Citric Acid Production by Yeast Grown on Glycerol ... - Semantic Scholar

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S.V. KAMZOLOVA et al.: Citric Acid Production by Yeast, Food Technol. Biotechnol. 49 (1) 65–74 (2011)

original scientific paper

ISSN 1330-9862 (FTB-2466)

Citric Acid Production by Yeast Grown on Glycerol-Containing Waste from Biodiesel Industry Svetlana V. Kamzolova1*, Alina R. Fatykhova1, Emiliya G. Dedyukhina1, Savas G. Anastassiadis2, Nikolay P. Golovchenko1 and Igor G. Morgunov1 1

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr-t Nauki 5, Pushchino, RU-142290 Moscow Region, Russia 2

Pythia Institute of Biotechnology, Avgi, GR-57002 Thessaloniki, Greece Received: March 3, 2010 Accepted: August 16, 2010

Summary The possibility of using glycerol and glycerol-containing waste from biodiesel manufacture as a carbon and energy source for microbiological production of citric acid has been studied. Acid formation on the selective media had previously been tested in 66 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis and Yarrowia). Under growth limitation by nitrogen, 41 strains (belonging mainly to species Yarrowia lipolytica) produced acids; unlike 25 strains of the genera Debaryomyces, Candida, Pichia, Saccharomyces and Torulopsis. Among the 41 acid-producing strains, mutant strain Yarrowia lipolytica N15 was selected since it was able to produce citric acid presumably in high amounts. The citric acid production by the selected strain was studied in dependence on the medium pH, aeration and concentration of glycerol. Under optimal conditions, the mutant Y. lipolytica N15 produced up to 98 g/L of citric acid when grown in a fermentor with the medium containing pure glycerol, and 71 g/L of citric acid when grown on glycerol-containing waste. The effect of growth phases on physiological peculiarities of the citric acid producer was discussed. Key words: Yarrowia lipolytica, citric acid production, biodiesel production, glycerol, glycerol-containing waste

Introduction In the 20th century, oil hydrocarbons were considered to be the main source of energy and the cheapest and readily available raw material for biotechnology. However, the ever-increasing rise in the cost of oil starting with the oil crisis of the seventies and the deterioration of the global ecological situation in the last years have made us turn to alternative energy sources, such as biodiesel produced from renewable plant raw materials. In 2010, the European Community (EU directive 2003/30/ EC) planned to raise the percent of biodiesel to 5.75 % of the total fuel. Biodiesel can be produced from various vegetable oils and animal fats. The technology consists

in that oil triglycerides are hydrolyzed and then methylated with the formation of methylated fatty acids, which are used just as biodiesel. One of the major wastes from this technological process is glycerol, which is formed in an amount of more than 1 kg per 10 kg of the biodiesel produced. In 2007, the amount of glycerol-containing waste in Europe reached 600 000 tonnes (1), which poses the problem of its utilization. Biodiesel waste, which contains glycerol (up to 80 %), oil residue, free fatty acids, sodium and potassium salts, and water, may serve as a raw material for various biotechnological processes. Several efforts were made for microbiological conversion of technological glycerol (crude glycerol) into valuable products: 1,3-propanediol

*Corresponding author; Phone: ++7 496 773 0742; Fax: ++7 495 956 3370; E-mail: [email protected]

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(1,2), microbial biomass and lipids (3–6), food-grade pigments (7), erythritol (8), mannitol (9), L-lysine (10), organic acids, in particular succinic acid (11) and citric acid (1,2,8,12–16). Citric acid (CA) has attracted increased interest due to its distinctive properties as an acidulate, flavouring agent and antioxidant, and it is used mainly in food and beverage industry (70 % of the total CA production). In recent years, the consumption of CA and its salt, trisodium citrate, has reached 1 400 000 tonnes with growth at 5 % per year (17). CA is an intermediate of tricarboxylic acid cycle and holds a key position in the metabolism of each microbial cell. However, under certain conditions of fermentation, fungi, bacteria and yeasts produce CA in excessive amounts. Traditionally, different strains of fungi, mostly belonging to Aspergillus niger, have been used in the commercial production of CA from molasses, sucrose or glucose. Alternatively, there is a great interest in various yeasts belonging to Candida (Yarrowia) lipolytica, which is capable of CA production from various carbon sources, such as n-alkanes (18–20), glucose (18,19,21–25), ethanol (26–28) and plant oils (29–33). The relevant literature data on attempts to use glycerol as carbon source for CA production are rare (1,4,8,12–14,32,34). The goal of the present work is: (i) to study CA production from pure glycerol and glycerol-containing waste from biodiesel industry as carbon sources by different yeast genera (Debaromyces, Candida, Pichia, Saccharomyces, Yarrowia and Torulopsis); (ii) to estimate the effect of cultivation conditions (the medium pH, oxygen supply, and concentration of carbon substrate) on CA production by the selected strain; and (iii) to develop a method for CA production.

Materials and Methods Yeast strains Screening for CA producer was carried out among 59 natural yeast strains belonging to the genera Debaryomyces, Candida, Pichia, Saccharomyces, Yarrowia and Torulopsis and 7 mutant strains with impaired ability to grow on acetate probably due to defects in the tricarboxylic acid cycle (35). Strains were obtained from the All-Russian Collection of Microorganisms (VKM) and from the collection of the Laboratory of Aerobic Metabolism of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino, Russia). The strains were maintained at 4 °C on agar slants with n-alkanes as the carbon source.

Chemicals All chemicals and enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glycerol-containing waste was provided from the Pythia Institute of Biotechnology (Thessaloniki, Greece). This is the product of transesterification reaction of rapeseed oil, methanol and NaOH at a molar ratio of 1:3:0.5, respectively. It contained (in %, by mass): glycerol 80, sodium salts 1, heavy metals 1, impurities of organic nature (oil residue, fatty acids) 5, methanol as a minor component, water 12.7. Crude glycerol was analysed enzymatically using bio-

chemical kit (Roche Diagnostics GmbH, Mannheim, Germany). Additionally, it was analysed for oil and fatty acid content. It was washed twice with n-hexane, the mixture was divided into two layers, of which the upper phase contained n-hexane and lipids. Hexane extract was collected into a glass flask with precision. The lipid extract was dried by passing it through a glass filter with anhydrous sodium sulphate; solvent was evaporated to constant mass of lipids.

Assessment of acid formation on solid media The acid formation was assessed under nitrogen limitation of yeast growth by measuring the zones of CaCO3 dissolution in Petri dishes with the agar medium containing (in g/L): glycerol 20, MgSO4·7H2O 0.7, Ca(NO3)2 0.4, NaCl 0.5, KH2PO4 1.0, K2HPO4 0.1, and trace elements as described by Burkholder et al. (36) with slight modifications (in mg/L): I– 0.1, B3+ 0.01, Fe2+ 0.05, Zn2+ 0.04, Mn2+ 0.01, Cu2+ 0.01, Mo2+ 0.01, yeast autolysate 8 mL/L (as a source of nitrogen and vitamins), and Bacto agar 20.0 g/L. Chemically pure powdered CaCO3 (6 g/L) was added into the heated medium directly before it was poured into the dishes. The cultures were plated onto cooled agar medium and incubated at (28±1) °C for 7 days.

Assessment of the acid formation by yeast in liquid cultures The acid formation in liquid medium was assessed after a 6-day cultivation of the studied strains under nitrogen deficiency in 750-mL flasks with 50 mL of the medium containing (in g/L): (NH4)2SO4 0.3, MgSO4·7H2O 0.7, Ca(NO3)2 0.4, NaCl 0.5, KH2PO4 1.0, K2HPO4 0.1, Burkholder trace elements, Difco yeast extract 0.5, and glycerol 30. Since growth was followed by a decrease in the pH of the medium, in order to maintain the medium at pH=4.5–5.5, 10 % NaOH was periodically added using pH paper strips.

Yeast cultivation in a fermentor The yeasts were cultivated in a 10-litre ANKUM-2M (SKB, Pushchino, Russia) with an initial working volume of 5.0 L. The medium contained (in g/L): (NH4)2SO4 3.0, MgSO4·7H2O 1.4, NaCl 0.5, Ca(NO3)2 0.8, KH2PO4 2.0, K2HPO4 0.2, Burkholder trace elements, Difco yeast extract 1.0 and thiamine 0.02. Glycerol (170 g/L) or glycerol-containing waste (100 g/L) were used as the sole carbon and energy source. The fermentation conditions were maintained automatically at the constant level: temperature was (28±0.5) °C; pH=4.5±0.1 was adjusted with 20 % NaOH; dissolved oxygen concentration (pO2) was 60 % (from air saturation); and agitation was 800 rpm. Pulsed addition of glycerol-containing materials was performed as the pO2 value increased by 5 %, indicating a decrease in respiratory activity of the cells due to the total consumption of carbon sources. Cultivation was performed as indicated in the text.

Measurement techniques Yeast growth was followed by measuring the absorbance of the culture at 540 nm with a Spekol 221 spectrophotometer (Carl Zeiss, Jena, Germany). The dry bio-


mass was estimated from the absorbance of the cell suspension using a calibration curve. Concentration of ammonium was determined potentiometrically with an Ecotest-120 ionometer using an Ekom-NH4 electrode (Econix, Moscow, Russia). Glycerol was analysed enzymatically using biochemical kit (Roche Diagnostics GmbH). The determination of glycerol was based on the measurement of NADH produced during the conversion of glycerol to L-lactate in coupled reactions; reactions were catalyzed by glycerol kinase, pyruvate kinase and L-lactate dehydrogenase. Concentration of organic acids was determined using high-performance liquid chromatograph (Pharmacia LKB, Uppsala, Sweden) on an Inertsil ODS-3 reversed-phase column (250×4 mm, Elsiko, Rostov-On-Don, Russia) at 210 nm; 20 mM phosphoric acid was used as a mobile phase with the flow rate of 1.0 mL/min; the column temperature was maintained at 35 °C. CA and, threo-D(S)-(+)-isocitric acid (ICA) was identified using the standard solutions (Roche Diagnostics GmbH). Moreover, diagnostic kits (Roche Diagnostics GmbH) were used for the assay of CA and ICA. The determination of CA was based on the measurement of the NADH produced during the conversion of CA to oxaloacetate and its decarboxylation product pyruvate, and following the conversion to L-malate and L-lactate. Reactions are catalysed by citrate lyase, malate dehydrogenase and L-lactate dehydrogenase. The determination of ICA was based on the measurement of the NADPH produced during the conversion of ICA to a-ketoglutarate, a reaction catalysed by isocitrate dehydrogenase. Protein was determined by the Lowry method, while carbon, hydrogen, and nitrogen were measured on a C/H/N element analyzer (Carlo Erba Instruments, Milan, Italy), and the ash content was determined by burning the sample in a muffle furnace. The oxygen content (O) was calculated from: O (in %)=100–(C+H+N+ash)

/1/

where C, H and N are the values of carbon, hydrogen, and nitrogen content (in %). Methyl esters of fatty acids were obtained by the method of Sultanovich et al. (37) and analysed by gas-liquid chromatography on a Chrom-5 gas chromatograph (Laboratorni pristroje, Prague, Czech Republic) with a flame-ionization detector. The column (2 m×3 mm) was packed with 15 % Reoplex 400 applied to Chromaton N-AW (0.16–0.20 mm). The temperature of the column was 200 oC. The lipid content in the biomass was determined from the total fatty acid content by using docosane (C22H46) as internal standard.

Calculation of fermentation parameters To take into account the medium dilution due to the addition of NaOH solution for maintaining the constant pH value, the total amount of CA in the culture broth was used for calculations of the mass yield of CA (YCA), volumetric citric acid productivity (QCA) and specific citric acid production rate (qCA). The mass yield of CA production (YCA), expressed in g of CA per g of glycerol, was calculated from:

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P /2/ S while the volumetric citric acid productivity (QCA), expressed in g/(L·h), was calculated from: YCA =

P QCA = . Vt

/3/

and the specific citric acid production rate (qCA), expressed in g per g of cell per h, was calculated from: P qCA = . Xt

/4/

where P is the total amount of CA in the culture liquid at the end of cultivation (g), S is the total amount of glycerol/crude glycerol consumed (g), V is the initial volume of culture liquid (L), t is the fermentation duration (h), and X is the average working biomass in the fermentor (g). Energy yield of CA from glycerol (hCA) is estimated as a fraction of energy content of the substrate (glycerol) which is incorporated into CA. It was calculated on the basis of mass and energy balance theory (38–40). The energy content in the biomass was calculated on the basis of mass and energy balance theory (38–40).

Statistical analysis All the data presented are the mean values of three experiments and two measurements for each experiment; standard deviations were calculated (S.D.