USE OF MULBERRY POMACE AS SUBSTRATE FOR CITRIC ACID PRODUCTION BY Aspergillus niger MT-4

: Mulberry pomace (MP) is a waste material obtained after the production of pekmez, a traditional Turkish food. This study was performed to test the usability of MP as substrate for citric acid (CA) production by Aspergillus niger MT-4 for the first time. In the study, some culture conditions were also optimized to increase CA production in MP-based medium. Moisture, total carbohydrate, water-soluble carbohydrate, protein, lipid and ash contents of MP were determined as 31.1, 47.1, 1.3, 13.4,


Introduction
Mulberry belongs to the Morus L. genus of the Moraceae family. The genus includes 24 species and one subspecies. M. alba L., M. nigra L. and M. rubra L. are the main species grown in Turkey. Mulberry cultivation in Turkey has long been known dating back to 400 years ago (Ercisli & Orhan 2007).
Mulberry fruits in Turkey are consumed as fresh or fresh fruits are processed to prepare traditional products such as pekmez, pestil and köme. Pekmez, which is consumed mainly in breakfast is made from different fruits but grape and mulberry are the most common fruits used in the process (Gunes & Cekic 2004, Cakmakci & Tosun 2010. For pekmez production, mulberry fruits are boiled in water until the sugars and other organic substances are passed into the water. After the boiling process, the mixture is filtered and the liquid fraction obtained is used for pekmez production. The remaining non-degrading solid fraction is referred to as pomace. Mulbery pomace (MP) is used as animal feed additives and no other use in Turkey is known. MP has also no important use in any application in the world.

R. Aidynova et al.
Citric acid (CA) is an organic acid commonly used in the food, pharmaceutical, cosmetic, detergent, chemical and textile industries (Dhillon et al. 2011, Torrado et al. 2011. Most of CA is used in food (about 70%) and pharmaceutical (12%) industries (Darouneh et al. 2009). The production of CA is carried out by extraction from plants or by chemical synthesis and microbial fermentation but about 99% of the production is achieved via microbial fermentation (Taskin et al. 2013, Arslan et al. 2016). Several microorganisms such as bacteria, filamentous fungi and yeasts are capable of producing CA. For example, filamentous fungi such as Aspergillus niger van Arslan et al. 2016). Microbial CA production is achieved by three different fermentation techniques as surface fermentation, submerged fermentation and solid-state fermentation. Submerged fermentation is performed using shaking flasks or fermenters. Shaking flask technique is usually used for the optimization of fermentation conditions (Show et al. 2015). CA production is affected by operational culture conditions such as pH, temperature, oxygen, incubation time, substrate concentration, minerals, carbon and nitrogen sources. In particular, high carbon source concentrations under the nitrogen and phosphorus-limited conditions are known to significantly increase CA synthesis (Soccol et al. 2006, Darouneh et al. 2009, Show et al. 2015, Arslan et al. 2016. Therefore, optimization of culture conditions in CA production is considered an important criterion.
Growth substrates make up the major part of the production costs in fermentation studies. Therefore, selection of a low-cost substrate is considered as a major aspect in microbial fermentation studies (Taskin et al. 2013). For example, with the aim of reducing the production cost in the production of CA, cheap agricultural wastes and/or byproducts such as beet molasses, black strap molasses, cane molasses, n-paraffin, glycerol, whey and waste oil are usually preferred as substrate (Soccol et al. 2006, Torrado et al. 2011Show et al. 2015, Arslan et al. 2016. However, to our best knowledge, there is no study on the use of MP as a substrate in microbial fermentations. Therefore, the present study was performed to produce CA from A. niger MT-4 in shaking flask culture using MP as substrate and to optimize some culture conditions for enhancement of CA production.

Materials and Methods
Microorganism, materials and chemicals Aspergillus niger MT-4 was obtained from Professor Mesut Taskin from the Department of Molecular Biology and Genetics, Atatürk University, Turkey (Taskin et al. (2013). MP was obtained from a commercial company producing mulberry pekmez in Erzurum province (Turkey). Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Merck (Germany). The other chemicals (Tween 80, phenol, sulfuric acid, acetylacetone, Ehrlich reagent, N-acetylglucosamine, citric acid, pyridine and acetic anhydride) were purchased from Sigma (USA).

Preparation of spore suspension
The fungal culture was left to sporulation at 30°C for 10 days on the slant containing PDA medium. At the end of this period, conidia were suspended in sterile physiological water including a surfactant (0.2 mL/L Tween 80). The slant was vortexed for approximately 5 min to distribute the spores homogeneously. The prepared suspension was then filtrated through three layers of muslin to eliminate hyphae and unsuspended conidia. The spore concentrations were determined by using a haemocytometer and the final spore count was adjusted to 10 6 spores per mL. During the experiments, 1 mL of the prepared spore suspension was used for inoculation of the production medium containing MP.

Determination of chemical composition of MP
The moisture content of MP was determined by the weight difference before and after drying in a oven for about 24 h at 100°C, up to constant weight. Total nitrogen content was determined using a micro-Kjeldahl apparatus, and the protein content was estimated by multiplying the nitrogen content by 6.25. Ash content was determined by combusting MP for 3 h in a muffle furnace (Thermolyne 62 700, Barnstead/Thermolyne Corp., Dubuque, IA, USA) at 550°C. The total lipid content was determined according to the Soxhlet extraction method using diethyl ether as solvent. Water-soluble carbohydrate and total carbohydrate contents were determined using phenolsulfuric acid method (Dubois et al. 1956). For analysis of water-soluble carbohydrates, MP was boiled in water for about 5 min. The suspension was then centrifuged to remove solid particles. Finally, the analysis of watersoluble carbohydrates in the obtained liquid fraction (supernatant) was performed by the phenol-sulfuric acid method.
CA production in mulberry pomace-based medium CA production using A. niger MT-4 was performed in 250 mL flasks containing 100 mL of sterile production medium. The production medium (pH 6.0) was prepared by adding MP into distilled water. Optimization of fermentation parameters was performed by using onefactor-at-a-time method. Initial experiments were performed to determine the most favorable concentration of dried MP. For this purpose, different concentrations of dried MP from 30 to 150 g/L were tested. The effects of different concentrations of KH2PO4 (0-1.5 g/L), MgSO4 (0-2.0 g/L) and (NH4)2SO4 (0-4 g/L) on CA production in MP-based medium were tested. Then, different initial pHs (pH 2-8) and incubation times (2-7 days) were tested to increase CA production in MP-based medium. All the experiments were performed at 30°C on a shaking incubator at 150 rpm.

Analytical methods
Final pH of the culture and the initial pH of the MPbased medium were measured using a pH meter (Ohaus Starter 3100). 10 mL sample taken from the culture after an appropriate cultivation was centrifuged at 5000 rpm for 5 min. The obtained supernatant was used for CA analysis. The concentration of CA was determined according to the acetic anhydride method (Marier & Boulet 1958). In brief, 1 mL of supernatant was first mixed with 1.3 mL of pyridine. Then, 5.7 mL acetic anhydride was added into the samples. After incubation at 32°C for 30 min, absorbance of CA was determined spectrophotometrically at 410 nm. Anhydrous CA (50-300 µg/mL) was used as standard. In case of very intense color formation, samples were diluted with distilled water. CA content of the samples was determined according to the standard graph prepared with anhydrous CA.
During the incubation period, some of the MP was not used as a substrate by the fungus. This part, which was not used as a substrate and remained in the culture, was named non-fermented MP. At the end of the cultivation period, it was observed that the nonfermented MP was attached to the fungal biomass (FB) and not separated from it by centrifugation. Therefore, the solid fraction (non-fermented MP + FB) consisting of non-fermented MP and FB was named as total biomass (TP).
For the determination of FB amount in TP, Nacetylglucosamine content in TP was measured. In brief, 1 mL of concentrated H2SO4 was added to 0.5 g of TP. Following this, acetylacetone reagent (1 mL) was added to the mixture. The mixture was incubated in a boiling water bath for 20 min and then cooled at room temperature. Then, 6 mL of ethanol and 1 mL of Ehrlich reagent were added to the cooled mixture. The final mixture was again left to incubation at 65 °C for 10 min and then cooled at the room temperature. Finally, optical density (OD) of the final mixture was determined at 530 nm (Velmurugan et al. 2011). The obtained absorbance was then used for the calculation of FB produced in MP-based medium. For this purpose, the following simple equation was used.
FB= (A1/A2) x TB A1: The absorbance assayed for N-acetylglucosamine content in per g of the dried TB (TB; FB + non-fermented MP) in MP-based medium at the end of cultivation period. A2: The absorbance assayed for N-acetylglucosamine content in per g of dried FB, which was produced in standard PDB medium at the end of cultivation period. TB: Total biomass (FB + non-fermented MP) in MPbased medium at the end of cultivation period.

Statistical analysis
Each analysis was repeated at least three times in two replicates. Statistical difference was analyzed with oneway ANOVA in the SPSS 15.0 package program at P <0.05 significance level.

Chemical composition of MP
The moisture content of raw MP was determined as 31.1%. Total carbohydrate, protein, lipid and ash contents of MP were determined as 47.1, 13.4, 1.8 and 1.4%, respectively. The ratio of water-soluble carbohydrates was found to be 1.3%. All measurements are mean ± standard deviations (±SD) of six determinations (n = 6).

Optimization of CA production from A. niger in MPbased medium
In the first step of the study, different concentrations of dried MP were tested for production of CA and FB from A. niger MT-4. Increased MP concentration increased both CA synthesis and fungal growth, and maximum concentrations of CA (10.6 g/L) and FB (14.3 g/L) were reached in medium containing 120 g/L MP (Fig. 1). Based on these results, the following experiments were performed on medium containing 120 g/L MP.  After determination of the optimum concentration of MP, the effect of different concentrations of KH2PO4 on cell growth and CA synthesis was investigated. The maximum CA production was achieved in the control medium (KH2PO4-free medium), whereas FB reached to the maximum (16.7 g/L) when 1.5 g/L KH2PO4 was added to MP-based medium (Fig. 2). Considering these results, the following experiments were carried out in the medium, which was not supplemented with KH2PO4. All tested concentrations of MgSO4 increased fungal growth (Fig. 2). In contrast to fungal growth, the maximum CA production (13.4 g/L) was achieved in the medium supplemented with 1 g/L MgSO4. However, excessive concentrations of MgSO4 gradually decreased CA production. For example, CA production decreased up to 11.5 g/L when 2 g/L MgSO4 was added to the medium. Therefore, the subsequent experiments were performed in the medium supplemented with 1 g/L MgSO4. The experiments revealed that 2 g/L (NH4)2SO4 caused maximum CA production (21.9 g/L) but higher (NH4)2SO4 concentrations decreased CA production. In contrast to CA synthesis, all tested concentrations of (NH4)2SO4 increased fungal growth and the maximum FB (19.2 g/L) was reached at the highest (NH4)2SO4 of 4 g/L ( Fig. 2). Taking into account these results, an ammonium sulfate concentration of 2 g/L resulting in the maximum CA production was chosen for subsequent experiments. When the effect of initial pH on CA synthesis and fungal growth was investigated, it was determined that maximum CA synthesis (23.4 g/L) occurred at an initial pH of 7.0, but FB reached to the maximum (18.6 g/L) at pH 6.0 (Fig. 2). Based on this result, the following experiments were carried out in the medium with an initial pH of 7.0.
The highest increase in CA production occurred in the first 4 days of incubation and CA concentration reached to the maximum (24.6 g/L) on day 5 (Fig. 3). FB reached the highest value (17.2 g/L) on day 4 and remained stable on day 5. But, reductions in both FB and CA concentrations were observed after the 5 th day. For example, at the end of day 7, FB and CA concentrations were measured as 13.8 and 18.5 g/L, respectively. Since maximum productions of CA (24.6 g/L) and FB (17.2 g/L) were achieved at 120 g/L MP concentration, Yp/s (gram CA produced/gram substrate), Yp/x (gram CA produced/ gram FB produced) and Yx/s (gram FB produced/gram substrate) were calculated as 0.2 g CA/g MP, 1.43 g CA/g FB and 0.14 g FB/g MP, respectively. When initial pH was adjusted at 7.0, there was a continuous decrease in culture pH up to the end of 5 th day, but an increase in culture pH was observed again after 5 th day. Lowest pH value (pH 3.3) was detected on day 5, by which the maximum CA concentration was reached.

Discussion
The previous studies demonstrated that hydrolysates, which are prepared from agricultural wastes and/or byproducts using chemical or enzymatic hydrolysis processes can be utilized as substrates for CA production in the culture of A. niger strains (Watanabe et al. 1998, Hu et al. 2014, Faruk et al. 2014, Muna et al. 2018. But, it is well known that when chemicals and/or enzymes are used for the preparation of hydrolysates, the production cost of CA increases. Therefore, researchers suggested using Aspergillus strains in CA production, which are capable of secreting hydrolytic enzymes (amylase, cellulose, pectinase etc.) and thereby growing directly on agricultural wastes and/or by-products (Andersen et al. 2011, Afify et al. 2012, Rehman et al. 2014). However, the potential of A. niger to produce CA on MP, which is not subjected to a pretreatment process such as chemical or enzymatic hydrolysis, has not been studied yet.
The chemical analyses revealed that dried MP contained vey low amount of water-soluble carbohydrates. This situation can be attributed to the boiling of mulberry fruits at high temperature for a long time during the production of pekmez (molasses). In other words, since water-soluble carbohydrates such as sucrose and glucose in mulberry fruits passed into pekmez, there was rather low soluble carbohydrate in MP. On the contrary, the experiments showed that the total carbohydrate content of MP was high. These carbohydrates may include pectin, starch, cellulose and soluble sugars. Considering that A. niger strains have hydrolytic enzymes, it is possible that insoluble carbohydrates such as pectin, starch and cellulose in MP can be used as carbon source by A. niger MT-4. Besides, it was determined in this study that MP was rich in ash. Considering the knowledge that ash consists of macro and micro-elements, it is possible to say that MP can also be used as a mineral source by this filamentous fungus. The experiments also indicated that MP contained protein and therefore it may be used as a nitrogen source in the medium.
Usability of non-pretreated MP as a substrate for CA production from A. niger MT-4 was tested in this study. In the first stage, MP was used as a sole source of all nutritional factors (minerals, carbon and nitrogen source) for production of FB and CA. The maximum CA and FB production were achieved in medium containing 120 g/L MP, while higher MP concentrations decreased both CA and FB production. This result might be attributed to the increase in solid/liquid ratio of the production medium. Namely, high solid ratio (high MP concentration) might have prevented the homogeneous mixing of the substrate and sufficient oxygen intake into the culture. Since oxygen is particularly important for CA synthesis (Max et al. 2010), the decrease in oxygen concentration might have limited CA synthesis in MP-based medium.
The experiments revealed that when KH2PO4 was used as phosphorus (P) and potassium (K) source, all concentrations caused inhibition on CA synthesis but a continuous increase in fungal growth. These results should not be so surprising, because it has been reported that excessive P concentrations can limit CA synthesis but increase fungal growth in A. niger (Jernejc et al. 1982, Chen 1996, Vandenberghe et al. 1999, Max et al. 2010, Angumeenal & Venkappayya 2013. These results indicate that P content of MP was sufficient to promote CA synthesis and no additional P source is required in MP-based medium for CA synthesis. When MgSO4 was added into the medium, its concentrations ≤ 1 g/L promoted CA synthesis but higher concentrations caused a slight inhibition. This finding is good agreement with the fact that although MgSO4 has a beneficial effect on CA synthesis in A. niger, its excessive concentrations can inhibit CA synthesis (Vandenberghe et al. 1999, Ikram-ul et al. 2004).
It has been documented that CA production is directly influenced by the nitrogen source, and (NH4)2SO4 is a good nitrogen source for CA production (Vandenberghe et al. 1999, Max et al. 2010. Considering this knowledge, the present experiments also focused on investigating the effect of (NH4)2SO4 as additional nitrogen source on CA production in MP-based medium. The experiments showed that CA production could be achieved in MPbased medium without (NH4)2SO4 (additional nitrogen source). This can be explained by the use of proteinous compounds in MP as nitrogen source in the medium. However, it was seen that (NH4)2SO4 concentrations ≤ 2 g/L caused increases in CA synthesis. This result revealed that the nitrogen in MP was insufficient for the production of high amounts of CA and additional nitrogen was required in the medium. The results also showed that (NH4)2SO4 concentrations over 2 g/L decreased CA synthesis in the culture. Conversely, there was a continuous increase in FB even at the highest (NH4)2SO4 of 4 g/L. This finding is in good agreement with the fact that excessive nitrogen concentration increases fungal growth but decreases the amount of CA produced (Hang et al. 1977, Vandenberghe et al. 1999 The results showed that initial pH values between 5 and 7 were more suitable for CA and FB production. Especially, initial pH of 7.0 was the superior for CA production. It was reported that although acidic pHs such as pH 2 and 3 are more suitable for CA synthesis in A. niger, the germination of A. niger spores requires pH > 5 (Soccol et al. 2006). Namely, the initial pH of the culture for CA synthesis in A. niger should be adjusted to pH levels > 5. Therefore, it should not be surprising that initial pHs of 5-7 lead to more CA synthesis in A. niger MT-4. The reduction in fungal biomass after day 5 may be attributed to cell lysis due to the depletion of nutritional compounds. The most likely cause of the decrease in CA concentration after day 5 may be the degradation of CA by the enzymes released into the culture when the fungal cells are lysed. A continuous drop in culture pH up to the end of the 5 th day could be ascribed to the production of organic acids, especially CA. Similar decreases in culture pH during CA production were also reported in the previous studies (Ali et al. 2002, Dienye et al. 2018. Increment in culture pH after the 5 th day might be attributed to the releasing into the culture of some alkaline compounds due to fungal cell lysis. In conclusion, this study revealed that MP alone could be used as a complex substrate for CA production with A. niger MT-4, but more CA production could be achieved when MP-based medium was supplemented with (NH4)2SO4 and MgSO4 at suitable concentrations. On the other hand, optimization of initial pH and incubation time could increase CA production in MP-based medium. When the optimal culture parameters are selected, 24.6 g/L CA could be produced in MP-based medium. The use of MP in CA production will contribute to the reduction of both fermentation cost and environmental pollution. In future studies, MP can be also tested as a substrate in the culture of microorganisms in the production of other substances such as lactic acid, acetic acid, single cell protein, ethanol, pigment and polysaccharide.