Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 11
Issue 1
Available Online: http://www.ejpau.media.pl/volume11/issue1/art-07.html


Anita Rywińska, Maria Wojtatowicz, Barbara Żarowska, Waldemar Rymowicz
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland



The purpose of the study was to determine growth and acid-forming activities, morphology and physiology of Yarrowia lipolytica acetate mutant A-101-1.31, used for citric acid (CA) production from glucose.

The repeated batch cultivation was carried out in 6 batches. The logarithmic phase of growth was observed only in batch I, while in the other batches, only linear and stationary phases occurred. The volumetric rate of biomass production (QX) was decreasing from 0.54 gL-1h-1 in batch I to 0.22 gL-1h-1 in the last batch. CA was produced at high specific rate (qCAg ~ 0.09 ÷ 0.11 gg-1h-1; qCAn-g ~ 0.04 ÷ 0.05 gg-1h-1) and yield (YCAP ~ 0.7 ÷ 0.8 gg-1) only in four batches, each lasting about 72 hrs. In the other batches, the efficiency of CA synthesis was markedly lower, which was correlated with rapid growth of clones forming S-colonies. Parallely to this phenomenon, a significant increase in the mean volume of cells was observed.

Key words: citric acid, Yarrowia lipolytica, repeated batch culture.


At present, world’s production of citric acid (CA) amounts to 1.4 million tonnes and the demand for this product is steadily increasing [21]. Extensive studies have been carried out on the technologies and mechanisms affecting CA biosynthesis in the media containing various carbon and energy sources as well as trace elements, such as iron, manganese and copper [9,11,13]. Advancement in genetic technologies has resulted in the extended number of strains capable of abundant production of this metabolite e.g. mutants with metabolic defects minimizing by-production of isocitric acid (ICA) and recombinants with enhanced capability of sugar utilization [20,27]. The kinetics of CA biosynthesis under different conditions of cultivation has been studied with the use of such carbon sources as: n–paraffins, vegetable oils, ethanol, glucose and glucose hydrol or molasses [10,14,15,23,25,29].

Most frequently, the data reported in literature are limited to CA biosynthesis by yeast Y. lipolytica in batch cultures [7,19,27,29]. This, however, does not give a possibility to make full use of technical equipment and metabolic potential of the cells. The technological effects are much better with continuous cultivation [2], although the population of microorganisms is more exposed to genotype changes, as compared to those observed in batch cultures. This applies especially to open batch cultures in which multiplied cells are maintained in a definite state for a long time, which can consequently result in degeneration of the strain. Such an adverse phenomenon was observed when CA was biosynthesized by Y. lipolytica A-101-1.31 under chemostat conditions. Stable CA biosynthesis was maintained for a short period of time (200 – 350 hrs), which was followed by selectivity of the mutants exhibiting reduced biotechnological properties [26].

The purpose of the present investigation was to determine growth and acid-forming activities as well as the morphology and physiological characteristics of yeast Yarrowia lipolytica, strain A-101-1.31, used for CA biosynthesis under repeated batch cultivation on glucose.


Microorganism. The yeast strain of Yarrowia lipolytica A-101-1.31 used in this study was from the collection belonging to the Department of Biotechnology and Food Microbiology at Wroclaw University of Environmental and Life Sciences, Poland. It was a mutant (acu-), unable to grow on acetate as the sole carbon and energy source, obtained from the strain A-101 by exposure to UV radiation. In contrast to its parent, the strain exhibited enhanced purity of citric fermentation on a glucose medium. The yeast strain was maintained on YM slants with paraffin at 4° C.

Media. The growth medium of the inoculum contained 40 g glucose, 1 g YE, 3 g NH4Cl, 0.5 g KH2PO4, 1 g MgSO4 x 7H2O, 2.5 mg ZnSO4 x 7H2O, 2 mg FeSO4 x 7H2O, 1 mg MnSO4 x H2O, 0.1 mg CuSO4 x 5H2O in 1L distilled water. The full growth medium contained 100 g glucose, 1 g YE, 8.6 g NH4Cl, 3 g KH2PO4, 1 g MgSO4 x 7H2O, 2.5 mg ZnSO4 x 7H2O, 2 mg FeSO4 x 7H2O, 1 mg MnSO4 x H2O, 0.1 mg CuSO4 x 5H2O and 200 μg thiamine-HCl (B1) in 1L distilled water. Citric acid production was conducted on a production medium containing 100 g glucose, 8.6 g NH4Cl, 1 g YE, 0.2 g KH2PO4, 1 g MgSO4 x 7H2O, 2.5 mg ZnSO4 x 7H2O, 2 mg FeSO4 x 7H2O, 1 mg MnSO4 x H2O, 0.1 mg CuSO4 x 5H2O, and 200 μg thiamine-HCl (B1) in 1L distilled water. Media were sterilized at 121° C for 20 minutes.

Cultivation methods

Inoculum cultures. The inoculum was grown in 0.25 flasks containing 0.025L of growth medium on an Elpan rotational shaker at 30° C for 3 days. The biomass of two shake flask cultures was inoculated into 1L of the production medium in the bioreactor.

In order to determine the elemental composition (C, H, N, P and S) of the biomass, the cultivation was performed in full medium on a shaker, under the conditions described above, for a period of 7 days.

Production cultures. Citric acid biosynthesis was performed in a 3.5L volume BIOFLO III bioreactor (New Brunswick), with 1L volume of the nutrient medium, at 30° C and the pH set at 5.5 (controlled automatically with 10M NaOH), at a stirrer speed of 500 rpm and an aeration rate of 0.2 vvm. At the beginning of the repeated batch cultivation (RBC), the process was performed under batch conditions, and next, after sugar concentration had been reduced to approximately 10 gL-1, 750 mL of the culture medium was collected from the bioreactor and replaced by 750 mL of fresh production medium. In this way, the second and subsequent batches of cultivation were launched (6 batches in total).

Total, budding and dead cell counts. The number of total (N), budding (NB) and dead (ND) cells was determined from the samples collected during cultivation, using the Thoma chamber. Each time, about 800 objects were counted in three microbiological preparations. The number of budding cells was compared with the total number of objects under investigation. The dead cells were counted in the preparations dyed with methyl blue (1:10000). The number of dark blue cells was compared with the total number of cells. The results were given in %. Measurements were carried out in three last samples (in the stationary phase) from each batch (see Fig. 1).

Fig. 1. Concentration of biomass (X), citric acid (CA), isocitric acid (ICA), and glucose (Glu) during repeated batch fermentation of CA with Y. lipolytica A-101-1.31 on glucose media

Measurements of cell volume. The volume of the cells was determined from the last three samples (in the stationary phase) collected during I, II, III, IV and V batch of cultivation. The length and width of the cells was measured using an eyepiece micrometer. Each time, 50 parent cells containing buds were measured. The volume of the cells was measured, using the formula for the volume of the ellipse. The cells were divided into 6 classes: class I <0.1 – 11.99 µm3>; class II <12 – 23.99 µm3>; class III <24 – 35.99 µm3>; class IV <36 – 47.99 µm3>; class V <48 – 59.99 µm3>; class VI (≥ 60 µm3) [8].

Reversion frequency of phenotype acu-. Stability of acu- mutants was determined at the end of each batch, using a plate method, on YNB agar-acet containing sodium acetate as a sole carbon source, according to the procedure described by Wojtatowicz & Rywińska [28].

Morphology of colonies. The last three samples (in the stationary phase) collected from the bioreactor under repeated batch cultivation were diluted, spread on YM agar and incubated at 30° C for 5 days. Stability of the phenotype of colonies was determined with regard to the occurrence of smooth colonies in relation to the total number of colonies on the plate.

Determination of biomass (X). In growth and production cultures, the biomass was determined gravimetrically after drying in a drier at 105° C.

Determination of citric acid (CA) production. CA was determined using pentabromoacetone method [22].

Determination of isocitric acid (ICA). ICA was determined using an enzymatic method, in the presence of isocitrate dehydrogenase [12].

Determination of glucose (Glu). Glucose was determined by enzyme analysis, using a ready-to-use diagnostic unit (Glucose et New, POCh Gliwice).

Determinations of carbon (C), nitrogen (N), hydrogen (H), sulphur (S) and phosphorus (P) in the biomass. The content of chemical elements in the biomass was analyzed at the end of each cultivation. C, H, N, and S were determined by gas chromatography, using a CHNS EA-1110 analyzer (CE Instruments). Phosphorus was analyzed, using an ICP-AES Liberty 220 spectrophotometer (Varian). Mineralization of the biomass samples was carried out at increased pressure in a CEM-MDS 2000 microwave mineraliser.

Statistics. One-way analysis of variance (STATGRAPHICS Plus, version 6.0) was used to determine significant differences between the results.

List of symbols
µmax = maximum specific growth rate (h-1)
QX = volumetric biomass productivity (gL-1h-1)
qCAg = specific rate of acid production in the yeast growth phase (gg-1h-1)
qCA n-g = specific rate of acid production after termination of yeast growth (gg-1h-1)
QCAg = volumetric citric acid productivity in the yeast growth phase (gL-1h-1)
QCA n-g = volumetric citric acid productivity after termination of yeast growth (gL-1h-1)
YCAP = yield of citric acid in the trophophase (gg-1)
YCAt = total yield of the process one batch of the process (gg-1)


Repeated batch cultivation containing Y. lipolytica A-101-1.31 was performed for a period of 481 hours. The process was conducted in 6 replications (batches) (Fig. 1). The length of the first batches (I, II, III) was the same, approximately 74 hrs, and next, the time was gradually extended to 95 hrs, which accounted for attenuated dynamics of growth and CA biosynthesis. Maximum biomass concentration in the first batch reached the level of 12.8 gL-1. In three subsequent processes, it reached 10 gL-1 because the media (in the second and subsequent batches) were 25% lower in major nutrients. However, a clearly increasing tendency in the biomass production was observed in batches V and VI (Table 1).

The yeasts produced CA and ICA in logarithmic growth phase as well as during stationary phase. Biosynthesis of citric acids in the first batch of cultivation started when the logarithmic growth of yeast ended, while in subsequent batches, the accumulation of acids was observed from the beginning of the process. The final CA concentration in the first four batches increased from 40.05 gL-1 to 53.9 gL-1, while its concentrations in batches V and VI were lower, 31.2 gL-1 and 34.1 gL-1, respectively. The concentration of ICA did not exceed 5% of the sum of citric acids.

Table 1. Experimental results of the CA production from glucose in RBC by Y. lipolytica A-101.1.31

Batch No.




Citric acid

Selectivity of fermentation
% CA







The curves of glucose consumption (Fig. 1) show that sugar utilization at the beginning of each batch cultivation was faster because it was used both for CA and biomass production.

The dry matter of the yeast biomass from the culture under investigation was twice lower in nitrogen (3.4%–4.3%) and about six-fold lower in phosphorus (0.3–0.4%) content as compared to that observed in the yeast growing in full medium (Table 2). This was a natural consequence of the medium content, low in nitrogen and with slight, but constant phosphorus deficiency [19].

Table 2. Comparison of the elemental composition of Y. lipolytica A-101-1.31 biomass accumulated during RBC (I and II period of culture) biosynthesis of CA related to growth conditions in full medium

Batch No.


Concentration (% in dry matter)




















Full medium (C : N : P : S ≈ 10 : 1: 0.2 : 0.1)






The results of the studies on the kinetics of yeast growth (Fig. 1) show that the logarithmic growth phase with constant, maximum µ value reached 0.23 h-1, occurred only in batch I (Table 3). In the other batches, only a linear (constant biomass gain in a time unit) and a stationary phase were observed. When the dynamics of growth was compared with respect to the volumetric biomass productivity (QX), significant differences between the batches were found. QX was decreasing from 0.54 gL-1h-1 in the first batch to 0.22 gL-1h-1 in the last one (Table 3).

Table 3. Major kinetic and yield parameters during biomass and CA production from glucose in RBC by Y. lipolytica A-101.1.31


Batch No.







µmax, h-1







QX, gL-1h-1







qCAg, gg-1h-1







qCA n-g, gg-1h-1







QCAg, gL-1h-1







QCAn-g, gL-1h-1







YCAp, gg-1







YCAt, gg-1







The dynamics of CA production by the yeast showed differences between the growth and stationary phases. A similar phenomenon was observed earlier by other researchers who studied CA biosynthesis by various yeast strains from glucose, molasses and vegetable oils [1,2,3,4,23,30]. In our study, the growing cells produced CA at a specific rate (qCAg) two-fold higher than non-growing ones (qCAn-g) (Table 3). The value of this parameter in the trophophase of batch I was 0.089 gg-1h-1. It was the highest (0.102–0.110 gg-1h-1) in batches II–IV, and next, rapidly decreased in batch V. This decreased value of qCAg remained low also in batch VI. The volumetric citric acid productivity (QCA) was also much higher in the trophophase (qCAg) than in the idiophase (qCAn-g). The non-growing cells produced CA at specific and volumetric rates slightly decreasing in batches I to IV; the decrease reached 50% in batches V and VI (Table 3).

The CA yield coefficient (g CA produced / g sugar consumption) in the trophophase (YCAP) was maintained at a high level (> 0.7 gg-1) in batches I, II, III and IV, and next rapidly decreased to 0.42–0.46 gg-1 in batches V and VI, which was strictly correlated with a decrease in final CA concentration, observed in these batches. Total CA yield (YCAt) was slightly lower (0.47 gg-1) in batch I, due to higher sugar consumption in conversion to the cell biomass. In the next 3 batches, it was maintained at a high, uniform level (0.56–0.58 gg-1), but later, in batches V and VI, CA yield markedly decreased to 0.33–0.35 gg-1.

Few data are available in the literature about CA biosynthesis by Y. lipolytica under repeated batch cultivation. As reported by Anastassiadis & Rhem [2], about 20 repeated batch experiments were sequentially carried out without any technical and microbiological stability problems at 100% conversion. Arzumanov et al., [3] described a process in which ethanol was used as a carbon source. They obtained very good results with a genetically modified strain of Y. lipolytica (VKM Y2373), which maintained high acid-forming activity (0.104–0.138 gg-1h-1) for 700 h. Much more information is available on repeated batch cultivations with biomass recycling [6,16,26]. In these processes, maximum yields (YCAt) ranged from 0.47 to 0.9 gg-1 and productivity from  0.475 to 1.36 gL-1h-1, depending on biomass concentration (16–28.9 gL-1). However, these processes were stable only for 4–10 days, therefore, for a period shorter than that observed in the present study.

The physiological state of Y. lipolytica A-101-1.31 during the entire process was good, despite unexpected presence of budding cells (NB) at a stationary phase in each batch; the percentages in the total number of cells were found within the range of 4–7.7% (Fig. 2). Taking into account the fact that no increment in biomass at this stage of culture was observed, it can be assumed that some of the cells underwent the process of autolysis, releasing the nutrients indispensable for cell growth and multiplication.

The yeast strain selected for the study exhibited good viability (Fig. 2). The dead cells dyed with blue methylene (ND) constituted a small fraction (from 1.5 to 2.1%) of the population. Other authors reported even a 25% decrease in viability of the populations in the cultures lasting longer than 500 hrs [6,16,17].

Fig. 2. Percentage of budding (NB) and dead cells (ND) during biosynthesis of CA in RBC by Y. lipolytica A-101-1.31 (in the stationary phase)

The yeast population was by no means uniform, regarding the size of cells. The range of volume in all the batches was enormous, from 3.2 to 134.7 µm3 (Table 4), which is a typical phenomenon of a stationary phase of growth of the microorganisms. On average, the volume of cells in batches I to IV was found within the range from 33.4 to 40.5 µm3; the cells in batch V were much larger (63.4 µm3) and significantly different from the cells at earlier stages of the process. For better understanding of the changes within populations, all the cells from each culture under repeated batch cultivation were divided into 6 classes, depending on the cell volume (Fig. 3). In batches I and II, 50% of the entire population was in class 1 and 2, but in batches III and IV, a considerable increase (49% of the population) was observed in cells of class 6 (≥ 60 µm3). In batches I to IV, the percentages of the cells of various classes did not influence the mean volume of the cells. On the other hand, the increased percentages of large cells in batch V, consequently increased the mean volume, which in this batch reached 63.4 µm3 (Table 4). However, the cells of the same strain under continuous cultivation in a membrane reactor, where CA is produced by non-growing cells, were much smaller. The percentages of the cells in these cultures, sized 40.1 µm3 to 50 µm3 were < 32% [18,30,30]. It seems quite likely that the conditions prevailing in these cultures, in a close system, were more favourable than those under our repeated batch cultivation.

Table 4. Volume of Y. lipolytica A-101-1.31 cells in the stationary phase during RBC biosynthesis of CA production

Batch No.



Average volume
m m3

Homogeneous groups
P = 0.0000·


























Probability level P < 0.05

Fig. 3. Percentage of volumetric classes of Y. lipolytica A-101-1.31 cells during RBC (in the stationary phase)

In batch V, the presence of untypical colonies (35.4%) with smooth surface was found (Fig. 4). In batch VI, the percentage of these colonies was > 40%. The occurrence of these forms coincided with a marked decrease in specific rate and yield of CA production and significant increase in biomass (Fig. 1). Smooth segregants of yeast Y. lipolytica were isolated earlier from one-stage continuous culture of A-101-1.31 strain, carried out in a similar medium [26]. The results obtained in those studies showed that these strains exhibited lower capability of CA synthesis as compared to the parent strain.

Fig. 4. Percentage of smooth colony during biosynthesis of CA in RBC by Y. lipolytica A-101-1.31 (in the stationary phase)

Table 5. Stability of the acu- mutation in Y. lipolytica A-101.1.31 in RBC

Batch No.

Frequency of acu+


< 2.59 x 10-7


< 5.52 x 10-7


< 1.99 x 10-7


< 2.8 x 10-7


< 2.35 x 10-7


< 1.9 x 10-7

The strain used in the present study was an acetate mutant, unable to grow on acetate used as a sole carbon source. The acu- mutation ensures the purity of citric fermentation, which is particularly important for CA producers. Stability of acu- phenotype was determined at the end of each batch process (6 replications) and it was high (Table 5), which was confirmed by constant and low production of ICA. The frequency of acetate phenotype reversion was < 5.52 x 10-7 of cells, therefore, it was lower than the frequency of spontaneous mutations (10-5 – 10-7) [5].


Summing up, the process under repeated batch cultivation ensured good parameters of kinetics and efficiency of CA synthesis at the time limited to 4 batches. In subsequent cultures, the yeast populations generated clones forming untypical S-colonies. For the entire process, the yeast were able to maintain the stability of acu- phenotype. The mean volumes of the bud-forming cells were increasing with increasing time of cultivation; in batch V, the cells were twice as large as in batch I.


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Accepted for print: 05.11.2007

Anita Rywińska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
email: anita.rywinska@wnoz.up.wroc.pl

Maria Wojtatowicz
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 25, 50-375 Wrocław, Poland
Fax. 4871- 3284124
Phone: 48-71-3205117
email: mwojt@wnoz.ar.wroc.pl

Barbara Żarowska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 25, 50-375 Wrocław, Poland
email: zarowska@ozi.ar.wroc.pl

Waldemar Rymowicz
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Norwida 25, 50-373 Wroclaw, Poland
Fax. 48-71-3284124
Phone: 48-71-3205143
email: rymowicz@ozi.ar.wroc.pl

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