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Microbiological Production of Citric and Isocitric Acids from ...

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54 S.V. KAMZOLOVA et al.: <strong>Production</strong> <strong>of</strong> <strong>Citric</strong> <strong>and</strong> <strong>Isocitric</strong> <strong>Acids</strong> <strong>from</strong> Sunflower Oil, Food Technol. Biotechnol. 46 (1) 51–59 (2008)where X 2 <strong>and</strong> X 1 are the biomass at the moment <strong>of</strong> timet 2 <strong>and</strong> t 1 , respectively.The biomass yield was calculated as follows:Y X/S =X/S /2/where X is the total amount <strong>of</strong> biomass in the cultureliquid at the end <strong>of</strong> fermentation (g/L) <strong>and</strong> S is theamount <strong>of</strong> the consumed oil (g/L).The specific rates <strong>of</strong> CA <strong>and</strong> ICA production (q CA orq ICA , respectively) were calculated using the followingequation:q=(P/X) ⋅ t /3/where P is the total amount <strong>of</strong> CA or ICA in the cultureliquid at the end <strong>of</strong> fermentation (g), X is biomass contentin a fermentor at the end <strong>of</strong> fermentation (g), <strong>and</strong> tis fermentation time (h).The mass yield coefficients <strong>of</strong> CA or ICA productionwere calculated as follows:Y=P/S /4/where P is the total amount <strong>of</strong> CA or ICA in the cultureliquid at the end <strong>of</strong> fermentation (g), <strong>and</strong> S is theamount <strong>of</strong> consumed oil (g).The volumetric productivity <strong>of</strong> the process was calculatedusing the following equation:C=(P/V) ⋅ t /5/where P is the total amount <strong>of</strong> CA or ICA in the cultureliquid at the end <strong>of</strong> fermentation (g), V is the volume <strong>of</strong>fermentor (L), <strong>and</strong> t is fermentation time (h).Results <strong>and</strong> DiscussionProperties <strong>of</strong> wild type strain Y. lipolytica VKMY-2373 <strong>and</strong> the development <strong>of</strong> regime <strong>of</strong> oil feedingThe wild type strain Y. lipolytica was cultivated inthe complete medium containing 10 g/L <strong>of</strong> sunfloweroil <strong>and</strong> 5.0 g/L <strong>of</strong> (NH 4 ) 2 SO 4 in a 10-litre fermentor withinitial working volume <strong>of</strong> 5 L; pH=5.0 was adjusted automaticallywith 10–15 % NaOH. Fig. 1 illustrates a typicaltime course <strong>of</strong> growth <strong>of</strong> Y. lipolytica. Cell growth wasaccompanied by a decrease in the content <strong>of</strong> nitrogen<strong>and</strong> oil in the medium; 10 g/L <strong>of</strong> biomass were accumulatedat the end <strong>of</strong> fermentation (27 h) (Fig. 1a).The maximum specific growth rate (m max ) calculated<strong>from</strong> the linear segment <strong>of</strong> the growth curve (Fig. 1)amounted to 0.230 h –1 ; a value comparable with literaturedata obtained for Y. lipolytica grown on fatty acids(8,9). The biomass yield <strong>from</strong> the consumed oil (Y X/S )was 1.1 g/L. The above findings support the potential <strong>of</strong>Y. lipolytica for the production <strong>of</strong> single-cell protein <strong>from</strong>fatty material. High Y X/S <strong>of</strong> (1.1±0.3) g/L has been reportedfor different strains <strong>of</strong> Y. lipolytica cultivated onvarious types <strong>of</strong> crude fat (8,9,30,31). Recently, in experimentswith Y. lipolytica grown on completely saturatedfatty acid mixture (9), it has been demonstrated that anincrease in dissolved oxygen concentration <strong>from</strong> 5–15 to60–70 % <strong>of</strong> air saturation resulted in the increase <strong>of</strong> Y X/S<strong>from</strong> 1.1 to 1.6, whereas the lipid production decreasedat the same time. Moreover, the growth parameters appearedto be critically influenced by fatty acid composition<strong>of</strong> the carbon source. It was indicated that yeaststrains (Saccharomycopsis lipolytica <strong>and</strong> Apiotrichum curvatum)did not grow sufficiently well on saturated fat dueto inadequate dispersion <strong>of</strong> these substrates into the liquidmedium; solid fat required considerable agitation(1200 rpm) for dispersal in the growth medium (32,33).In other reports, it was observed that Y. lipolytica demonstratedsignificant growth without differentiations, regardless<strong>of</strong> the fatty acid composition <strong>of</strong> the culture medium(saturated free fatty acids, rapeseed oil <strong>and</strong> mixture <strong>of</strong>these two substrates) (8).The utilization <strong>of</strong> sunflower oil involved its hydrolysisby lipase that resulted in the formation <strong>of</strong> di- <strong>and</strong>monoglycerides, glycerol, <strong>and</strong> free fatty acids. The glycerolconcentration rapidly increased during the first 9 h<strong>of</strong> cultivation (up to 0.125 g/L) (Fig. 1b) <strong>and</strong> then remainedat this level. It should be noted that residual glycerolremained at the trace level as sunflower oil wasconsumed <strong>and</strong> no accumulation <strong>of</strong> glycerol was observed.Also, that a ratio among di-, monoglycerides, <strong>and</strong>free fatty acids in the culture broth remained at the constantlevel in the course <strong>of</strong> cultivation (data not shown).Analysis <strong>of</strong> free fatty acid composition revealed that inthe course <strong>of</strong> cultivation, the fractions <strong>of</strong> oleic acid(D 9 C 18:1 ) <strong>and</strong> linoleic acid (D 9,12 C 18:2 ) decreased <strong>from</strong> 36 to27 % <strong>and</strong> <strong>from</strong> 52.1 to 35.0 % (by mass), respectively,while the amount <strong>of</strong> stearic acid (C 18:0 ) increased <strong>from</strong>4.0 to 26.0 % (by mass). It may be considered that thestrong fatty acid specificity <strong>of</strong> Y. lipolytica was probablyg(biomass, oil)/(g/L)g(glycerol)/(g/L)141210864200.50.40.30.20.10OilNH 4+(a)Biomass0 3 6 9 12 15 18 21 24 27t/hC 18:2(b)C 18:1C 18:0C 16:0Glycerol0 3 6 9 12 15 18 21 24 271200t/hFig. 1. The growth <strong>of</strong> Y. lipolytica VKM Y-2373. Variation <strong>of</strong> sunfloweroil, biomass, nitrogen concentrations (a), glycerol <strong>and</strong> freefatty acids (b) with time. Cultivation conditions: 10 g/L <strong>of</strong> sunflower<strong>and</strong> 5.0 g/L <strong>of</strong> (NH 4 ) 2 SO 4 ; pH=5.09006003000706050403020100g(NH )/(mg/L)4 +w(fatty acids)/%

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