Converting Orange Acreage in Beef Cattle
Braz J Microbiol. 2011 January-Mar; 42(1): 394–409.
Citric Acid Production from Orange Peel Wastes by Solid-State Fermentation
Ana María Torrado
oneDepartamento de Ingeniería Química. Universidad de Vigo (Campus Ourense), Edificio Politécnico. As Lagoas. 32004 Ourense, Spain
Sandra Cortés
1Departamento de Ingeniería Química. Universidad de Vigo (Campus Ourense), Edificio Politécnico. As Lagoas. 32004 Ourense, Spain
José Manuel Salgado
1Departamento de Ingeniería Química. Universidad de Vigo (Campus Ourense), Edificio Politécnico. As Lagoas. 32004 Ourense, Kingdom of spain
Belén Max
twoLaboratory of Agro-food Biotechnology, CITI-Tecnópole, Parque Tecnológico de Galicia, San Cibrao das Viñas, Ourense, Spain
Noelia Rodríguez
twoLaboratory of Agro-food Biotechnology, CITI-Tecnópole, Parque Tecnológico de Galicia, San Cibrao das Viñas, Ourense, Spain
Belinda P. Bibbins
twoLaboratory of Agro-food Biotechnology, CITI-Tecnópole, Parque Tecnológico de Galicia, San Cibrao das Viñas, Ourense, Spain
Attilio Converti
iiiSection of Chemical and Process Engineering, Genoa University, Via Opera Pia 15, 16145 Genoa, Italian republic
José Manuel Domínguez
1Departamento de Ingeniería Química. Universidad de Vigo (Campus Ourense), Edificio Politécnico. As Lagoas. 32004 Ourense, Spain
Received 2010 Jun 24; Revised 2010 Sep 24; Accepted 2010 November 3.
Abstruse
Valencia orange (Citrus sinensis) pare was employed in this piece of work every bit raw material for the production of citric acid (CA) by solid-state fermentation (SSF) of Aspergillus niger CECT-2090 (ATCC 9142, NRRL 599) in Erlenmeyer flasks. To investigate the effects of the main operating variables, the inoculum concentration was varied in the range 0.5·103 to 0.seven·108 spores/chiliad dry orange peel, the bed loading from ane.0 to 4.8 g of dry out orange peel (corresponding to 35-80 % of the full volume), and the wet content betwixt 50 and 100 % of the maximum water retentiveness capacity (MWRC) of the textile. Moreover, additional experiments were washed calculation methanol or water in unlike proportions and ways. The optimal weather for CA production revealed to be an inoculum of 0.5·10half dozen spores/k dry orange peel, a bed loading of 1.0 g of dry orange peel, and a humidification blueprint of lxx % MWRC at the start of the incubation with posterior addition of 0.12 mL H2O/g dry orange skin (corresponding to 3.3 % of the MWRC) every 12 h starting from 62 h. The addition of methanol was detrimental for the CA production. Nether these atmospheric condition, the SSF ensured an effective specific production of CA (193 mg CA/g dry orangish peel), corresponding to yields of production on total initial and consumed sugars (glucose, fructose and sucrose) of 376 and 383 mg CA/g, respectively. These results, which demonstrate the viability of the CA production by SSF from orange skin without addition of other nutrients, could be of interest to possible, future industrial applications.
Keywords: Orange peel, citric acid, Aspergillus niger, solid-country fermentation
INTRODUCTION
Today, citrus is unrolling in about all regions of the world within the strip bounded by a line of breadth 40 degrees Northward and S. The citrus processing industry yearly generates tons of residues, including peel and segment membranes, from the extraction of citrus juice in industrial plants. The management of these wastes, which produce odor and soil pollution, represents a major problem for the food manufacture (33). Orange pare contains soluble sugars and pectin as the main components. According to Rivas et al. (42), the orange peel is in fact constituted by soluble sugars, 16.9 % wt; starch, iii.75 % wt; fiber (cellulose, 9.21 % wt; hemicelluloses, ten.five % wt; lignin 0.84 % wt; and pectins, 42.5 % wt), ashes, three.50 % wt; fats, 1.95 % wt; and proteins, 6.50 % wt.
The total saccharide content of orange peel varies betwixt 29 and 44 % (21), soluble and insoluble carbohydrates beingness the most abundant and economically interesting constituents of this rest (26). Approximately 50 % of the dry weight of orangish is soluble in alcohol (47), and soluble sugars are the major components also of this fraction. Glucose, fructose and sucrose are the chief sugars, although xylose tin can besides be found in small quantities in orange skin. Insoluble polysaccharides in orange skin are composed of pectin, cellulose and hemicelluloses. Pectin and hemicelluloses are rich in galacturonic acid, arabinose and galactose, but they too contain small amounts of xylose, glucose, and perhaps rhamnose (xvi,33). Glucose is the dominant sugar in the cellulosic fraction, which also contains some quantities of xylose and arabinose, traces of galactose and uronic acids, and in some instances mannose. On the other hand, lignin seems to be absent in these tissues. Consequently, a mixture of cellulases and pectinases is needed to complete the conversion of all polysaccharides to monosaccharides (15,16).
Citric acid (CA), an intermediate of the tricarboxylic acrid cycle, is found in a multifariousness of acidic fruit juices, particularly in the citric ones, although its extraction from natural sources, primarily lemon, was gradually replaced by biological procedures, mainly based on the use of microfungi, which are currently the about widely used. The production of CA was described in 1893 by Wehmer as a result of the metabolism of the mucus Penicillium glaucum (49). In 1913, it was obtained the commencement patent in the United States for a method of producing CA past Aspergillus niger in carbohydrate solutions. Recent estimates put the global product of CA in over 1.4 million tons per year (49) with rise trend in need. More than 50 % of this book is being produced in Cathay. It is traditionally used in the food industry thanks to its loftier solubility, extremely depression toxicity, and palatability; moreover, examples are given of some recent CA applications in the industry of detergents and cosmetics, or equally the active ingredient in some bathroom and kitchen cleaning solutions (56).
The depression cost and the high carbohydrate content and susceptibility to fermentation make citrus byproducts attractive raw materials for CA biotechnological production (42). In almost cases, the industrial production of CA by fermentation is done using A. niger strains, but also many other microorganisms are capable of accumulating CA, including other species belonging the same genus, Penicillium janthinellum, Penicillium restrictum, Trichoderma viride, Mucor pirifromis, Ustulina vulgaris and various species of the genera Botrytis, Ascochyta, Absidia, Talaromyces, Acremonium and Eupenicillium (25). There are some processes that apply various species of yeast (mainly belonging to the genus Candida) or leaner and a wide range of carbon sources, including sucrose, glucose, molasses, alcohol, fatty acids, natural oils, acetate, and hydrocarbons (4). Additionally, some attempts have been made to induce CA overproduction by mutations of unlike microorganisms, particularly A. niger strains (31,46). Aravantinos-Zafiris et al. (5) examined iii unlike strains of A. niger and establish that the strain NRRL 599 was the best CA producer, followed past NRRL 364 and NRRL 567, respectively.
CA has been successfully produced using submerged, liquid surface or solid state fermentation (SSF), with the all-time results being obtained in this last case (36). In spite the SSF was the first process proposed for the production of CA using dissimilar absorbing materials (beet pulp, carbohydrate cane bagasse, pineapple pulp) with embedded solutions of carbohydrates (mainly sucrose-rich solutions), CA has been conventionally produced by submerged fermentation, mainly by A. niger. Nonetheless, considering of several advantages over the submerged fermentations such as solid waste matter management, biomass energy conservation, product of high value products and piffling risk of bacterial contagion (44), the SSF methods take recently gained attending using agroresidues similar sugarcane or cassava bagasse (29, 30, 38, 46), carob pod (44), areca husk (36), beet molasses (1), soy residues (27), sugar cane bagasse, coffee husk and cassava bagasse (55) and waste of nutrient processing industries including pineapple wastes (6, 11, xviii, 22, 52, 53), apple pomace (twenty, 48), grape pomace (19), or dissimilar fruit peels, including kiwi (17), orangish (43) or prickly pear (12).
The primary characteristics of SSF that differentiate it from submerged cultures are the low water content, which is usually related to depression values of h2o activity, especially for hydrophylic supports, and the enhanced aeration. The O2 and CO2 exchange between the gas phase and the substrate depends on the intra- and inter-particle mass transfer in SSF systems, which is influenced by various factors (8,xiv): a) the matrix porosity that depends on its physical characteristics and water content; b) the pore size and particle diameter that influence the surface area of interchange; c) the system geometry; and d) the aeration and the agitation, particularly when the fermentation is advanced.
Following a previous study (42), where the bioproduction of CA by A. niger NRRL 599 was studied in submerged civilization using a medium prepared later on sugars solubilization past orange peel autohydrolysis, in this piece of work we investigated the potential of such a residue as a substrate for CA production by solid-state fermentation by the same microorganism. To this purpose, we investigated the effects of inoculum concentration, bed loading, and water and methanol addition on CA production and civilization performance. In comparison to other related works, no nutrients were added to the fermentation broth in club to minimize the costs of production. Finally, the results of CA production past SSF were compared with those previously obtained in submerged culture.
MATERIALS AND METHODS
Raw cloth
Samples of Valencia orange (Citrus sinensis) skin obtained from a national citrus processing plant were dried at 40 °C to reach a final moisture lower than 10 %, milled to a particle size less than 2 mm, homogenized in a single lot to avert whatever variation in composition, and stored at 4 °C in a common cold bedchamber until use.
Microorganism
Aspergillus niger CECT-2090 (ATCC 9142, NRRL 599), obtained from the Spanish Drove of Type Cultures (Valencia, Spain), was used in this work.
Inoculum
The fungus was grown on slants of potato dextrose agar (Scharlau Chemie, Barcelona, Kingdom of spain) at 33 °C for 5 days. A spore inoculum was prepared past adding sterile distilled water to the slant, shaking vigorously for 1 min with the help of sterile glass balls to prepare the spore suspension, and filtering to eliminate mycelium particles. Spores were quantified by optical density measurement at 750 nm using a calibration curve.
Maximum h2o memory capacity
Earlier SSF, an experiment was carried out in triplicate to determine the maximum water retentivity capacity (MWRC) of the dry orange skin nether saturated weather condition, which resulted to be iii.vi ± 0.1 mL of h2o captivated per gram of dry out cloth. This upshot was taken into account when the liquid phase was added to the substrate to promote the microbial growth.
Culture media and sterilization
Stale and milled samples of orange peel were dispensed into 50 mL Erlenmeyer flasks provided with aluminum caps with 24-26 mm diameter, model Sero-Tap (Selecta, Abrera, Spain), without any additional nutrients. Different bed loadings from 1.0 to 4.8 g/Erlenmeyer were assayed according to the experimental design described later, which corresponded to minimum and maximum loadings of 35 and 80 % of the flask working volume, respectively. The cloth was moistened by two-step addition of the liquid stage. In a first step, and for all cases, 1.6 mL of h2o/yard, respective to 45 % of the MWRC, was added before sterilization to protect the cloth from thermal degradation. And so, the remainder of h2o necessary to accomplish the level of wet desired for each experiment was added together with the inoculum. Sterilization was made by autoclaving at 100 °C for i h.
Incubation
Flasks were incubated at 30 °C in a stationary incubator where a saturated NaCl solution allowed to maintain the level of air moisture needed to prevent drying of the material. Each flask was stirred every 24 h with a sterile spatula inside a laminar catamenia bedroom to reduce compactation and improvidence restrictions. All experiments were washed in triplicate.
In selected experiments performed with variable degrees of humidification, sterile distilled water was added at dissimilar rates and proportions, as indicated in Table one. For the study of the outcome of methanol on citric acrid production, experiments were done adding methanol at the beginning of the cultivation at variable proportions (0, 2, iv, and 6 % v/due west). Methanol, of analytical grade, was purchased from Sigma (Switzerland). In both cases, the content of each flask was shaken by a sterile spatula later on every addition.
Table 1.
Conditions of SSF with variable water addition.
| Runs | Amount added in each add-on (mL/yard) | Frequency of add-on (h) | Time of addition (h) | Number of additions | mL water/g dry out solid accumulated at the end of incubation | increase of % saturation at the end of incubation |
|---|---|---|---|---|---|---|
| A (command) | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 0.08 | 12 | 48 | 6 | 0.48 | 13.three |
| C | 0.12 | 12 | 48 | 6 | 0.72 | xx.0 |
| D | 0.12 | 12 | 62 | 5 | 0.lx | 16.seven |
Sampling
The whole content of a flask was used for each sample. The fabric was homogenized carefully with the help of a spatula or even of a mortar, particularly when the arable cell growth hampered the homogenization of the samples. Amounts of about 0.5-1.0 one thousand were used to determine the moisture content in oven at 105 °C. According to the sample consistency, aqueous extracts were obtained from the remaining sample past addition of distilled water upward to a 10:1 (5/w) ratio. The extraction was assisted mechanically with an Ultra-Turrax homogenizer, model T25 (IKA-Labortechnik, Staufen, Germany) for 30 s, and centrifugation at 8,000 rpm for 10 min to eliminate the solid particles.
Analytical methods
Samples of the aqueous extracts were filtered through 0.45 μm-pore membranes and assayed for glucose, fructose, sucrose, citric acid, and galacturonic acid concentrations past HPLC, model 1100 (Agilent, Palo Alto, CA) with a Refractive Index detector. Standards were prepared from the corresponding reagents purchased from Sigma (Switzerland). Separation was performed using a ION-300 column (Transgenomic Inc., San Jose, CA), thermostated at l °C and eluted with 0.01 M H2So4 at 0.iv mL/min flow rate. All analyses were carried out in triplicate, and the error was less than 3 %.
Total sugars (including neutral and acrid sugars) were analyzed past the method of Dubois et al., modified according to Strickland and Parsons (51), which is based on the phenol-sulfuric acid reaction that allows determining the reducing sugars after acid hydrolysis of polysaccharides. Glucose, from Sigma (Switzerland), was used as a standard.
Experimental design
The effect of the bead loading and h2o content on orangish peel SSF was studied past means of a 2d-lodge rotatable experimental design with α?= 1.414 and five replicates in the center of the domain, co-ordinate to Akhnazarova and Kafarov (2) and Box et al. (7). Experimental domain and coding criteria are given in Tabular array ii. The significance of the coefficients of the models was calculated using Educatee's t exam (α?< 0.05) as the acceptance criterion. Model consistency was verified by Fisher's F test (α?< 0.05) applied to the following mean foursquare (QM) ratios:
-
model/full error (QMM/QME) (F ≥ Fden num)
-
(model + lack of fitting)/model (QM(M + LF)/QMM) (F ≤ Fden num)
-
total error/experimental mistake (QME/QMEe) (F ≤ Fden num)
-
lack of plumbing fixtures/experimental error (QMLF/QMEe) (F ≤ Fden num)
Table two.
Experimental domain and codified of the independent variables analyzed by means of a second-order rotatable factorial blueprint applied to citric acid production by SSF on orange peels.
| Actual values | |||
|---|---|---|---|
| Coded values | Water content(% of saturation) | H2o content(mL/g dry out solid) | Bed loading(g/flask) |
| -1.414 (-α) | 50.0 | 1.lxxx | 1.0 |
| -1 | 57.three | two.06 | 1.half-dozen |
| 0 | 75.0 | 2.seventy | 2.ix |
| +1 | 92.7 | three.34 | 4.2 |
| +1.414 (+α) | 100.0 | 3.60 | 4.viii |
RESULTS AND DISCUSSION
Preliminary experiments: inoculum concentration
A preliminary fix of solid-state fermentations was performed using orangish skin equally substrate and Aspergillus niger at different inoculum concentrations. The objective of these runs was to evaluate the suitability of this organisation for citric acid production and to get a start insight of the kinetics of this fungus on this substrate in view of hereafter optimization of this production.
Considering the aeration requirements of this microorganism, a low bed loading of ii g of dry out orange skin (corresponding to 46.8 % of the flask working book) was selected as a status of reduced depth of the matrix in the flask able to provide an aerobic environment suitable for cell growth. Taking into account that inoculum concentrations in the range 10iii-ten8 spores/yard substrate are usually employed for CA production by A. niger (32,45) and that an increase in the inoculum level is well known to reduce the lag stage, iii different spore concentrations were tested, namely 0.5·10iii, 0.5·106 and 0.7·108 spores/k dry orange pare, the concluding existence the maximum value that could be easily achieved using the procedure described in Materials and Methods. The solid was moistened to reach 75 % saturation, and incubations were done at 30 °C.
The results illustrated in Fig. one demonstrate the feasibility of using orange skin as a substrate for CA product by A. niger by SSF, with no need of supplying whatsoever additional nutrient and using the sterilization as the only pretreatment. Moreover, they clearly show that the intermediate inoculum level (0.5·106 spores/chiliad of orange peel) ensured the highest product concentration (170.5 mg of CA/g dry orangish peel).
Effect of the inoculum concentration on CA product from orange pare by A. niger SSF. Inoculum concentration (spores/g dry out orangish peel): 0.five·103 (?), 0.v·10half dozen (A), 0.5·tenviii (?). Results are the average of three contained experiments. Standard deviations were beneath two.ii % of the hateful.
It should exist noted that the lag stage was very long at the lowest inoculum level (0.5·103 spores/thousand of dry out orangish pare). Both the consumption of sugars and the production of CA (Fig. 1) did in fact start no sooner than 60 h. As a consequence, after 86 h the CA concentration was only 65 mg/g dry orangish pare, and a large portion of sugars remained unconsumed in the medium. Also the highest inoculum level (0.seven·108 spores/grand dry orange peel), used with the aim of accelerating the fermentation, revealed to be unadvisable from an industrial point of view, owing not just to the long time required to produce CA, but also to the concluding CA concentration reached (100.4 mg/g dry orange pare subsequently 72 h), which was considerably lower than that obtained at the intermediate inoculum level.
In social club to go a more detailed view of the kinetics, Fig. two illustrates the evolution of the solubilized sugars and CA production during the best run (0.five·106 spores/g of orange peel). It is noteworthy the occurrence of two distinct phases of the culture, determining a fermentation pattern quite different from the trophophase-idiophase one unremarkably observed in submerged civilisation. A first phase, which occurred approximately between 23 and 47 h, was characterized by a) a low charge per unit of CA product coinciding with the disappearance of sucrose, b) an initial increase in glucose and fructose concentrations as the mainly result of sucrose hydrolysis at higher charge per unit than its metabolization, and c) the cyberspace solubilization of sugars. A second one corresponded to an intense CA production and the net consumption of sugars.
In addition to sucrose, glucose and fructose, also pectins were analyzed since they are the main component of orange pare (42). Pectins are a heterogeneous group of acidic structural polysaccharides, consisting mainly of galacturonic acid units. There are many references describing the ability of this microorganism to produce pectinases that catalyze the fractional or full hydrolysis of pectins, leading to their solubilization and the release of galacturonic acrid. The production of pectinases is referenced either in solid-country or in submerged cultures (34,50), and even in semisolid lemon pulp culture (ten), which is like to the orange pare SSF. Information technology is also described the production of pectinolytic enzymes by A. niger fifty-fifty on materials with low pectin content including wheat bran and soy (9). Whereas the addition of sucrose, glucose, or galacturonic acid reduced the production of pectinases in submerged cultures, during SSF the improver of these sugars even increased their levels in the broth (50).
With the aim of evaluating the possible solubilization and hydrolysis of pectins from orange peel cultures, the concentration of total sugars (TS) was followed throughout the fermentation by the phenol-sulfuric method that allows quantifying neutral and acidic sugars from pectins together (Fig. 2). It tin can be observed that TS was really college than the sum of neutral sugars determined by HPLC. Such a difference became more evident at 48 h, when TS achieved a maximum value coinciding with the beginning of the internet consumption of glucose and fructose and the intensive product of CA, which suggests the occurrence of a meaning solubilization of pectines. After 48 h, TS decreased drastically together with the levels of neutral monosaccharides. Nevertheless, the rate of TS reduction was lower than that of neutral sugars, reflecting a gradual aggregating of pectins and/or their products of hydrolysis. Co-ordinate with that, galacturonic acid (GA) started to accrue progressively in the medium coinciding with the increase of total sugars at 48 h and until the cease of the culture. These data seem to provide an indirect confirmation of the power of A. niger to produce pectinases in these weather condition.
Time behavior of sugars consumption, CA product and galacturonic acid release during SSF of orange peel by A. niger using an inoculum concentration of 0.v·10six spores/g dry orangish peel. Sucrose (◊), glucose (△), fructose (□), sucrose + glucose + fructose (×), total sugars (○), galacturonic acrid (▲), citric acid (•). Results are the average of 3 independent experiments. Standard deviations were below ii.iv % of the mean.
Finally, it must be highlighted the progressive acidification of the medium during these runs from nearly iv.v to approximately 2.7, as the consequence of CA accumulation. Although A. niger has higher tolerance to low pH than other microorganisms, backlog acidification is well known to affect both growth and CA production. Rivas et al. (2) did in fact plant an increase in the CA production from four.9 to 8.3 k/L when the pH was controlled by the addition of CaCOiii during the submerged culture of this microorganism. On the opposite, the command of pH during the SSF procedure is difficult; therefore, this variable was only monitored in this report.
CA yields accomplished in SSF of fig fruits practically remained constant when initial pH values ranged from iv to 8 (45), whereas Selvi et al. (46) found the all-time results in terms of CA yield using sugarcane bagasse as a substrate at initial pH of iv. Withal, information technology has been reported that CA is accumulated in significant amounts but when the pH is below 2.5 (28). Although the reasons for the requirement of a low pH are not clear, information technology is known that at pH>4 the gluconic acid produced by the reaction catalyzed past glucose oxidase accumulates at the expense of CA (xl). Moreover, due to its extracellular localization, this enzyme is directly susceptible to the external pH and is inactivated at pH < iii.5 (57). On the footing of these considerations, we think that the low pH values reached in this work likely favored the CA production.
Influence of the bed loading and water content
The O2 supply is a limiting factor of submerged aerobic cultures considering, due to its low solubility in water, its concentration in a saturated aqueous medium is usually lower than the microorganism requirements. This aspect is of a great concern for CA product because A. niger is an aerobic microorganism, and the oxygenation is essential for its growth (35). Although in SSF the oxygen availability is many orders of magnitude college than that plant in submerged cultivations, several reports demonstrated the importance of aeration also in SSF. For case, even though forced aeration at the beginning of the solid-land fermentation of Kumara past A. niger favored the CA production in a packed-bed reactor compared to flasks, besides high air menses rates exerted adverse shear stress to the fungus (32). Vandenberghe et al. (55) reported that an air menstruum rate of lx mL/min improved cassava fermentation by A. niger, achieving 265 1000 citric acid/kg dried cassava.
According to Lu et al. (32), the bed loading (B) is the most of import cistron affecting the CA production by SSF because it influences the degree of aeration in the arrangement. It is too related to heat transfer, which is afflicted past higher restrictions in the solid land than in submerged cultures. Optimal B is therefore necessary to ensure the suited supply of oxygen and heat exchange necessary for efficient growth and CA production.
The water content (Due west) of the medium is another important operating variable for CA production by SSF considering information technology influences growth and metabolism of the microorganism also every bit the mass transfer phenomena primarily related to the diffusion of nutrients, oxygen and toxic metabolites (41). In addition, it causes swelling of the substrate, facilitating the penetration of the mycelium for its effective utilization (39), and affects rut transfer since its molecules occupy the interparticular spaces and/or causes aggregation of the solid particles. Therefore, the optimal moisture content depends on the specific requirements of the microorganism, the desired production, and the nature of the textile, with item concern to its hydrophilicity and porosity.
Because all of that, a 2nd order factorial design was done, every bit described in Materials and Methods, to better investigate the effect of these two operational variables on CA production. The best inoculum concentration (0.5·106 spores/g dry orange peel) was applied, and the fourth dimension of incubation was fixed at 62 h to avert possible substrate limitations under some of the tested weather.
As a result, the post-obit significant model (Table three), expressed in codified values, was obtained:
CA (mg/g) = 92.0 − v.5W − nineteen.0B − 9.0 W2 + 7.1B2,
[1]
whose response surface within the experimental domain is illustrated in Fig. 3.
Table 3.
Experimental results and assay of variance of the model describing the effect of the water content (W) and the bed loading (B) on SSF citric acid (CA) production by A. niger on orange peels. (SS: sum of squares, FD: liberty degrees, QM: mean squares).
Response surface obtained from equation [1], showing the result of the bed loading (B) and the h2o content (W) of the solid on the citric acid (CA) product from orange peel by SSF.
The absolute values of the coefficients of the equation, representing 44 % of the absolute value of the independent term, confirm, starting time of all, the stiff outcome of these two operating variables considered. The maximum predicted response (133.eight mg CA/chiliad), corresponding to codified values of W=-0.305 and B=-1.414 and natural values of W=70 % of saturation and B=ane g/flask, was in fact two.5-fold the minimum predicted one (53.five mg CA/k).
Considering now the behaviour of these two variables inside the experimental domain (Fig. three), it is possible to make a mechanistic interpretation of the model in terms of the real variables: aeration and water action (aw). The convex profile of the CA response surface with regard to W, with a maximum inside the domain, reflects the demand of increasing the amount of water to ameliorate aw and, consequently, fungal metabolism and nutrients diffusion. From this maximum threshold, aeration restrictions become relevant equally a result of the subtract of the substrate porosity and the interparticular void space.
With respect to B, and taking into account the influence of this operating variable on gas transfer, the high value of the offset social club B term indicates the very important effect of aeration on this organization and reduces the relative importance of the quadratic term.
Water addition during incubation
Although the model allowed enhancing CA production at 62 h of incubation, at this time there were all the same sugars remaining that could lead to higher levels of CA if the culture were continued until substrate depletion. When thinking about prolonging the fourth dimension of incubation in solid state cultures, it must be taken into account that h2o activity can decrease as a consequence of water evaporation and consumption by the fungus metabolism, and reach low and harmful levels. As a issue, the optimum W value defined by the model for 62 h of incubation could not be directly extrapolable to longer times.
To allow the incubation to keep without water limitations in order to consume the substrate and increase CA production, it seemed necessary to supply h2o forth the culture without affecting negatively the aeration by an excess of the liquid. Consequently, four fermentations were performed starting at the best atmospheric condition divers by the model (W=70 % of saturation and B=1 g/flask) and adding water in different proportions and rates, as specified in the Materials and Methods section, whose results, in terms of CA production, are illustrated in Fig. 4.
Evolution of CA throughout SSF after water addition: A) control without addition of water (υ); B) addition of 0.08 mL/flask every 12 h starting from 48 h (?); C) addition of 0.12 mL/flask every 12 h starting from 48 h (?); and D) addition of 0.12 mL/flask every 12 h starting from 62 h (A). Results are the average of three independent experiments. Standard deviations were beneath 2.ane % of the mean value.
The highest CA production (193.2 mg CA/grand dry) was obtained at 86 h of incubation adding water every 12 h starting from 62 h (run D), a value 17 % higher than that obtained in the reference run (A). Moreover, it is interesting to highlight that, in spite of the negative event at 62 h on CA production expected in serial C past the utilize of W values higher than the optimum predicted past the model, the productions of run C and D at 86 h were practically ancillary, as the likely consequence of backlog water evaporation at this fourth dimension. However, run B was the worst i e'er, probably due to an backlog of h2o at 62 h and to an bereft water content at longer incubation times. These results strongly propose the need of calculation water progressively along the SSF and then controlling carefully the maintenance of adequate aw and aeration.
Effect of methanol (MeOH) improver on citric acrid production by SSF
The addition of methanol at low concentrations can improve the yield of citric acid in cultures of A. niger, although contradictory effects have been reported. For example, Zhang (59) added 2 % MeOH to the solid residue of an orangish juice factory in fermentations carried out past A. niger 999. Meanwhile, Kang et al. (24) found the optimal atmospheric condition in terms of maximum yield of citric acid (fourscore.4 %) from skins of mandarin past A. niger, using semisolid fermentations, with the addition of 0.ii % NHfourNO3, 0.one % of MgSOiv·7H2O, 2.5 % MeOH or i.v % of ethanol. Tran et al. (52), obtained the highest CA yield (194 g/kg) adding 3 % methanol and 5 ppm de Iron2+ during fermentations conducted using pineapple waste matter and A. niger ACM 4992. De Lima et al. (11) added 4 % methanol using A. niger ATCC 1015 and pineapple waste in solid-state fermentation to achieve the highest CA production (132 g/kg). Recently, Rodrigues et al. (43) obtained the all-time results (445.4 grand of CA/kg of citric pulp) with sugarcane molasses and 4 % methanol (5/w) in SSF by A. niger LPB BC mutant. Finally, Roukas (44) reported that the addition of 6 % (westward/w) methanol into the substrate increased the concentration of citric acid from 176 to 264 g/Kg dry pod during SSF. The same stimulatory effect of methanol was observed by Kumar et al. (30) using a mixture of different fruit wastes and bagasse in SSF by A. niger DS1. Conversely, some authors have reported a decreased synthesis of CA after methanol addition. For instance, Hang et al. (17) observed that the supplementation of 0.74 mmol methanol/L diminishes the CA production during the SSF of kiwifruit skin by A. niger ATCC 9142, and Tsay and To (54) reported that methanol inhibited mycelial growth of A. niger TMB 2022 as well as CA product. Similar findings were reported by Navaratnam et al. (37), Ali et al. (three) and Xie and West (58).
In a previous study, Rivas et al. (42), adding 4 % (v/w) methanol to an orangish peel aqueous extract as culture medium, increased 20-fold the maximum CA production with the same strain of Aspegillus niger in submerged fermentation, although this was accompanied by an increase in the duration of the lag phase. To study the possibility of improving the product also in this organisation, 4 fermentations were done in the presence of methanol nether the all-time atmospheric condition previously defined by the model, with water add-on according to the protocol earlier defined for run D. To this purpose, methanol was added at the beginning of the incubation and after sterilization in proportions (0, 2, 4, and 6 % five/w) ordinarily applied in submerged and solid state cultures (18, 43). Even so, in all the cases methanol had a negative consequence on the cultivation, leading not only to an increase in the lag time, but too to a potent decrease in the maximum CA production for fermentations with 4 % and 6 % (v/west) methanol.
These results advise that the positive effects attributed to methanol due to the reduction of the toxicity of some metal ions, the positive amending of the cell wall and the membrane, and the modification of the fungal morphology (23), are not relevant in this system. The intrinsic event of the weather defined by the solid state culture system on fungal morphology and metabolism could explain the lack of an important favorable effect of methanol on CA production in this case. On the other hand, the depression values of aw that usually narrate the solid state cultures performed with low water contents and hydrophilic supports as orange skin, could have emphasized the methanol toxicity as a consequence of a loftier local methanol activity.
Comparison between SSF and submerged culture for CA production
Recently, there accept been an increasing number of reports on the use of SSF processes because they exhibit a series of advantages over submerged fermentations. Since the culture conditions are more similar to the natural habitat of filamentous fungi, these are in fact able to abound and excrete big amounts of hydrolytic enzymes and, consequently, product concentrations after extractions are usually higher, and the amounts of liquid and solid wastes generated are lower (sixteen, 17, 30). Furthermore, in SSF the caste of aeration is higher, the depression h2o action reduces the risk of bacterial contaminations, and the energy requirements are lower. On the other hand, SSF also shows some disadvantages such equally a greater claiming for control of some important operating variables such as pH and temperature.
In a previous work, Rivas et al. (42) submitted orange peel to autohydrolysis at 130 °C at liquid/solid ratio of 8.0 g/g. Without boosted nutrients, 40 g of the liquors generated were employed equally media for submerged CA production by A. niger in 100 mL-Erlenmeyer flasks at thirty °C and 200 rpm. Tabular array 4 allows comparison the results of the best fermentations of dry orangish peel in terms of CA produced by the aforementioned strain in submerged fermentation (in the presence of 40 mL/kg methanol and twenty yard/50 calcium carbonate) and in SSF (Due west = 70 %; B = 1 g; addition of 0.12 mL H2O/Erlenmeyer flask every 12 h starting from 62 h). All the information refer to the fourth dimension of the highest CA concentration in each fermentation. The first remarkable differences between these two civilisation systems were the dissimilar land of the substrate and the carbohydrate availability, which made unavoidable the use of dissimilar units. Yet, to make easier the comparing betwixt them, the concentrations of sugars and CA in the submerged culture were expressed as mg per g of dry orange peel considering the early on-mentioned 8.0 g/k water/solid ratio employed for preparing the extract. All the sugars contained in the solid substrate were available for SSF, whereas merely those solubilized by autohydrolysis were and so for the submerged fermentation. Thus, sucrose was the well-nigh arable saccharide in the SSF and fructose in the submerged one. It is also important to notice that the authoydrolysis led to more diluted fermentation media in comparison with SSF.
Tabular array 4.
Comparing of the primary results of submerged fermentation and SSF of dry out orange peel by A. niger.
| Reference | Submerged civilization (g/L)Rivas et al., 2008 | SSF (mg/g)This piece of work |
|---|---|---|
| Initial sucrose | 6.6 | 156.6 |
| Initial glucose | ix.half-dozen | 137.3 |
| Initial fructose | 13.6 | 145.2 |
| TS0 | 29.viii | 439.1 |
| Residuum sucrose | 0.0 | 0.0 |
| Residuum glucose | 4.1 | 7.6 |
| Residual fructose | 8.3 | 0.0 |
| Total residual sugars | 12.4 | 7.half dozen |
| TSc | 17.4 | 431.5 |
| CaCOthree | 20 | - |
| Methanol | 40 | - |
| Fermentation time (h) | 72 | 85 |
| Citric acid | nine.2 | 223.two |
| Y TSc (1000 citric acid/g TSc) | 0.53 | 0.52 |
| Y TSo (g citric acid/g TS0) | 0.31 | 0.51 |
Some other notable difference betwixt the two methods concerned the time behavior of the concentrations of the different sugars. In both cases sucrose was the starting time sugar to disappear. However, while in submerged culture glucose and fructose were gradually consumed throughout the cultivation, the levels of monosaccharides in SSF increased slightly during sucrose depletion. Several species belonging to the genus Aspergillus are reported to hydrolyze extracellular sucrose to be used later every bit a carbon source in the form of glucose and fructose (13). These results suggest that the rate of sucrose hydrolysis was higher than that of consumption of the resulting monosaccharides, thus leading to their net accumulation at the kickoff of cultivation in the case of the SSF.
The SSF and the submerged cultures also differed markedly in terms of the yield of CA per gram of dry orange peel, which reached a value in SSF (193.2 mg/m dry orange peel) near iii-fold that observed in submerged culture (73.half dozen mg/g dry out orange pare). In addition, it was comparable with those reported for dry carob pod (176 mg/grand) (44) and higher than for kiwifruit peel (100 mg/k) (17), and dry out fig (64 mg/thousand) (45).
On the other hand, the yields of CA referred to saccharide consumption (Y TSc = 0.61 and 0.58 g/1000 in SSF and submerged culture, respectively) were comparable for the ii systems, suggesting that the metabolic dysfunctions responsible for CA accumulation were ensured to the same extent in both culture modalities. However, the potential of SSF is highlighted past the fact that no methanol is required to stimulate CA production as for the submerged culture (42). This seems to be confirmed past the values of the yield referred only to the neutral sugars available at the showtime. This parameter was in fact essentially lower in submerged civilization (Y TSo = 0.32 m/thou) than in SSF (Y TSo = 0.59 g/k), likely due to the pause of CA production when sugars were not yet completely consumed.
CONCLUSIONS
The results of this written report point out the viability of Valencia orange (Citrus sinensis) peels as substrate for the production of citric acrid (CA) by Aspergillus niger CECT-2090 in solid-country fermentation (SSF). Compared to previous results obtained in submerged culture, the SSF proved to exist very versatile and did not need whatsoever additional nutrients or treatment too sterilization. The highest CA concentration (193.ii mg/thousand dry orange skin) was obtained at 85 h of incubation using an inoculum concentration of 0.v·x6 spores/g of dry orange peel, a bed loading of one.0 one thousand/Erlenmeyer, an initial water content of two.52 mL/chiliad of orange peel, corresponding to a seventy % saturation, and a water addition of 0.12 mL H2O/Erlenmeyer flask every 12 h starting from 62 h. Methanol improver did not testify to improve CA production. During SSF the microorganism likely produced pectinases, but pectins were not metabolized in any appreciable extent. Finally, SSF ensured yields of product on full initial sugars and consumed sugars of 0.59 g CA/1000 TS0 and 0.61 grand CA/k TSC, respectively. These results are considerably ameliorate than those previously obtained in submerged culture.
ACKNOWLEDGMENTS
Nosotros are grateful to the Spanish Authorities (project CT Q2006-02241/PPQ), which partially financed this work through the FEDER funds of the European Spousal relationship, the FPI grant to Belén Max and the MAEC-AECID grant to Belinda P. Bibbins.
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