Citric Acid Production by Aspergillus Niger Grown on Orange Peel Medium Fortified with Cane Molasses

Ann Microbiol (2013) 63:267–278 DOI 10.1007/s13213-012-0470-3

ORIGINAL ARTICLE

Citric acid production by Aspergillus niger grown on orange peel medium fortified with cane molasses

Hossam S. Hamdy

Received: 20 January 2012 /Accepted: 13 April 2012 /Published online: 8 May 2012 # Springer-Verlag and the University of Milan 2012

Abstract Citric acid production (CAP) by Aspergillus niger 2011b). This acid possesses many important qualities, such was obtained following culture on an orange peel medium as palatability, high solubility and an extremely low toxicity to (OPM) fortified with cane molasses. The key physico- human and mammals (Rodriguez et al. 2009). The global chemical parameters influencing CAP, such as bed loading, production of CA has recently been estimated to be over 1.4 moisture levels, volume and age of inoculum, initial pH, million tons per year (Kamzolova et al. 2011b), and its con- incubation temperature and duration, agitation rate, sugar sumption is escalating gradually, witnessing a high annual concentration, addition of nitrogen and phosphorus sources, growth rate due to more advanced and versatile applications, treatment of molasses and the addition of different low levels such as in the food manufacturing, detergent and cosmetics of alcohols, were assessed. The suitability of molasses to industries (Wang and Liu 1996; Gurpreet et al. 2011). In increase the concentration of sugar in the fermentation medi- addition, CA and its salts are used extensively in medicine as um without previous treatments with EDTA or ferro-cyanide ingredients in buffers and pharmaceutical syrups (Kamzolova was indicated. Maximum amounts of CA (640 g/kg orange et al. 2008). peel) were obtained after 72 h of incubation on an OPM Traditional methods for extracting CA from natural fruits, moisturized to 65 %w/v, with bed loading of 20 %, an initial but especially lemons, have gradually been replaced by bio- pH of 5, a temperature of 30 °C, an agitation rate of 250 rpm, production technologies. The first patent for CA production with fortification of the medium with molasses at a final sugar (CAP) by Aspergillus niger utilising sugar solutions was concentration of 14 % in the presence of 3.5 % methanol. reported in 1913 (Torrado et al. 2011). Traditionally, different strains of fungi, but especially A. niger, Penicillium Keywords Citric acidproduction .Orangepeels .Solid-state janthinellum, P. restrictum, Trichoderma viride, Mucor pir- fermentation . Optimization of cultural conditions ifromis and Ustulina vulgaris, have been used for CAP from molasses, sucrose or glucose (Kapoor et al. 1983; Gurpreet et al. 2011; Torrado et al. 2011). Recently, however, both Introduction natural and transformed strains of Yarrowia lipolytica have been shown to be capable of producing CA when cultivated Citric acid (CA) is an intermediate of the tricarboxylic acid on various traditional and renewable carbon sources, such as cycle (TCA) and holds a key position in the metabolism of all glucose, ethanol, methanol, n-alkanes, plant oils and glycerol- microbial cells. However, under certain conditions of drastic containing wastes of biodiesel industry (Papanikolaou et al. nutrient imbalance, fungi, yeast, and bacteria produce CA in 2002; Papanikolaou and Aggelis 2009;Gurpreetetal.2011; excessive amounts (Soccol et al. 2006; Kamzolova et al. Kamzolova et al. 2011a, b). Considerable interest has also focused on the utilisation of H. S. Hamdy (*) various agro-industrial wastes and their by-products in CAP, Biological Sciences & Geology Department, Faculty of Education, including pineapple waste, bagasse of sugarcane and cassava, Ain Shams University, molasses of sugarcane, beet and soy, fruit pulp and peels, Roxy, Heliopolis, Cairo 11757, Egypt especially of citrus fruits and lemons, corn cobs, apple and e-mail: [email protected] grape pomace, industrial solid potato wastes, glycerol- 268 Ann Microbiol (2013) 63:267–278 containing biodiesel waste, cooking olive oil waste and GRAS (generally regarded as safe) by the U.S. Food and Drug wheat bran (Kumar and Jain 2008; Rodriguez et al. Administration (Schuster et al. 2002). 2009; Kareem et al. 2010; Afifi 2011; Kamzolova et al. The aim of the study reported here was to investigate the 2011a, 2011b; Papanikolaou et al. 2011). However, despite potential of A. niger to produce CA by utilising OP fortified the fact that the agricultural wastes and industrial by-products with a low-cost carbon source, such as molasses, and opti- are rich in nutrients and good substrates for both the growth mising the key physico-chemical parameters for CAP. and activity of microorganisms, they usually require pre- treatments, such as alkali or acid hydrolysis (Khosravi-Darani and Zoghi 2008), or the addition of various carbon and nitro- Materials and methods gen sources (Rodriguez et al. 2009; Kareem et al. 2010). Citrus wastes, however, especially orange peel (OP), do not Materials and microorganism require such pre-treatments because they contain a negligible amount of lignin and considerable amounts of economically Aspergillus niger van Tiegh 1867 (LEG; MB284309) was important fermentable constituents, such as soluble sugars, previously isolated from the peel surface of the orange fruit starch, cellulose, hemicellulose, pectin, fats and proteins and identified by the CBS-KNAW (Fungal Biodiversity (Rivas et al. 2008). Moreover, OP has also been used in Centre, Utrecht, The Netherlands). The fungus was main- various biotechnological applications to produce animal feed, tained on a potato dextrose (PD) agar medium and was fertilisers, essential oils, pectin, ethanol, methane, single-cell routinely sub-cultured every other week. proteins and industrial enzymes, such as macerating enzymes OP was obtained from fresh mature Navel orange fruits and pectin-lyase (Hamdy 2005;Joseetal.2010). purchased from a local market. The peels were air dried, The utilisation of inexpensive agro-industrial wastes and then oven dried at 55 °C for 24 h until a constant weight was their by-products in solid-state fermentation (SSF) systems achieved. The dried OP was then finely ground and passed is a potential route for CAP in comparison to submerged through a 50-μm sieve. The powder was stored in dry flasks liquid fermentation systems. There are many advantages to under dark conditions at room temperature. Maximum water the former, including the low risk of bacterial and yeast retention capacity (MWRC), which is a measure of the contamination, biomass energy conservation and the presen- maximum amount of water absorbed by the dry peel, was tation of a similar natural habitat for the microorganism, determined using distilled water and was found to be 35 % enabling higher yields, low energy requirements and low (v/wt). levels of polluting effluents (Gurpreet et al. 2011; Mienda et OP was analysed for cellulose, hemicellulose and pectin al. 2011). content, as described by Jermyn (1955). Total sugars, in- Aspergillus niger was selected for this study due to the cluding neutral and acid sugars, were determined using the easy manipulation, low cost of fermentation, and capacity of method of Dubois et al. (1956), while glucose and fructose this species to produce a high yield of CA (Vandenberghe et were determined using the anthrone method (Bonting 1954) al. 1999). Its high potential for converting various raw materi- (Table 1). als to valuable products, such as lipase, oxalic acid and mi- Cane molasses was kindly supplied by the Sugar Distill- crobial lipids, has been demonstrated in previous studies ery Company of Egypt and treated as described by Panda et (Ruijter et al. 1999; Edwinoliver et al. 2009;Papanikolaou al. (1984): the initial sugar level was diluted from 52 (w/v) et al. 2011). In addition, CA and many other products of A to 25 %, boiled for 30 min with 1 N H2SO4 (3.5 %, v/v), niger have been in use for many decades and are considered cooled, neutralised with lime-water (CaO) and left to stand

Table 1 Chemical composition of orange peel

Chemical compositiona

Insoluble sugars Soluble sugars (SS) Protein and nitrogen

Cellulose Hemicellulose Pectin Lignin Glucose Fructose Total SS Soluble Insoluble Total Total (TSS) nitrogen nitrogen nitrogen protein

Residual 17.3 13.5 37.8 0.2 12.4 10.5 32 0.38 1.64 2.02 12.62 yield (%) a Cellulose, hemicellulose, pectin and lignin contents were determined as described by Jermyn (1955). TSS were determined as described by Dubois et al. (1956), while glucose and fructose were determined using the anthrone method (Bonting 1954). Total nitrogen was estimated using the conventional micro-Kjeldahl method (Lexander et al. 1970; Pirie 1955) Ann Microbiol (2013) 63:267–278 269 overnight for clarification. The treated molasses was pre- Determination of CA pared for use by adding EDTA and ferro-cyanide to the clarified molasses at a final concentration of 200 ppm (Ali The fermented material of each flask was made up to 100 ml et al. 2002). with distilled water and vigorously shaken for 15 min; the All chemicals used were of analytical grade in order to content was then filtered through Whatman no. 1 filter paper avoid the possible interference of metals. supported by a Buchner funnel. CA was determined by titration against 0.1 N NaOH (freshly prepared) using phe- nolphthalein as an indicator (AOAC 1995), followed by Citric acid production spectrophotometric determination using the modified acetic-pyridine method with a sensitivity of 25 μgCA Triplicate sets of 250-ml Erlenmeyer flasks containing bed (Marier and Boulet 1958; Imandi et al. 2008). Pyridine loads of 10 g OP moisturised to 50 % of MWRC with (1.3 ml) was added to the colorimetric tubes containing distilled water were sterilised by autoclaving at 121 °C for either 1 ml of sample, CA as a standard or water as a blank, 15 min. The orange peel medium (OPM) was then inocu- followed by the addition of 5.70 ml of acetic anhydride. lated with mycelial pellets or a mycelial mat of A. niger. Tubes were immediately placed in an oven at 32 °C for 30 Mycelial pellets and mats were prepared by inoculating 250- min to allow the development of colour, which remains ml Erlenmeyer flasks containing 50 ml of sterilised PD stable for 30 min. Absorbance was determined at 420 nm medium with 1 ml of a 7-day old spore suspension and CA amounts were calculated and expressed as grams (107spore/ml, estimated using a haemocytometer). The CA per kilogram OP. The CA content of a non-inoculated flasks were incubated at 30±1 °C for different lengths of OPM was used as the control. The accuracy of the results time (12–60 h). The flasks containing the mycelial pellets was confirmed by comparison to results obtained by high- were shaken at an agitation rate at 150 rpm in a GFL orbital performance liquid chromatography [HPLC; standard devi- skaker, and those containing the mycelial mat remained ation (SD)±3.18 %]. static. CA and gluconic acid levels in the experiments were determined by studying the kinetic profile of CAP using a Optimisation of CAP HPLC system (Knauer, Berlin, Germany) equipped with a UV detector (210 nm). A 10-μl aliquot of the sample was Studies aimed at optimising CAP were performed by inves- applied to a Luna C 18 column (300 × 3 mm) and eluted tigating the effects of two related variables in one experi- with 5 mM H2SO4 (pH 2.81) at a flow rate of 0.56 ml/min ment, and the optimum conditions obtained were applied in and column temperature of 20 °C. all subsequent studies. The optimisation of process parameters included investigating the effects of different bed load- Statistical validation of treatment effects ings of OP (10–25 g) at different levels of moisture (50– 75 % of MWRC), which were obtained by adding distilled To determine the significance of the optimization steps, the water at two steps. At step 1, the amount necessary to obtain mean, standard deviation, Tukey’s test (“t”) and probability 50 % of the MWRC was added to all flasks before steri- ("P") values of three independent determinations of the lisation to prevent thermal degradation of the OP and to investigated parameters and the control were calculated. ensure that the effect of autoclaving was similar in all treat- Calculations were performed according to the mathematical ments. At step 2, water amounts necessary to obtain differ- principles that results were considered highly significant, ent moisture levels in addition to any other materials to be significant or non-significant when P was <0.01, between added, such as molasses and alcohols, was added aseptically 0.01 and 0.05, and >0.05, respectively. Moreover, to vali- with the inoculum. Beakers containing saturated solutions of date the significance of all the experimental work, analysis NaCl were placed inside the incubator to prevent the media of variance (ANOVA) on the means was also performed from drying out. The effects of different volumes of myce- (Glantz 1992). lial pellets (2.5–12.5 % of the 250-ml flask volume) of different ages (12 h–60 h), different initial pH values (3–6, adjusted with 0.1 N HCl), different incubation temperatures Results and discussion (25–32.5 °C), different incubation periods (48–84 h) at different agitation rates (150–300 rpm), various total sugar Previous studies have indicated that CAP by A. niger and Y. concentrations (12–16 %, w/w) using molasses (M), EDTA- lipolytica requires a fermentation medium with a high sugar treated molasses (ETM) and ferrocyanide-treated molasses concentration (up to 22 % in some cases), metal limitation, (FTM) and different low alcohols concentrations (2–4.5 %, nitrogen (N) and phosphorous (P) depletion, and initial pH v/wt) were investigated. value of <5. Conversely, such conditions, which shift the 270 Ann Microbiol (2013) 63:267–278 metabolism towards CAP, negatively affect biomass produc- increase compared to the previous amount of CA produced tion, which in turns reduces the amount of CA produced (195 g/kg OP; Fig. 1). It has been reported that the pelleted (Papagianni 2007; Papanikolaou et al. 2008; Gurpreet et al. form is more suited for CAP than other forms (Khadijah and 2011; Kamzolova et al. 2011a). As a result, the OPM used in Mohd 2011), while the inoculum volume of pellets showed this study was inoculated with either 48-h-old mycelium wider variations, ranging from 2 (Yigitoglu 1992)to10% pellets or a 48-h-old mycelium mat, with the results indicat- (Vandenberghe et al. 1999). The effect of inoculum age on ing the superiority of inoculation with pellets. This result CAP has attracted little interest in the past, yet 10 % of a 40- could be attributed to the lower viscosity of the pellet culture h culture containing mycelial pellets 0.2–0.3 mm in diame- which may improve the bulk mixing and aeration conditions ter was reported as the optimum inoculum age (Hossain et and lower oxygen consumption (Gomez et al. 1988). al. 1984). Other studies have reported the use of spore The effect of bed loading and its moisture level on CAP suspensions as an inoculum, and a significant effect of was investigated, and the maximum CAP of 195 g/kg OP inoculum size was also indicated where a range of 103– was obtained at a bed loading of 20 g and a moisture level of 108spores/g substrate was employed (Lu et al. 1997; Roukas 65 % (Fig. 1). Bed loading and moisture levels are important 2000). Contrary to these results, Kristiansen (1976) indicat- factors for CAP in SSF systems because they affect fungal ed that CAP is independent of the inoculum size as long as metabolism by influencing the degree of aeration, heat and the size was kept at <106spore/ml culture. mass transfer, diffusion and solubility of nutrients, oxygen Increasing the initial pH of the OPM and the incubation and toxic metabolites and swelling of the substrate, as well temperature resulted in a slight increase in CAP, with maxi- as by facilitating penetration of the mycelium, all of which mum amounts obtained for pH 4.5 (317.5 g/kg OP) and pH 5 are important delimiters of CAP (Lonsane et al. 1985;Luet (320 g/kg OP) at 30 °C (Fig. 3). As this difference was non- al. 1997). The optimal moisture content and bed loading significant (P>0.05), the initial value of pH 5 was selected for depend on the specific requirements of the microorganism subsequent experiments as this was closer to the original pH and the nature of the material, with hydrophilicity and porosity of the OPM (5.4), meaning that a minimum adjustment was being of particular importance (Torrado et al. 2011). The required. At higher pH values or temperatures, CAP decreased relatively low level of initial moisture required for the medium significantly (Fig. 3). Similar results have been reported in (65 %) in this study is in agreement with many other research previous studies where the optimum temperatures for CAP, findings where the substrates used in SSF for CAP were mainly by A. niger and Y. lipolytica, were between 28 and moistened to approximately 70 % depending on the substrate 30 °C (Soccol et al. 2006; Papanikolaou et al. 2009). Con- absorption capacity (Vandenberghe et al. 1999;Kareemetal. versely, a wide variation in the optimum pH value for CAP has 2010). been reported, and Hossain et al. (1984) found that controlling These results confirmed the significant effects of both the the pH did not affect the CAP in SSF systems, which contra- volume and the age of the inoculum on CAP. The maximum dicts the findings of many other studies which suggest an amount of CA (267 g/kg OP; Fig. 2) was obtained using important role for pH in CAP. Papagianni (1995) found that 10 % of 48-h-old pellets, which representsing a 36.92 % increasing the pH from 2 to 4.5 reduced the final yield of CA

Fig. 1 Effect of varying bed loading at different levels of moisture on citric acid (CA) production. Inoculum comprising 5 % (v/v) of 48-h-old mycelium pellets of Aspergillus niger was used to inoculate 250- ml Erlenmeyer flasks with a different bed loading of orange peel (OP; g/250-ml flask) moisturised at the indicated levels [% of maximum water retention capacity (MWRC)]. The initial pH was 5.5, agitation rate was 150 rpm and incubation was at 30 °C for 72 h. Data presented in all figures (this and all subsequent experiments) are the aver- age of 3 independent determinations; error bars standard deviation of the mean Ann Microbiol (2013) 63:267–278 271

Fig. 2 Effect of varying the volume of the inoculum at different ages on CA production. The indicated volumes of mycelial pellets of A. niger (2.5–12.5 % of the flask volume) at the indicated ages (12–60 h) were used to inoculate 250-ml Erlenmeyer flasks, with a bed loading of 20 g OP, moisturised to 65 % of MWRC, at an initial pH 5.5 and incubation at 30 °C for 72 h

by about 80 %, which has been confirmed and explained in other organic acids were present, which would result in a other studies where pH levels of 4.5–5 were found to shift the lowering of the pH of the fermentation medium, was assessed metabolism towards the production of oxalic acid instead of using descending paper chromatography. The chromatogram CA (Ruijter et al. 1999; Rymowicz et al. 2006;Papanikolaou revealed that CA was the dominant acid, but trace amounts of et al. 2011). The maintenance of the pH value of the medium gluconic acid were also detected; however, no oxalic acid was at pH 5 or slightly higher (approx. 6.0) has been considered to detected. This result can possibly be explained by the gradual be of crucial importance for a successful CA fermentation shift in the pH value, as it reached 2.5 after 72 h of fermenta- process by A. niger and Y. lipolytica yeasts (Ruijter et al. tion which was sufficiently long for the synthesis and accu- 1999;Papanikolaouetal.2002, 2008; Khosravi-Darani and mulation of CA (Fig. 7). Other studies reported that CAP was Zoghi 2008; Afifi 2011). In our study, the final pH of the accompanied by a strong shift in pH values to less than 3 after fermentation medium, regardless of the initial pH value, the first day of incubation, following which the pH remained changed to 2.3–2.5, primarily as a result of the accumulation almost constant until the last day (Soccol et al. 2006;Afifi of CA (data not shown). Papanikolaou et al. (2002)reported 2011). A drop in the pH was found to depend on both the that a rapid drop in the pH value of the fermentation medium substrate and the microorganism. The pH of the medium shifts the metabolism towards the synthesis of other organic dropped very quickly to less than 3 during CAP by Aspergil- acids. To better understand this finding, the possibility that lus sp., Penicillium sp., and Rhizopus sp., while it was more

Fig. 3 Effect of varying the initial pH value and incubation temperature on CA production. 250-ml Erlenmeyer flasks with a bed loading of 20 g OP moisturised to 65 % of MWRC were inoculated with 48-h-old mycelial pellets of A. niger at 10 % of the flask volume and incubated at the indicated temperature and pH values at an agitation rate of 150 rpm. A solution of 0.1 N HCl and NaOH was used to control the pH value. The pH of the substrate was measured using a solid:liquid ratio of 10 % (w/v) with distilled water 272 Ann Microbiol (2013) 63:267–278 stable at between 4 and 5 for other fungi, such as Tricho- the only variable among such findings was the substrate used, derma, Sporotrichus and Pleurotus sp. (Raimbault et al. suggesting that the substrate is a detrimental factor affecting 1997). Taken together, it can be concluded that the optimum the optimum conditions. initial pH of the fermentation medium is strongly correlated Potential interest has been reported in other studies with with the nature of the available substrates and that it is a regards to both the type and concentrations of carbon source detrimental factor in terms of the dominant metabolic activity. as critical factors affecting CAP by A. niger and Y. lipolytica. Moreover, comparisons of the optimum initial pH value of the In general, conditions of carbon excess were found to favour medium should be performed to take into account whether the the secretion of CA into the growth medium (Papanikolaou et medium is only used for CAP or also for development of the al. 2009), whilst no CA was obtained in media with less than mycelium. 2.5 % sugar (Papagianni 2007) and concentrations of up to Results indicated that increasing both the agitation rate 22 % were generally required (Honecker et al. 1989). In our and incubation period resulted in a significant increase in study, the concentration of total soluble sugars available in the CAP and that the maximum amount of 373.4 g/kg OP (Fig. OPM after autoclaving was 12 % (w/w) which enabled A. 4) was obtained after 72 h of shaken incubation at 250 rpm. niger to produce 375 g CA/kg OP (Fig. 5). The effect of This represented a 16.7 % gain in comparison with the increasing the sugar concentration in OPM on CAP was previous amount of 320 g/kg OP (Fig. 3). The concurrent investigated using corn-steep liquor (CSL) and molasses be- increase in CAP with an increase in the agitation rate could cause of their low cost, high sugar content, and their proven be explained by increased shear stress that results in the suitability as carbon sources (Vandenberghe et al. 1999). The breaking of filaments, disruption of pellets and the develop- sugar contents of molasses (52 %,w/v) and CSL (3.4 %, w/v) ment of short, thick and highly branched filaments, all of were determined after autoclaving and added to the OPM after which result in recurring cycles of mycelial fragmentation being either diluted or concentrated to obtain final sugar and regrowth. These cycles are beneficial to CAP because concentrations of 13–16 % (w/w). The results indicate the the most susceptible mycelia to fragmentation are the inac- superiority of cane molasses over CSL in supporting higher tive, old heavily vacuolated parts of the filaments, while the CAP (data of corn-steep not shown). However, the final sugar new tips generated from fragmentation give rise to new and concentrations following the addition of either molasses or active filaments (Papagianni et al. 2007). The optimum CSL were similar; it should be noted, however, that sucrose is incubation periods for CAP by A. niger reported in the main sugar in molasses whilst glucose is the main sugar in other research studies range from 72 h utilising corn- CSL. Sucrose has been reported to be more effective than cobs (Hang and Woodams 1998) to 96 h utilising solid glucose in supporting CAP by A. niger (Kubicek and Rohr potato wastes (Afifi 2011),andevenupto14days 1989; Xu et al. 1989; Ruijter et al. 1999). Moreover, the (336 h) utilising pure sugars (Hossain et al. 1984). profile of the other constituents, particularly minerals, may The optimum CAP by Y. lipolytica utilising rapeseed oil was play an additional critical role in favouring cane- olasses over obtained after 168 h (Kamzolova et al. 2011b). Once again, CSL. It is also of interest that the addition of CSL to reach a

Fig. 4 Effect of varying agitation rate (rpm) on CA production. A. niger (48 h-old mycelial pellets at 10 % of the flask volume) was used to inoculate 250-ml Erlenmeyer flasks, with a bed loading of 20 g OP moisturised to 65 % of MWRC and incubated at 30 °C. The initial pH was 5, and the indicated incubation periods (48–84 h) and agitation rates (150–300 rpm) were assessed Ann Microbiol (2013) 63:267–278 273 final sugar concentration of 13–16 % (w/w) would also result caused by the simultaneous uptake of the monosaccharides in the addition of significant quantities of nitrogen into the after hydrolysis (Hossain et al. 1984). This process was con- medium, which would shift metabolism towards biomass firmed by Begum et al. (1990) who found that a combination instead of CA production (Gurpreet et al. 2011;Kamzolova of sugars, such as sucrose, glucose, sorbitol and xylose, im- et al. 2011a). These results also confirm the significant effect proved CAP by A. niger. Interestingly, the results of a study on of an increased initial sugar concentration on CAP; the max- the ability of Y. lipolytica to utilise two different types of imal amount of 512 g/kg OP (Fig. 5) was obtained at a 14 % carbon sources for CAP, including carbohydrates and fatty sugar concentration, representing a 36.5 % gain over the acids, such as glucose, glycerol, oleic acid and hexadecane, previous amount (375 g/kg OP; Fig. 4). It has been previously show that carbohydrates are generally more easily metabol- shown that the more carbon substrate available in the medium, ised and that the consumption of fatty acids usually starts after the more the microbial metabolism is directed towards CA a significant depletion of carbohydrates has occurred, from biosynthesis (Papanikolaou et al. 2009). On the other hand, 10%tolessthan2.5%(Kamzolovaetal.2011b). In addition lower CAP at low concentrations of sucrose could be due to to sugars, CAP by Y. lipolytica utilising non-traditional carbon oxalic acid formation (Papanikolaou et al. 2011), while at sources, such as raw lipid materials, including rapeseed oil, sucrose concentrations of more than 14 % it could be attrib- olive-mill wastewater and glycerol-containing waste of the uted to polyalcohol formation (Gutierrez-Rozas et al. 1995). biodiesel industry, has been demonstrated (Papanikolaou et With regards to CAP utilising OPM fortified with cane mo- al. 2008; Rymowicz et al. 2010; Kamzolova et al. 2011b). lasses (M-OPM), the process is somewhat complicated when Molasses contains some metal salts, such as iron, calci- it involves the utilisation of multiple sugars that are either um, magnesium, manganese and zinc, which retard CA directly available in the medium as natural constituents of the synthesis (Soccol et al. 2006), and these are usually treated OP and molasses or which result from mild hydrolysis during with EDTA, ferro- or ferry-cyanide (Yigitoglu 1992; Van- the preparation of the medium (such as pre-treatment of mo- denberghe et al. 1999). In the present work, the added lasses and autoclaving the media) or from the activity of the molasses was treated with EDTA (ETM) and ferro-cyanide hydrolytic enzymes secreted by A. niger. The presence of (FTM) and it was found that these treatments had a non- multiple sugars appears to be beneficial for CAP, as it has significant effect (P>0.05) on CAP, as 512, 517 and 515 g been shown that, in fermentations involving sucrose, a com- CA/kgOPwereproducedinthepresenceof14%molasses, plete hydrolysis into glucose and fructose takes place before ETM and FTM, respectively (Fig. 5). These results are in CAP starts, which facilitates a higher rate of sugar utilisation agreement with the findings reported by Hossain et al. (1984),

Fig. 5 Effect of varying sugar concentrations added as molasses (M), adding different amounts of sterilised molasses to the OP prior to EDTA-treated molasses (ETM) and ferrocyanide-treated molasses inoculation. The various amounts of molasses were mixed with the (FTM) on CA production. A. niger (48-h-old mycelial pellets at 10 % remaining amount of water added at step 2 in order to obtain the of the flask volume) was used to inoculate 250-ml Erlenmeyer flasks, desired level of moisture as described in the Materials and methods with a bed loading of 20 g OP moisturised to 65 % of MWRC and and were separately sterilised by autoclaving. ETM and FTM were incubated at 30 °C, an initial pH 5 and an agitation rate of 250 rpm, for prepared by adding EDTA and ferrocyanide at 200 ppm 72 h. Various concentrations of total soluble sugars were obtained by 274 Ann Microbiol (2013) 63:267–278

Fig. 6 Effect of adding different concentrations of low alcohols on CA production. A. niger (48- h-old mycelial pellets at 10 % of the flask volume) was used to inoculate 250-ml Erlenmeyer flasks, with a bed loading of 20 g OP moisturised to 65 % of MWRC, fortified with molasses at a final sugar concentration of 14 %, and incubated at 30 °C, an initial pH 5 and agitation rate of 250 rpm, for 72 h. Alcohols at the indicated proportions were mixed with the remaining amount of water added at step 2 (as described in Materials and methods) and added immediately before inoculation

Shankaranand and Lonsane (1994)andXieandWest effect on citrate synthase and is accompanied by a decrease (2006), all of whom observed a different response of in the activities of aconitate hydratase and NAD-isocitrate A. niger to metal ions and minerals in SSF and sub- dehydrogenase, which are favourable conditions for inten- merged systems. These authors concluded that SSF sys- sive CAP (Kamzolova et al. 2011b). tems were able to overcome the adverse effects of high Attempts to increase CAP by adding various low levels concentrations of trace metals and, as a result, pre- of alcohol at different concentrations (2–4.5 %, v/w) treatments of substrates with chelating agents and/or revealed that methanol at 3.5 % (v/w) significantly increased potassium ferro-cyanide were of no use, which is a CAP and resulted in the formation of 640 g/kg OP, repre- prime advantage of the SSF culture system. In another senting a 25 % gain over the control (512 g/kg OP) (Fig. 6). study, the addition of phytate to molasses-containing Iso-propanol significantly inhibited CAP, but ethanol did medium at the beginning of fermentation resulted in a not have any significant effect. Enhancement of CAP by threefold increase in CAP due to the ability phytate to adding low levels (up to twofold) of alcohols, such as overcome the negative effect of metals (Lu et al. 1998). methanol, ethanol and isopropanol, has been previously Fortunately, phytate is a natural plant constituent already reported (Rodriguez et al. 2009; Afifi 2011), and CAP was present in OP, again suggesting the suitability of using found to depend on the strain of A. niger, the composition of non-treated molasses. Moreover, CAP requires a medi- the medium and the type of alcohol used (Vandenberghe et um with a controlled concentration of metals rather than al. 1999; Demirel et al. 2005). The usual concentration of a total metal deficiency, with Mg (Kapoor et al. 1983), the added alcohol ranged from 1 to 5 % (Soccol et al. 2006), Zn (Shankaranand and Lonsane 1994)andasmallex- and CAP was found to decrease at higher concentrations. cess of copper ions (Soccol et al. 2006)favouringCAP. The inductive effect of low alcohols on CAP was attributed Attempts to enrich the M-OPM with different sources of to their effect on the ability of A. niger to directly ferment nitrogen, such as NH4NO3,(NH4)2SO4 or urea, or with the carbohydrates available in crude substrates, such as sources of phosphorous, such as K2HPO4 or KH2PO4, did grains, OP, liquor and brewery wastes, as well as molasses not significantly affect the level of CAP (data not shown). (Roukas and Kotzekidou 1987; Vandenberghe et al. 1999; This result is consistent with previous findings where the Kareem et al. 2010). Low levels of alcohols also inhibit need for nitrogen and phosphorus in submerged systems CAP in synthetic media containing pure chemicals (Ingram was not applicable in SSF systems (Papagianni 2007), pos- and Buttke 1984) and affect the permeability of the cell sibly due to the low water activities in SSF that decreases membrane by altering the phospholipid composition and the diffusion rates of the added nutrients. Moreover, CAP by the spatial organization of the cytoplasmic membrane A. niger and Y. lipolytica is a non-growth associated process (Orthofer et al. 1979; Ingram and Buttke 1984), resulting which is principally induced under conditions of nitrogen in citrate overflow (Rodriguez et al. 2009). It has also been and phosphorous depletion (Vandenberghe et al. 1999; suggested that low levels of alcohols decrease the negative Papanikolaou et al. 2009; Gurpreet et al. 2011). The induc- effects of heavy metals either by increasing the tolerance of tive role of nitrogen depletion in the fermentation media of the fungus or by reducing heavy metal uptake (Dasgupta et Y. lipolytica on CAP has been attributed to its inductive al. 1994; Rodriguez et al. 2009). Ann Microbiol (2013) 63:267–278 275

A summary of the data on the optimisation of CAP by A. inhibited at pH values of ≤3.5 (Mischak et al. 1985; Roukas niger is depicted in Table 2. A successful increase in CAP to and Harvey 1988). This precisely describes the conditions of 377.61 % of the original amount of CA produced was the fermentation medium used in this study, where the pH achieved. The results obtained in this study identify sugar value after 48 h of fermentation was 4, which is the point at concentration as the most significant parameter for CAP, which the maximum GA was observed. This pH then de- while all other nutritional effects, such as nitrogen and clined to 3.2 and 2.3 after 60 and 72 h, respectively, at which phosphorus, had a non-significant effect. Meanwhile, bed time the concentration of GA decreased to 61 and 39 g/kg load and moisture levels, as well as the volume of inoculum OP, respectively (Fig. 7). An important question was raised and its age, were the most significant physical factors and by Papagianni et al. (2007) as to whether A. niger can utilise were able to enhance CAP to 45.52 and 36.92 %, respec- GA to synthesise CA at later stages in the fermentation tively, over the initial level of production. process. This question has not been answered to date and The kinetics of CAP at the optimised conditions in the deserves to be studied. Our results show that both sugar presence of 3.5 % methanol was studied over 84 h. The consumption and CAP start after 24 h of incubation, which results reveal that the lowest concentration of total sugars is relatively early in comparison to the corresponding period (2.8 %) and the lowest pH value (2.3) were associated with reported in other studies (Hossain et al. 1984; Rodriguez et the maximum CAP (640 g/kg OP) after 72 h of incubation. al. 2009; Afifi 2011). This is one of the main advantages of Quantitative determinations of gluconic acid (GA), which is inoculating the fermentation medium with pre-formed my- usually produced simultaneously with CA, were performed celial pellets, as no time is needed for the germination of by HPLC, and their concentrations relative to fermentation spores and formation of mycelium. Moreover, the conver- time are shown in Fig. 7. The maximum amount of GA sion rate of sugar into CA is higher, whilst almost all of the (75 g/kg OP) was detected after 48 h of fermentation, available sugar is used in CAP (Khadijah and Mohd 2011). followed by a sharp decline synchronised with a sharp These results suggest that the carbohydrate hydrolysing increase in CAP. As CA accumulation reached 640 g/kg activity continues for the first 48 h (pH of approx.4), after OP, the concentration of GA decreased to 39 g/kg OP, and which the sharp decrease in pH value is thought to be a further decrease was detected at 84 h of fermentation inhibitory. The kinetic profile of CAP by A. niger grown (24 g/kg OP). This result confirms that the observed de- on OPM is similar to that of another strain of A. niger used crease in the pH of the fermentation medium is principally for CAP using citrus pulp (Rodriguez et al. 2009). However, due to the accumulation of CA. The observed decrease in CAP in our system (640 g/kg OP) is significantly higher pH as fermentation proceeds is possibly one reason for the than that of the other strain (441 g/kg citrus pulp). observed decrease in the accumulation of GA as it has been A comparison of data on CAP by A. niger with previously reported that the glucose oxidase enzyme, which corresponding data on other strains of A. niger and Y. lip- is responsible for the conversion of glucose into GA, is olytica obtained in previous studies is given in Table 3.

Table 2 Data on the enhancement of CAP production by different optimisation steps

Optimisation step Optimal condition CAP (g/kg)a % increase compared with the:

Before optimum After optimum. Previous step First step

Bed load and moisture level 20 g, 65 % of MWRC 134 ± 4.21 195 ± 5.26 45.52 45.52 Volume and age of mycelial pellets 10 % of flask volume, 48-h-old 195 ± 5.26 267 ± 9.56 36.92 99.25 mycelial pellets pH and temperature pH 5, 30 °C 267 ± 9.56 320 ± 10.62 19.85 138.80 Incubation period and agitation rate 72 h, 250 rpm 320 ± 10.62 373.4 ± 11.68 16.7 178.36 Concentration of total sugars provided 14 %, untreated molasses 373.4 ± 11.68 512 ± 17.86 36.5 282.09 by different sources Concentration of different low levels 3.5 % methanol 512 ± 17.86 640 ± 18.21 25 377.61 of alcohols

CAP, Citric acid production; MWRC, maximum water retention capacity Values are presented as the mean ± SD where appropriate a Statistical analysis was performed at each step by comparing the amount of CA produced before and after optimisation by thet test and all results were found to be highly significant (P <0.01). The significance of the mean values of CA obtained throughout all of the experimental work in comparison to each other was checked by analysis of variance (ANOVA) tests. The high F value (936.841) and low P value (3.66 E-69) indicates a highly significant relationship between the results 276 Ann Microbiol (2013) 63:267–278

Fig. 7 Kinetic profile of CA production by A. niger at the optimised conditions over an 84-h fermentation period

These data show an apparent yield of 5.71 g/g, which is only pomace, sugar cane bagasse, orange wastes and coffee based on the apparent sugar consumption and neglects the husks), the yield of CA per sugar consumed has been neither other sugars released and consumed throughout fermenta- determined nor discussed in several studies (as reviewed by tion as a result of the various hydrolytic activities of A. niger Vandenberghe et al. 1999; Soccol et al. 2006). This defi- on the various constituents of OP, especially with regards to ciency may suggest an interesting point of research based on celluloses and hemicelluloses (Table 1). For this reason, in studying the enzymatic profiles and other metabolic activi- spite of the promising CAP (up to 883 g/kg) obtained by A. ties accompanying the process of CAP. However, the yield niger utilising various agro-industrial wastes (such as apple values for various strains of A. niger and Y. lipolytica were

Table 3 Comparison of data concerning CAP by Aspergillus niger to the corresponding data of A. niger and Yarrowia lipolytica obtained in other studies

Microorganism Substrate Fermentation CAPa Yieldb (g/g)

Aspergillus niger van Tiegh. 1867 [LEG; MB284309] OP+molasses SSF 640 5.71c (present study) A. niger ATCC 9142 NRRL 599 (Torrado et al. 2011) OP wastes+various sugars SSF 223.2 0.52 A. niger GCBT7 (Ali et al. 2002) Cane molasses SMF 99.56 0.929 A. niger DS1 (Kumar and Jain 2008) Sugarcane bagasse+sucrose SSF 160.76 0.56 A. niger (Hang et al 1987) Kiwi fruit peel SSF 104 0.60 A. niger NRRL (strain No. 2001, 2270, 599, 328, 567) Apple pomace SSF 124-766 0.80 and strain BC1 (Soccol et al. 2006). A. niger NRRL 567 (Soccol et al. 2006). Grape pomace SSF 413-600 0.88 A. niger (Kareem et al. 2010) Pineapple waste+sucrose SSF 60.61 0.9 Yarrowia lipolytica (strains wt W29, JMY 798, JMY 794) Glucose SMF 32.3-49 0.71-0.85 (Papanikolaou et al. 2009) Y. lipolytica strain 187/1 (Kamzolova et al. 2005) Rapeseed oil SMF 135 1.55 Y. lipolytica (Kamzolova et al. 2011b) Glycerol containing waste SMF 71 1.55

SSF, Solid state fermentation; SMF, submerged fermentation, CAP, citric acid production; N.D. not determined a CAP is given as g/kg substrate in SSF or g/l in SMF b Yield,g of CA produced per g of carbon substrate consumed (maximum values). c Represents the apparent yield, which is not a real value as it depends on the difference (11.2 %, w/v) between the initial sugar concentration of orange peel medium (14 %, w/w) and the final sugar concentration (2.8 %, w/w) and neglects the other amounts of sugars released into the medium during fermentation due to the known activities of the hydrolytic enzymes of A. niger on the various polysaccharides detected in the orange peel Ann Microbiol (2013) 63:267–278 277 within the range of 0.55–0.90, and higher values up to 1.55 Bonting SL (1954) Differential determination of glucose and have also been reported (Papanikolaou et al. 2009;Kamzolova fructose in microgram quantities. Arch Biochem Biophys 52 (1):272–279 et al. 2011a). 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