular circumstances of the cycloheximide-sensitive mutants without methanol were similar to those of WU-2223L with methanol, and that the impairment of protein synthesis contributed to increased citric acid accumulation by the mutants in the absence of methanol. Received: 21 November 1994 / Received last revision: 10 July 1995 / Accepted: 26 July 1995     

Improvement in citric acid production by haploidization of Aspergillus niger diploid strains

Segregation of diploid strains by a haploidizing agent was used to improve citric acid producing strains of Aspergillus niger. Stable diploid strains were obtained via protoplast fusion between two citric acid-producing strains from different genealogies, one for shaking culture and the other for solid culture. Diploid strains were treated by benomyl as a haploidizing agent, and many segregants were obtained. Prototrophic segregants were selected and their haploidy was confirmed by their conidial size and DNA contents. The prototrophic segregants were very variable in their citric acid productivities, some of them better either in shaking culture or in solid culture than both the parental strains. The presence of methanol stimulated citric acid production by the parental and the diploid strains. However, all prototrophic segregants derived from one diploid strain had higher productivities in solid cultures without methanol than in those with methanol.     

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.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

Earthworm Communities in the Pineapple (Ananus comosus) and Mixed Fruit Plantations of West Tripura, India

Present studies on the community characteristics of earthworms revealed the occurrence of 11 species of earthworms in the pineapple (Ananus comosus) and 14 species in the mixed fruit plantations of west Tripura (India). While 9 species of earthworms namely Drawida assamensis, Drawida papillifer papillifer, Drawida nepalensis, Kanchuria sp., Metaphire houlleti, Eutyphoeus gigas, Eutyphoeus scutarius, Eutyphoeus comillahnus and Pontoscolex corethrurus are of common occurrence to both the pineapple and the mixed fruit plantations, two and five earthworm species namely Kanchuria sumerianus, Eutyphoeus sp. and Metaphire posthuma, Perionyx excavatus, Lampito mauritii, Amynthus alexandri, Eutyphoeus gammiei are restricted to the pineapple and the mixed fruit plantations respectively. Earthworms were found mostly within 15 cm depth of soils having temperature 25–25.8 °C, moisture 18.8–22.4 %, water holding capacity 26–31.7 % and organic matter content 2.4–4.0 %. Mean earthworm densities (158 ind. m^?2) was significantly higher (p < 0.01, t = 9.67) and biomass (36.67 g m^?2) significantly lower (p < 0.01, t = ?5.98) in the pineapple plantation than the mixed fruit plantation (density 93 ind. m^?2, biomass 56 g m^?2). High density value of earthworms in pineapple plantation is linked with dominance of D. assamensis and high biomass value in mixed fruit plantation was due to the higher relative abundance of larger species like E. gigas, E. scutarius, E. comillahnus and E. gammiei. Compared to the mixed fruit plantation, significantly (p < 0.05) higher index of dominance, lower index of diversity, species richness index and species evenness were recorded in the pineapple plantation.     

Antimutagenicity of some citrus fruits in Salmonella typhimurium

The antimutagenic effect of 10 citrus fruit juices was observed against the mutagenicity of N-nitro-o-phenylenediamine (NPD) in TA97a and sodium azide in TA100 tester strains of Salmonella typhimurium using the Ames test. It was noticed that the juices of all these fruits reduced significantly the NPD and sodium azide induced revertant colonies. The inhibitory activity was enhanced if the mutagen and juice were co-incubated for about 30 min at 37 degrees C prior to performing the mutagenicity assay. Dilution with distilled water led to the reduction in the inhibitory activity. The antimutagenic activity of synthetic ascorbic acid or citric acid or combined ascorbic acid and citric acid was also seen. But the results with fruit juices tempted us to believe that in addition to ascorbic acid and citric acid, the presence of other factor(s) possessing antimutagenic properties cannot be ruled out.     

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.