Lipid production by yeasts growing on biodiesel-derived crude glycerol: strain selection and impact of substrate concentration on the fermentation efficiency.

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Article Type: Original Article

Lipid production by yeasts growing on biodiesel-derived crude glycerol: strain selection and impact of substrate concentration on the fermentation efficiency

S.S. Tchakouteu1, O. Kalantzi1, C. Gardeli1, A.A. Koutinas1, G. Aggelis2,3 and S. Papanikolaou1

1 Department of Food Science and Human Nutrition, Agricultural University of Athens, Athens, Greece 2 Unit of Microbiology, Department of Biology, Division of Genetics, Cell and Development Biology, University of Patras, Patras, Greece 3 Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

Correspondence S. Papanikolaou, Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, Athens, Greece. E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12736 This article is protected by copyright. All rights reserved.

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Running title: Single cell oil from waste glycerol fermentation S.S. Tchakouteu et al.

Abstract Aims: To screen yeasts in relation to the potential to produce single cell oil (SCO) from biodiesel-derived glycerol and to enhance SCO production in Lipomyces starkeyi and Rhodosporidium toruloides yeasts. Methods and Results: Yarrowia lipolytica, Cryptococcus curvatus, R. toruloides and L. starkeyi were grown in nitrogen-limited flask cultures. Y. lipolytica strains produced citric acid and mannitol. L. starkeyi DSM 70296 and R. toruloides NRRL Y-27012 showed potential for SCO production, and were cultivated at increasing initial glycerol concentrations with the initial nitrogen concentration remaining constant. Significant biomass and SCO production was reported even in cultures with high initial glycerol concentrations (i.e. 180 g l-1). Lipid quantities of c. 12 g l-1 (lipid in dry cell weight 35–40%) were obtained for both L. starkeyi and R. toruloides, quite high values compared with literature values for oleaginous microorganisms growing on glycerol. However, these strains presented different kinetic profiles for the synthesis of intracellular polysaccharides. L. starkeyi produced a significant quantity of polysaccharides (c. 7 g l-1). The yeast lipids contained mainly oleic and palmitic and to a lesser extent linoleic and stearic acids. Conclusions: L. starkeyi and R. toruloides are potential SCO producers from crude glycerol. Significance and Impact of the Study: Very scarce numbers of reports have indicated the production of SCO by L. starkeyi and R. toruloides growing on glycerol. We report here that these yeasts are able efficiently to convert raw glycerol into SCO, while L. starkeyi also synthesizes intracellular polysaccharides in marked quantities.

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+B: Growth of Rhodosporidium toruloides NRRL Y-27012 and Lipomyces starkeyi DSM 70296 with increasing initial glycerol As seen in the previous paragraph, R. toruloides NRRL Y-27012 showed a clearly higher biomass production and relatively higher lipid accumulation compared with R. toruloides DSM 4444. Likewise, L. starkeyi DSM 70296 had an appreciable DCW and lipid production during its cultivation on crude glycerol. In the next step, it was desirable to enhance the production of single cell oil in these micro-organisms; hence, R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296 were cultivated on media containing higher initial glycerol concentrations by maintaining the same initial nitrogen quantity (utilization of peptone at 0.75 g l-1 and yeast extract at 0.5 g l1),

in order to formulate culture media presenting a higher excess of carbon, and, therefore, to

favour the process of lipid accumulation (Ratledge 1997; Papanikolaou and Aggelis 2011a). The results are shown in Tables 2 and 3, for R. toruloides and for L. starkeyi, respectively. For R. toruloides NRRL Y-27012 this cultivation clearly enhanced the production of total biomass and lipid up to an initial glycerol concentration of c. 120 g l-1. Significant quantities of lipids (12 g l-1) were produced (see Table 2). However, it is interesting to note that within the range of Glol0 concentrations tested 50–120 g l-1, although in absolute values the quantities of produced DCW and lipid increased with the increment of Glol0 concentration, the lipid dry weight (YL/DCW) values decreased. Moreover, another interesting feature of the microbial behaviour was related to the fact that growth did not cease in the trial in which a very high Glol0 concentration was tested (180 g l-1). Even with this trial, a non-negligible production of DCW and lipid was observed. Interestingly, at this very high Glol0 concentration, the YL/DCW value was the highest obtained in this study by this micro-organism, being c. 54% w/w, while marked assimilation of glycerol occurred (i.e. 360 h after inoculation c. 45% w/w of glycerol had been consumed by the micro-organism; further incubation did not improve the assimilation of glycerol). However, it was apparent that substrate inhibition occurred due to the increasing Glol0 concentrations, since the higher the Glol0 concentration in the medium, the greater the


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quantities up to c. 85% w/w) in several algal species such as Dunaliella sp. (Clomburg and Gonzalez 2013). In some of the above-mentioned cases, quantities of glycerol up to 7.8 mol l-1 (equivalent to c. 720 g/l of glycerol in water) can occur (Clomburg and Gonzalez 2013). It may be assumed therefore, that conversion of this low- or even negative-value product to higher added-value compounds by the means of chemical or fermentation technology currently attracts high and continuously increasing interest; as far as biotechnological conversions are concerned, in several cases, prokaryotic micro-organisms have been implicated in the fermentative conversion of (crude) glycerol into compounds such as bioalcohols (principally 1,3-propanediol and to a lesser extent 2,3-butanediol, ethanol and butanol), poly-(hydroxylakanoates), biosurfactants, dihydroxyacetone and succinic acid (Yazdani and Gonzalez 2007; Celińska and Grajek 2009; Clomburg and Gonzalez 2012; Kachrimanidou et al. 2013). Likewise, a significant number of reports, appearing in most cases in the past few years, indicates the potential of eukaryotic micro-organisms (yeasts, molds and heterotrophically grown algae) to convert (crude) glycerol into a plethora of metabolic compounds of added-value, such as microbial lipids (also called single cell oils, SCOs) citric acid, microbial mass, enzymes and polyols (for reviews see: Papanikolaou and Aggelis 2009; Rivaldi et al. 2009; Wen et al. 2009a, 2009b; Rywińska et al. 2013a; Abghari and Chen 2014). Single cell oils are of great interest for industrial biotechnology, since they can be used either as replacements of high-added value oils and fats rarely found in the plant or animal kingdom, or they can constitute precursors of the synthesis of ‘2nd’ or ‘3rd’ generation biodiesel (Papanikolaou and Aggelis 2009, 2011a, 2011b; Abghari and Chen 2014). Although (crude, biodiesel-derived) glycerol has been used in many studies as a substrate for the production of SCOs by several eukaryotic microbial strains (Meesters et al. 1996; Papanikolaou et al. 2002; Chi et al. 2007; Pyle et al. 2008; André et al. 2009, 2010; Liang et al. 2010a, 2010b; Makri et al. 2010; Chatzifragkou et al. 2011a; Ethier et al. 2011; Saenge et al. 2011; Dedyukhina et al. 2012; 2014; Fontanille et al. 2012; Chang et al. 2013; Cui et al. 2012; Duarte et al. 2013a, 2013b; Kitcha and Cheirslip 2013; Louhasakul and Cheirslip 2013; Wensel et al. 2014), or as a substrate for the


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production of metabolic compounds (such as citric acid, acetic acid, polyols, etc) by yeast strains (Papanikolaou et al. 2002, 2008, 2013; Rymowicz et al. 2008, 2010; André et al. 2009; Rywińska et al. 2009, 2011; Rywińska and Rymowicz 2010; Chatzifragkou et al. 2011a, 2011b; Kamzolova et al. 2011; Morgunov et al. 2013; Petrik et al. 2013), data concerning the growth and lipid production by strains of yeasts Rhodosporidium toruloides and Lipomyces starkeyi growing on glycerol are scarce in the international literature (Moreton 1988; Xu et al. 2012; Uçkun Kiran et al. 2013; Yang et al. 2014). The aim of the current study was to investigate the potential of biodiesel-derived waste glycerol conversion into metabolic compounds of added-value (i.e. yeast biomass, SCOs, intracellular polysaccharides) by yeast strains. After a first initial selection of yeast strains cultivated on biodiesel-derived waste glycerol utilized as a carbon source under nitrogen-limited conditions (conditions that favour the accumulation of storage lipid by microorganisms) two microbial species not often studied with regard to their potential for producing lipid from crude glycerol, namely Lipomyces starkeyi (strain DSM 70296) and Rhodosporidium toruloides (strain NRRL Y-27012) were studied in depth.

+A: Materials and Methods +B: Microorganisms and media The following strains were used in this study: Yarrowia lipolytica ACA-YC 5029 (Papanikolaou et al. 2009), Yarrowia lipolytica ACA-YC 5033 (Papanikolaou et al. 2009), Cryptococcus curvatus NRRL Y-1511, Rhodosporidium toruloides DSM 4444, Rhodosporidium toruloides NRRL Y-27012 and Lipomyces starkeyi DSM 70296. The NRRL Y strains were provided by the NRRL culture collection (Peoria, USA), while the DSM strains were provided by the DSMZ culture collection (Leibniz, Germany). All strains were maintained on yeast peptone dextrose agar (YPDA) at 4°C and sub-cultured every 4 months in order to maintain their viability. All experiments were performed in submerged shake-flask cultures. The culture medium


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salt composition was (g l-1): KH2PO4 7.0; Na2HPO4 2.5; MgSO4×7H2O 1.5; CaCl2×2H2O 0.1; FeCl3×6H2O 0.15; ZnSO4×7H2O 0.02; MnSO4×H2O 0.06 (Papanikolaou et al. 2002). Peptone (0.75 g l-1) and yeast extract (0.5 g l-1) were used as nitrogen sources. The peptone and yeast extract contained 14% w/w and 7% w/w of nitrogen, respectively. Prior to any inoculation in the liquid growth medium the strains were regenerated so that the inoculum would be 3 days old. The preculture was prepared as follows: 250 ml Erlenmeyer flasks were filled with 50 ± 1 ml medium containing mineral salts and glucose at a concentration of 10 g l-1 and were inoculated aseptically from the principal freshly regenerated strain. Precultures were incubated in an orbital shaker (for 24 ± 2 h at 180 ± 5 rpm, 28 ± 1°C; in the case of L. starkeyi, the duration of incubation was 48 h). In many of the trials, the inoculation volume of the preculture to the principal culture was 1 ml (2% v/v inoculum; c. 106 cfu; initial dry cell weight (DCW0) of the inoculum c. 0.08 g l-1). However, in the trials with R. toruloides and L. starkeyi strains, in which the initial concentration of glycerol (Glol0) was higher than 30 g l-1, the inoculation volume of the preculture to the principal culture was 5 ml (10% v/v inoculum; c. 5×106 cfu; DCW0 c. 0.4 g l-1). The carbon source used in the principal cultures for the current investigation was biodiesel-derived crude glycerol provided by Pavlos N. Pettas Fats and Oils SA, Patras, Greece, derived from trans-esterification of various blends of edible oils in order to generate biodiesel. The purity of the feedstock was as follows: glycerol 91–93% w/w; impurities composed of 2–4% w/w water, 2–4% w/w potassium salts, 1% w/w free-fatty acids (Kachrimanidou et al. 2013). In order to carry out experiments with various Glol0 concentrations, appropriate calculations were performed to take into consideration the purity of the feedstock used. Ιn all calculations, the actual concentration of Glol0 (measured at the beginning of all fermentations) was taken into consideration. The initial pH for all media before and after sterilization (performed at 115°C, 20 min) was 6.0 ± 0.1. No acid addition was required, because although the pH of the glycerol feedstock was somewhat elevated (c. 10.0), the dilution of glycerol into the medium presented a satisfactory buffering capacity resulting in a decrease of the pH of the culture medium to c. 6.0. All experiments were performed in shake-flask cultures performed in 250 ml non-baffled conical


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flasks filled with 50 ± 1 ml of culture medium, previously sterilized. Flasks were inoculated with 1 or 5 ml of preculture (see above), incubated in a rotary shaker (New Brunswick Sc, USA) at 28 ± 1°C and 180 ± 5 rpm and periodically removed from the incubator for further analyses.

+B: Analytical methods The kinetics of dry biomass production, glycerol consumption, organic acids or polyols secretion (if appropriate) and intracellular lipids and polysaccharides production were assessed. A Jenway 3020 pH meter was used for pH measurements. As indicated, the initial pH of the cultures was 6.0 ± 0.1. If during culture, (specifically for trials with Y. lipolytica strains) there was an accumulation of organic acids in the medium, and the pH value in the culture medium dropped markedly, the medium pH was maintained in the range 5.0–6.0 by adding (periodically and aseptically) small quantities (e.g. 500–600 μl) of 5 mol l-1 KOH to the flasks (see: Papanikolaou et al. 2002). The dissolved oxygen tension (DOT in %, v/v) was measured with the aid of a selective electrode (oxi200 Sensodirect, Lovinbod) according to previous literature (Papanikolaou et al. 2004). All trials were conducted under fully aerobic conditions (DOT > 20% v/v for all growth steps). Before determination of the substrate (glycerol) or the metabolic compounds, after the measurement of DOT and the correction of pH, the volume of the collected cultures was corrected to 50 ± 1 ml (or 55 ± 1 ml when a 5 ml inoculum had been used). Due to water evaporation the volume of medium in the flasks after harvesting was 47 ± 2 ml). Yeast cells harvested by centrifugation (Hettich Universal 320-R, Germany) at 10000 × g for 15 min, were washed twice with distilled water and recentrifuged as above. The biomass concentration (in g l1)

was determined by dry cell weight (DCW) determination (90 ± 5°C until constant weight, in

most cases c. 28 h). Glycerol (Glol, in g l-1), organic acids and polyols (in g l-1) were determined with the aid of a HPLC device as described previously (Kachrimanidou et al. 2013). Total cellular lipids (L, expressed as g l-1 and % of DCW-YL/DCW) were extracted from the dried biomass with a mixture of chloroform/methanol 2/1 (v/v) and determined


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gravimetrically (Papanikolaou et al. 2002, 2011). For L. starkeyi, a quantity of dried cells (i.e. c. 100 mg) was acidified first by adding 10 ml HCl (2.5 mol l-1) at 100°C for 30 min and then a mixture of chloroform/methanol 2/1 (v/v) was used to extract total lipids from the acidified biomass. This was necessary because without the prior acidification step, the extraction of total cellular lipids completely failed; for example, in trials in which the Glol0 concentration was adjusted to c. 30 g l-1, for a DCW concentration achieved of 9.0 g l-1, the YL/DCW value obtained without previous boiling and acidification was c. 8.0% w/w (L = 0.72 g l-1), compared with c. 33.0% w/w (L = 2.95 g l-1) after boiling and acidification. On the other hand, in the other strains (Y. lipolytica ACA-YC 5029, Y. lipolytica ACA-YC 5033, C. curvatus NRRL Y-1511, R. toruloides DSM 4444 and R. toruloides NRRL Y-27012) both extraction methods (extraction of lipids from DCW with or without previous boiling and acidification of the DCW) gave almost equivalent results with regard to the quantity of total lipids produced. Lipids were converted to their fatty acid methyl-esters (FAMEs), analysed in a GC apparatus (Fisons 8000 series) equipped with a FID (Fisons) according to Zikou et al. (2013) and identified by comparison with authentic standards. In some of the performed trials the total intracellular polysaccharide production was assessed. Total intracellular polysaccharides (IPS, expressed as g l-1 and % of DCW-YIPS/DCW) were measured using a modified protocol proposed by Liang et al. (2010b). Briefly, c. 50 mg of DCW was acidified by adding 10 ml HCl (2.5 mol l-1). The acidified solution was then hydrolysed at 100°C for 30 min and neutralized to pH 7 with c. 10 ml KOH (2.5 mol l-1), filtered through Whatman filter paper and, finally, the reducing sugar content was determined using the 3,5-dinitrosalicylic acid method (Miller 1959). Experiments for all micro-organisms tested were performed in duplicate using different inocula. All the experimental points presented in the tables and the figures are the mean value of two independent determinations, with a standard error ≤ 10%.


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+A: Results +B: Initial screening of yeast strains on crude glycerol-based media In the first part of this work, the six strains were tested on media composed of crude glycerol at an initial concentration adjusted to c. 30 g l-1 under nitrogen-limited conditions (utilization of peptone 0.75 g l-1 and yeast extract 0.5 g l-1; initial molar ratio c. 100 moles moles-1) in order to favour the accumulation of storage lipids and (potentially for the Yarrowia lipolytica strains) the secretion of secondary metabolites (e.g. citric acid, mannitol, etc) useful for the food industry. The results obtained for DCW and lipid production are shown in Table 1. Indeed, as has already been reported previously for several strains of Y. lipolytica (Papanikolaou et al. 2002, 2008, 2013; Makri et al. 2010; Chatzifragkou et al. 2011a) batch cultures on glycerol, despite the significant nitrogen limitation imposed, were not accompanied by a marked accumulation of lipids per unit of DCW. What was interesting in the current study and coincided with previous information concerning the growth of several strains of Y. lipolytica on hydrophilic carbon sources (e.g. glycerol, glucose, etc) (Makri et al. 2010; Chatzifragkou et al. 2011a; Sarris et al. 2011; Papanikolaou et al. 2013) was the fact that YL/DCW values in the first stages of culture increased, despite the fact that nitrogen was found in excess in the growth medium (YL/DCW reached c. 21% w/w for the strain ACA YC 5033). Thereafter, cellular lipids were subjected to degradation, accompanied by secretion into the medium of citric acid (for ACA-YC 5033) and a mixture of citric acid and mannitol (for ACA-YC 5029). Patterns of changes of DCW (g l-1), citric acid (g l-1) and lipid in DCW (%, w/w) for the strain ACA-YC 5033 are illustrated in Fig. 1, in which, in agreement with the current literature (Papanikolaou et al. 2002, 2009; Rymowicz et al. 2006, 2010; Sarris et al. 2011) citric acid was produced almost exclusively at the stationary growth phase, while, as previously stressed, its secretion coincided with a significant decrease in the quantity of lipids per unit of DCW (Makri et al. 2010; Sarris et al. 2011). As far as the other yeast strains screened in this work are concerned, Cryptococcus curvatus NRRL Y-1511 presented an interesting biomass formation (11.8 g l-1) with a


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concomitant dry biomass yield per unit of glycerol consumed (YDCW/Glol) of c. 0.46 g g-1. Nevertheless, despite the significant nitrogen limitation imposed, under the present culture conditions this micro-organism accumulated relatively low quantities of total lipids (YL/DCW = 12.5% w/w; Table 1). Moreover, the micro-organism Rhodosporidium toroloides DSM 4444 presented a satisfactory biomass production (DCW ≈ 9 g l-1; YDCW/Glol ≈ 0.25 g g-1) but it accumulated moderate quantities of lipid (YL/DCW = 24.9% w/w). Higher quantities of total biomass (DCW = 11.0 g l-1; YDCW/Glol ≈ 0.36 g g-1) were produced by the strain R. toruloides NRRL Y-27012, accumulating slightly higher quantities of total lipids compared with DSM 4444 (YL/DCW = 25.7% w/w). Likewise, Lipomyces starkeyi DSM 70296 produced marked quantities of total biomass (DCW = 9.0 g l-1; YDCW/Glol ≈ 0.43 g g-1) that contained interesting quantities of lipids (YL/DCW = 32.7% w/w; L = 2.95 g l-1). In the latter fermentation, although some non-negligible quantities of glycerol remained unconsumed in the growth medium (at 192 h, Glol ≈ 6 g l-1), the micro-organism showed a significant degradation of its previously accumulated lipids resulting in a further increase of the DCW value achieved (Table 1). Besides the strains of Y. lipolytica, in the other yeasts screened (C. curvatus, R. toruloides and L. starkeyi), the pH of the culture medium showed an insignificant drop (initial 6.0 ± 0.1, final 5.7 ± 0.1), indicating a small secretion of organic acids into the culture medium. Indeed, HPLC analysis of the supernatant at the end of culture of C. curvatus, R. toruloides and L. starkeyi showed that citric acid in small concentrations (0.8 ± 0.2 g l-1) was the sole extracellular metabolite detected. It is evident that, unlike the two Y. lipolytica strains tested in which glycerol was mainly converted into citric acid (ACA-YC 5033) and blends of citric acid and mannitol (ACA-YC 5029), in all the other microorganisms (C. curvatus, R. toruloides and L. starkeyi) the carbon flow was mainly channeled towards the synthesis of biomass (presenting maximum absolute values 45–93% higher than the maximum 6.1 g l-1 for ACA YC 5033) and lipid (presenting maximum absolute values that were 72–343% higher than the maximum obtained of 0.86 g l-1 for ACA YC 5033) (Table 1).


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+B: Growth of Rhodosporidium toruloides NRRL Y-27012 and Lipomyces starkeyi DSM 70296 with increasing initial glycerol As seen in the previous paragraph, R. toruloides NRRL Y-27012 showed a clearly higher biomass production and relatively higher lipid accumulation compared with R. toruloides DSM 4444. Likewise, L. starkeyi DSM 70296 had an appreciable DCW and lipid production during its cultivation on crude glycerol. In the next step, it was desirable to enhance the production of single cell oil in these micro-organisms; hence, R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296 were cultivated on media containing higher initial glycerol concentrations by maintaining the same initial nitrogen quantity (utilization of peptone at 0.75 g l-1 and yeast extract at 0.5 g l1),

in order to formulate culture media presenting a higher excess of carbon, and, therefore, to

favour the process of lipid accumulation (Ratledge 1997; Papanikolaou and Aggelis 2011a). The results are shown in Tables 2 and 3, for R. toruloides and for L. starkeyi, respectively. For R. toruloides NRRL Y-27012 this cultivation clearly enhanced the production of total biomass and lipid up to an initial glycerol concentration of c. 120 g l-1. Significant quantities of lipids (12 g l-1) were produced (see Table 2). However, it is interesting to note that within the range of Glol0 concentrations tested 50–120 g l-1, although in absolute values the quantities of produced DCW and lipid increased with the increment of Glol0 concentration, the lipid dry weight (YL/DCW) values decreased. Moreover, another interesting feature of the microbial behaviour was related to the fact that growth did not cease in the trial in which a very high Glol0 concentration was tested (180 g l-1). Even with this trial, a non-negligible production of DCW and lipid was observed. Interestingly, at this very high Glol0 concentration, the YL/DCW value was the highest obtained in this study by this micro-organism, being c. 54% w/w, while marked assimilation of glycerol occurred (i.e. 360 h after inoculation c. 45% w/w of glycerol had been consumed by the micro-organism; further incubation did not improve the assimilation of glycerol). However, it was apparent that substrate inhibition occurred due to the increasing Glol0 concentrations, since the higher the Glol0 concentration in the medium, the greater the


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decrease in the yield of DCW and total lipid produced per unit of glycerol consumed (YDCW/Glol and YL/Glol) (see Table 2). Specifically, when Glol0 was 50 g l-1 the YL/Glol value was 0.17 g g-1 (close to the maximum achievable 0.21 g g-1 – see Papanikolaou and Aggelis 2011a), this value decreased to c. 0.10–0.11 g g-1 when the Glol0 concentration rose to c. 120 or 180 g l-1 (Table 2). The YDCW/Glol value clearly dropped from 0.36 to 0.20 g g-1 with the rise of glycerol concentration from 50 to 180 g l-1, clearly demonstrating the negative effect of the high glycerol concentration on the biosynthetic ability of R. toruloides NRRL Y-27012. Similarly, L. starkeyi DSM 70296 had a significantly increased production of DCW and SCO with increments in the Glol0 concentration until the initial substrate concentration was c. 120 g l-1 (see Table 3). Significant quantities of lipids (12.5 g l-1) and total biomass (34.4 g l-1), the highest values reported in this investigation, were produced by L. starkeyi in the trial with Glol0 ≈ 120 g l-1 (see Table 3). The production of SCO in absolute values (g l-1) clearly increased with the rise of Glol0 concentration in the medium (up to c. 120 g l-1), whereas in accordance with most of the findings of the international literature (Papanikolaou and Aggelis 2011b), YL/DCW equally increased with the increase of glycerol concentration. Moreover, in contrast to the trials with R. toruloides NRRL Y-27012, the yields with L. starkeyi DSM 70296 were improved by the increase of Glol0 concentration up to the threshold of 120 g l-1; this statement can be justified by the fact that the yields of YDCW/Glol and YL/Glol were higher in the trial in which the concentration of carbon substrate was c. 120 g l-1 (= 0.30 and 0.11 g g-1, respectively), being clearly higher than those obtained in the trials with Glol0 ≈ 50 and 100 g l-1. Finally, as observed in R. toruloides NRRL Y-27012, the strain L. starkeyi DSM 70296 presented appreciable biomass production, notable glycerol assimilation and non-negligible SCO production in a fermentation in which a significantly high Glol0 concentration had been added to the medium (c. 180 g l-1). However, in any case, the yields of YDCW/Glol and YL/Glol were lower for this micro-organism at this initial substrate concentration within the range of Glol0 concentrations tested in the current study, a fact that suggests substrate inhibition due to the very high Glol0 quantity in the medium. It should also be pointed out that in this trial, in accordance with the results obtained for R.


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toruloides, further incubation after 310 h did not improve glycerol assimilation and biomass and SCO production by L. starkeyi. In addition to the production of SCO by both R. toruloides and L. starkeyi it was also desirable to study the production of intracellular polysaccharides (IPS) as a function of the fermentation time. The results for DCW (g l-1), SCO (g l-1), IPS (g l-1), glycerol (g l-1), YL/DCW (% w/w) and YIPS/DCW (% w/w) at one Glol0 concentration are shown for R. toruloides (Fig. 2a, b) and L. starkeyi (Fig. 3a, b). Different kinetic profiles were seen in the production of IPS and lipids: for R. toruloides, intracellular polysaccharides in significant quantities were principally synthesized at the early growth steps (Figs 2a, b). It is interesting to note that significant quantities of IPS per unit of DCW (i.e. YIPS/DCW ≈ 40% w/w) were synthesized up to 50 h after inoculation, when nitrogen was found in the growth medium (extracellular nitrogen analysis not presented). Thereafter, the YIPS/DCW quantities constantly decreased (final YIPS/DCW value ≈12% w/w, after c. 400 h of culture), while in absolute values (g l-1), after significant biosynthesis had occurred at the first growth steps, c. 50 h after inoculation and until the end of the culture the concentration of intracellular polysaccharides remained practically constant. On the other hand, after virtual assimilable nitrogen depletion from the medium (c. 50 h after inoculation) significant quantities of lipids were produced with a threshold of lipid in DCW (YL/DCW) being c. 40% w/w throughout the culture (Fig. 2b). L. starkeyi showed a different kinetic profile; IPS per unit of DCW remained practically constant with a value c. 30% w/w throughout the culture, while in absolute values (g l-1), the concentration of intracellular polysaccharides increased constantly until the end of the culture (with a significant IPS quantity ≈ 7 g l-1 at c. 350 h after inoculation). Likewise, the total lipids in both absolute (g l-1) and relative (%, w/w) terms increased constantly, with a significant onset of lipid production found after nitrogen limitation, until virtual glycerol depletion from the growth medium (Fig 3a, b).


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+B: Fatty acid composition of lipids produced by Rhodosporidium toruloides NRRL Y27012 and Lipomyces starkeyi DSM 70296 Total lipids of R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296 cultivated at several Glol0 concentrations were converted into their respective FAMEs and analysed at the early and the late growth steps of the cultures (R. toruloides, Table 4; L. starkeyi Table 5). From the results it can be seen that the fatty acid (FA) composition of the cellular lipids varied as a function of the fermentation time and with the Glol0 concentration, in the case of R. toruloides, higher quantities of unsaturated FAs (in % w/w), mainly Δ9C18:1 and Δ9,12C18:2 were detected at the early compared with the late growth phase (Table 4). Likewise, the results indicated that the higher the Glol0 concentration in the medium, the greater the quantity of saturated FAs (mainly C16:0 and to a lesser extent C18:0) in the lipids of R. toruloides (Table 4). On the other hand, a different kinetic profile of the FA composition of the lipids of L. starkeyi was identified, both with regard to the fermentation time and the Glol0 concentration in the medium; the cellular lipids of L. starkeyi were more saturated at the early growth steps than the later, whereas the greater the Glol0 concentration in the medium the more cellular unsaturated FAs (Table 5). In any case, the SCO of both R. toruloides and L. starkeyi was mainly composed of C16:0 and Δ9C18:1 and to lesser extent of the FAs C18:0 and Δ9,12C18:2 fatty acids, constituting in accordance with the literature (Li et al. 2007; Zhao et al. 2008; Xu et al. 2012), a perfect material amenable to be converted into 2nd generation biodiesel.

+A: Discussion Yeast strains Yarrowia lipolytica, Cryptococcus curvatus, Rhodosporidium toruloides and Lipomyces starkeyi were initially screened in nitrogen-limited media composed of biodieselderived waste glycerol (at Glol0 concentration adjusted to c. 30 g l-1) for their potential to produce (mostly) SCO and probably other valuable metabolites such as citric acid, polyols, etc. The past few years, a continuously increasing number of researchers have been interested in


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screening studies related to the selection of yeast strains capable of processing glycerol into metabolites of significance for industrial biotechnology, such as pigments, yeast biomass, organic acids, polyols, ribonucleotides, SCO, etc (Chatzifragkou et al. 2011a; Rivaldi et al. 2012; Taccari et al. 2012; Duarte et al. 2013a; Juszczyk et al. 2013; Kitcha and Cheirslip 2013; Petrik et al. 2013). On the other hand, many screening studies of (mainly) yeasts capable of producing SCO growing on several carbon sources (e.g. glycerol, molasses, lignocellulosic sugars, etc) have been performed the past years and a large number of strains have been tested, due to the continuously increasing interest of the scientific community towards the discovery of new strains capable of storing high levels of SCOs, amenable to being converted into 2nd generation biodiesel (Duarte et al. 2013a; Sitepu et al. 2013, 2014; Vieira et al. 2014). In the present research paper a relatively small number of strains was tested, so as to include data concerning the effect of glycerol concentration on yeast growth and lipid production that would be of interest for researchers and engineers. On the other hand, in the current investigation R. toruloides and L. starkeyi yeasts were included, since strains of these species have shown excellent capacities of lipid production during growth on hydrophilic carbon sources (in some cases glycerol was amongst the substrates tested) (Moreton 1988; Xu et al. 2012; Uçkun Kiran et al. 2013; Vieira et al. 2014; Yang et al. 2014). In the current study, from this initial screening test, C. curvatus NRLL Y-1511 was capable of producing non-negligible quantities of DCW (c. 12 g l-1) with a YDCW/Glol of 0.46 g g-1, constituting an interesting value reported for the production of yeast biomass from crude glycerol-based media (Chatzifragkou et al. 2011a; Taccari et al. 2012; Juszczyk et al. 2013). It is interesting to note that, despite the nitrogen-limited conditions imposed, the above-mentioned micro-organism did not produce significant quantities of SCO, in contrast to the results reported for other strains of the same species (i.e. C. curvatus ATCC 20509). This strain (ATCC 20509) has been revealed capable of producing marked quantities of SCO during growth on media composed of (pure or crude) glycerol; specifically, when cultivated in fed-batch mode with pretreated crude glycerol employed as a substrate, this strain produced c. 22 g l-1 of lipid with a


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concomitant YL/DCW value of c. 49% w/w (Cui et al. 2012). During growth on hydrolysed spent yeast waste blended with glycerol, ATCC 20509 strain had a lipid production of c. 19 g l-1 with a respective YL/DCW of c. 38% w/w (Ryu et al. 2013). In fed-batch bioreactor trials performed with different feeding strategies, the above-mentioned strain managed to produce SCO in the range 13.7–17.4 g l-1 with respective YL/DCW values of 44.6 and 52.9% w/w (Liang et al. 2010a). Finally, the same strain (ATCC 20509) when cultivated in fed-batch cultures with pure glycerol as a substrate produced c. 30 g l-1 of lipid with YL/DCW ≈ 25% w/w (Meesters et al. 1996). The two strains of Y. lipolytica employed in the current investigation (ACA-YC 5029 and ACA-YC 5033) produced some quantities of citric acid during their growth on glycerol. Both the maximum quantity of citric acid produced and the conversion yield of citric acid produced per glycerol consumed are (relatively or much) lower than the respective values reported in the international literature for flask or bioreactor cultures of Y. lipolytica strains cultivated on glycerol (maximum values 50–155 g l-1 with concomitant yields 0.55–0.90 g g-1 in the literature) (Rymowicz et al. 2006, 2010; Papanikolaou et al. 2008, 2013; Rywińska and Rymowicz 2010; Rywińska et al. 2010, 2011; Kamzolova et al. 2011; Morgunov et al. 2013). Moreover, an interesting result associated with the growth of ACA-YC 5033 cultivated on crude glycerol was that this strain secreted mannitol in non-negligible quantities into the growth medium (up to 6.0 g l-1). This polyol was synthesized almost in equal quantities to citric acid (citric acid produced of c. 7.0 g l-1). No other polyols (i.e. erythritol) were produced. In a relatively scarce number of reports, the secretion of polyols into the culture medium together with citric acid, when glycerol was utilized as the sole carbon source in nitrogen-limited submerged experiments, has been reported (Rymowicz et al. 2008; Rywińska and Rymowicz 2009). On the other hand, the production of erythritol, in some cases in very high quantities (e.g. > 45 g l-1 or even > 80 g l-1) has been reported when crude or pure glycerol was used as a fermentation substrate by wild or mutant Y. lipolytica strains (Tomaszewska et al. 2012; Rywińska et al. 2013b).


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In accordance with reports in the literature for various strains of this species (André et al. 2009; Papanikolaou and Aggelis 2009; Papanikolaou et al. 2009, 2013; Makri et al. 2010; Sarris et al. 2011), the two Y. lipolytica strains during growth on glycerol, a substrate for de novo lipid accumulation, under nitrogen-limited conditions, did not present features typical of classical oleaginous microorganisms; in the first growth step, and during the balanced growth phase (nitrogen-excess conditions), both strains accumulated some storage lipid (for ACA YC 5033 the YL/DCW was > 20% w/w, considered a threshold that categorizes a micro-organism as ‘oleaginous’; Ratledge 1997; Papanikolaou and Aggelis 2009). Thereafter, and despite the presence of significant glycerol in the medium and progressive and almost complete exhaustion of nitrogen from the medium, the YL/DCW values were depleted while simultaneously lowmolecular weight metabolites (citric acid and mannitol) were secreted into the medium. The concentration of available nitrogen is important for SCO production in Y. lipolytica, since, as demonstrated in the literature (Fontanille et al. 2012; Papanikolaou et al. 2013), some quantities of nitrogen seem indispensable for lipid accumulation, whereas when the nitrogen concentration drops below a threshold value, secondary metabolites, and notably citric acid, are produced, with lipid biodegradation being observed (Papanikolaou et al. 2013). Likewise, in agreement with the current investigation and unlike typical oleaginous strains, during growth on glycerol other Y. lipolytica strains (e.g. ATCC 20460) have been reported to show increased YL/DCW at the beginning of the culture (YL/DCW ≈ 32% w/w 48 h after inoculation) (Sestric et al. 2014), while a restricted number of strains have been reported to show a high accumulation of lipid (e.g. > 10 g l-1 with simultaneous YL/DCW > 30% w/w) during growth on glycerol in batch or fed-batch fermentations (Fontanille et al. 2012; Celińska and Grajek 2013). R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296 when cultivated at several Glol0 concentrations under constant nitrogen availability showed significant DCW and SCO production; interestingly both strains presented marked biomass production and glycerol assimilation even at very high Glol0 concentrations added to the medium (180 g l-1), although a conversion yield reduction due to the high Glol0 concentration was observed. In similar types of


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experiments, the strain C. curvatus ATCC 20509, when it was flask-cultured on various initial (crude or pure) glycerol concentrations presented significantly decreased microbial growth when the Glol0 concentration > 60 g l-1 (Meesters et al. 1996; Liang et al. 2010a). For this reason, and in order to increase the quantity of glycerol assimilated by the strain so as to enhance the production of SCO, fed-batch fermentation strategies were used (Meesters et al. 1996; Liang et al. 2010a; Cui et al. 2012). Other yeast strains showed similar physiological behaviour to the strains investigated in the current study; for instance, Y. lipolytica ACA-DC 50109 showed significant biomass and citric acid formation and notable glycerol assimilation in media in which the Glol0 concentration was adjusted to c. 170 g l-1 (Papanikolaou et al. 2008). Likewise, in a recent development a newly isolated strain, Metschnikowia pulcherrima, when cultivated in flasks, was capable of biomass production and glycerol assimilation even in media containing extremely high Glol0 concentrations of c. 250 g l-1 (Santamauro et al. 2014). As far as oleaginous fungi are concerned, strains belonging to the genus Mortierella have been shown capable of remarkable growth, SCO production and glycerol assimilation in media with Glol0 concentrations of >100 g l-1 with little inhibition being observed due to the increment of glycerol concentration in the medium (Papanikolaou et al. 2008; Dedyukhina et al. 2012); for instance, two M. alpina strains cultivated on media in which increasing Glol0 concentrations had been used, showed the highest DCW and glycerol assimilation values for Glol0 concentration values > 110 g l-1 (Dedyukhina et al. 2012). R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296 showed presented notable production of DCW and microbial lipids during growth on biodiesel-derived glycerol. The conversion of (pure or crude) glycerol into SCO has rarely been seen in R. toruloides strains (Moreton 1988; Xu et al. 2012; Uçkun Kiran et al. 2013), whereas the current study is the first to report the production of SCO by L. starkeyi growing on glycerol-based media. The reported maximum values of SCO produced for both strains (>12 g l-1) as well as the concomitant YL/DCW values (35–47% w/w) are among the highest reported for shake-flask fermentations of microorganisms growing on glycerol, and compare favourably with many of the results achieved in


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bioreactor experiments. A summary of the results of DCW and SCO production by several oleaginous microorganisms cultivated on glycerol-based media in various fermentation configurations and their comparison with the present investigation is shown in Table 6. Two different physiological patterns with regard to the accumulation of storage materials have been seen in R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296; as far as R. toruloides is concerned, it has been seen that during the first microbial growth phases, and despite the presence of nitrogen in the culture medium, significant quantities of intracellular polysaccharides were synthesized (YIPS/DCW ≈ 40% w/w was observed; Fig. 2b). Thereafter, the YIPS/DCW values were reduced while in absolute quantities (g l-1), after the significant biosynthesis that had occurred at the first growth steps, c. 50 h after inoculation and until the end of the culture the concentration of intracellular polysaccharides remained practically constant, whereas a simultaneously significant accumulation of storage lipids (rise of both L and YL/DCW values) was observed. A similar interplay of polysaccharides and lipids biosynthesis of R. toruloides has been reported for an oleaginous Chlorella sp. strain growing autotrophically under constant illumination in an open-pond simulating photo-bioreactor (Bellou and Aggelis 2012). On the other hand, L. starkeyi presented a different physiological profile since IPS per unit DCW remained practically constant with a value c. 30% w/w throughout the culture, while in absolute values (g l-1), the concentration of intracellular polysaccharides increased (c. 7 g l-1 was reported at the end of the culture). Worth mentioning was the fact that high quantities of IPS (YIPS/DCW ≥ 30% w/w) were produced even at the early growth steps for both micro-organisms tested. This is a rather unusual result; on a biochemical basis, the events that lead to IPS and/or storage lipid production are theoretically triggered after nitrogen depletion from the culture medium, which leads to a rapid decrease of intracellular AMP, resulting in intra-mitochondrial accumulation of citric acid, since the key enzyme NAD+-isocitrate dehydrogenase is allosterically activated by the presence of AMP, that now is in low concentrations (Ratledge 1997; Ratledge and Wynn 2002; Papanikolaou and Aggelis 2011a). Citric acid, after exceeding a critical value, is excreted to the cytoplasm. Thereafter, citric acid will be cleaved to acetyl-CoA and oxaloacetate,


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a reaction catalysed by ATP-citrate lyase (ATP-CL) and the acetyl-CoA produced will constitute the precursor for the synthesis of cellular acyl-CoAs, reactions catalysed by the FA-synthetase complex. Finally, the acyl-CoAs synthesized will be stored in the cytoplasm, mostly in the form of triacylglycerols (TAGs) formed presumably through the Kennedy pathway (Ratledge and Wynn 2002; Fakas et al. 2009a; Papanikolaou and Aggelis 2011a). When micro-organisms grow on sugars under nitrogen-limited conditions, in the imbalanced growth phase (absence of nitrogen in the medium) and in the absence of ATP–CL enzymatic complex, citric acid will be either excreted into the culture medium (in the case of many Y. lipolytica strains – see Papanikolaou et al. 2002, 2008, 2009, 2013), or will provoke the inhibition of 6-phosphoro-fructokinase (6-PFK). The above-mentioned event, in association with the decreased activity of 6-phosphoroglucose isomerase (PGI), will result in intracellular accumulation of polysaccharides (Galiotou-Panayotou et al. 1998; Zhong and Tang 2004). On the other hand, the IPS biosynthetic pattern from glycerol revealed in this paper is interesting; passive diffusion is used for glycerol uptake, and therefore the uptake rate of this sugar-alcohol depends on its concentration in the growth medium (Papanikolaou and Aggelis 2011a). Therefore, the fact that intracellular polysaccharides are accumulated for both R. toruloides and L. starkeyi inside the cells in the early growth steps, a period in which glycerol is found in the medium in high concentrations, suggests that the glycerol uptake rate occasionally exceeds the catabolic capacity of the cell and therefore high glycerol quantities, which cross the plasma membrane, are used for IPS synthesis. Later, in the case of R. toruloides, when the glycerol concentration in the growth medium drops, IPS in DCW values significantly decrease, suggesting a possible use of IPS for generating energy. Biosynthesis of other cellular compounds, i.e. reserve lipid, also might occur using IPS as an intracellular substrate, together with the assimilated extracellular glycerol. In contrast, as far as L. starkeyi is concerned, this micro-organism constantly produces intracellular polysaccharides, since their absolute values constantly increase, indicating that the enzymes of gluconeogenesis (e.g. fructose biphosphate aldolase, fructose biphosphatase, etc) were active in both the balanced and the imbalanced growth


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phases. Likewise, several higher fungi such as Flammulina velutipes, Pleurotus pulmonarius, Morchella esculenta and Volvariella volvacea have been reported to produce significant quantities of IPS during the balanced growth phase (Diamantopoulou et al. 2012, 2014). In any case, the production of IPS by oleaginous yeasts (and in significant quantities, c. 7 g l-1, as in the current investigation) has rarely been reported, whereas the current investigation is among the first in the international literature to deal with the conversion of waste glycerol into intracellular polysaccharides. The analysis of the fatty acid composition of SCOs produced by both R. toruloides and L. starkeyi varied as a function of both the fermentation time and the Glol0 concentration used; for R. toruloides, higher quantities of unsaturated FAs were detected at early compared with late growth phases, while the greater the Glol0 concentration in the medium, the greater the quantity of saturated FAs. For L. starkeyi, the cellular lipids were more saturated at the early growth steps than later, whereas the greater the Glol0 concentration in the medium, the more cellular FAs were unsaturated. In all cases and in accordance with the literature in which L. starkeyi and R. toruloides were cultivated on sugar-based or similarly metabolized materials (implying de novo lipid accumulation), mainly C16:0 and Δ9C18:1 and to lesser extent C18:0 and Δ9,12C18:2 fatty acids were synthesized, indicating that SCOs produced by the above-mentioned microorganisms can constitute perfect precursors for the synthesis of 2nd generation biodiesel (Li et al. 2007; Zhao et al. 2008; Xu et al. 2012). Differences in the fatty acid composition of the storage lipids produced by L. starkeyi and R. toruloides seem to be strain dependent; neither the fermentation time nor increasing the concentration of glycerol (or glucose) in the culture medium had any systematic common effect on the modification of cellular fatty acids in the cells of oleaginous yeasts (Papanikolaou and Aggelis 2009; Papanikolaou et al. 2002, 2009; Makri et al. 2010; Chatzifragkou et al. 2011b). Likewise, there was no systematic effect concerning the composition of cellular fatty acids due to the addition of natural compounds in the culture medium, since in other cases the synthesis of saturated fatty acids is favoured (Moreton 1988; Chatzifragkou et al. 2011b) whilst in others mostly unsaturated fatty acids are predominant in


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the cellular lipids (Sarris et al. 2011). On the other hand, utilization of fatty materials and partial or complete implication of the ex novo mechanism for the synthesis of cellular lipids results in the production of intracellular fatty materials that, in general, have similar composition to the fats used as substrates (Papanikolaou et al. 2003, 2011; Papanikolaou and Aggelis 2011a). It is evident that the ex novo mechanism of lipid accumulation presents fundamental differences to that of the de novo, whereas in the former case, fermentation of the fatty material is performed mainly in order to ‘up-grade’ and ‘ameliorate’ the quality of the hydrophobic carbon source employed as substrate (Papanikolaou et al. 2003, 2011; Papanikolaou and Aggelis 2011a). In conclusion, six yeast strains were screened for their ability to assimilate crude glycerol, waste deriving from biodiesel production units, and produce metabolic compounds of significance for industrial biotechnology. Two of the tested strains, R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296, were revealed as satisfactory candidates of glycerol consumption and during growth on high Glol0 content media presented notable production of biomass and microbial lipid. Single cell oil production comparable to some of the highest in the international literature for micro-organisms growing on glycerol was reported. A scarce number of reports have indicated the production of SCO by R. toruloides growing on crude glycerol, whilst this is the first report to deal with the conversion of this residue to SCO by L. starkeyi. Finally, this is one of the first reports in the literature to indicate the conversion of glycerol into intracellular polysaccharides.

Acknowledgements The State Scholarship Foundation (Athens, Greece) is gratefully acknowledged for the scholarship to Sidoine Sadjeu Tchakouteu. Financial support was attributed by the project entitled ‘New bioprocess for microbial oil from crude glycerol and cellulosic sugars’ (Acronym: BIO4OIL, project code 2359) financed by the General Secretariat for Research and Technology,


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Ministry of National Education and Religious Affairs, Greece (project action: ‘Bilateral S&T cooperation between Greece and Germany 2013–2015’).

Conflict of interest None declared.

References Abghari, A. and Chen, S. (2014) Yarrowia lipolytica as an oleaginous cell factory platform for production of fatty acid-based biofuel and bioproducts. Front Energy Res, in press, doi: 10.3389/fenrg.2014.00021. André, A., Chatzifragkou, A., Diamantopoulou, P., Sarris, D., Philippoussis, A., Galiotou-Panayotou, M., Komaitis, M. and Papanikolaou, S. (2009) Biotechnological conversions of bio-diesel derived crude glycerol by Yarrowia lipolytica strains. Eng Life Sci 9, 468–478. André, A., Diamantopoulou, P., Philippoussis, A., Sarris, D., Komaitis, M. and Papanikolaou, S. (2010) Biotechnological conversions of bio-diesel derived waste glycerol into added-value compounds by higher fungi: production of biomass, single cell oil and oxalic acid. Ind Crops Prod 31, 407–416. Bellou, S. and Aggelis, G. (2012) Biochemical activities in Chlorella sp. and Nannochloropsis salina during lipid and sugar synthesis in a lab-scale open pond simulating reactor. J Biotechnol 164, 218–329. Bellou, S., Moustogianni, A., Makri, A. and Aggelis, G. (2012) Lipids containing polyunsaturated fatty acids synthesized by zygomycetes grown on glycerol. Appl Biochem Biotechnol 166, 146– 158.


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Celińska, E. and Grajek, W. (2009) Biotechnological production of 2,3-butanediol-Current state and prospects. Biotechnol Adv 27, 715–725. Celińska, E. and Grajek, W. (2013) A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica. Microb Cell Factor 12, 102. Chang, G., Luo, Z., Gu. S., Wu, Q., Chang, M. and Wang, X. (2013) Fatty acid shifts and metabolic activity changes of Schizochyrium sp. S31 cultured on glycerol. Bioresour Technol 142, 255–260. Chatzifragkou, A., Makri, A., Belka, A., Bellou, S., Mavrou, M., Mastoridou, M., Mystrioti, P., Onjaro, G., et al. (2011a) Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36, 1097–1108. Chatzifragkou, A., Petrou, I., Gardeli, C., Komaitis, M. and Papanikolaou, S. (2011b) Effect of Origanum vulgare L. essential oil on growth and lipid profile of Yarrowia lipolytica cultivated on glycerol-based media. J Am Oil Chem Soc 88, 1955–1964. Chi, Z., Pyle, D.J., Wen, Z., Frear, C. and Chen, S. (2007) A laboratory study of producing docosahexanoic acid from biodiesel-waste glycerol by microalgal fermentation. Proc Biochem 42, 1537–1545. Clomburg, J.M. and Gonzalez, R. (2013) Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol 31, 20–28. Cui, Y., Blackburn, J.W. and Liang, Y. (2012) Fermentation optimization for the production of lipid by Cryptococcus curvatus: Use of response surface methodology. Biomass Bioenerg 47, 410– 417. Dedyukhina, E.G., Chistyakova, T.I., Kamzolova, S.V., Vinter, M.V. and Vainshtein, M.B. (2012) Arachidonic acid synthesis by glycerol-grown Mortierella alpine. Eur J Lipid Sci Technol 114, 833–841.


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Dedyukhina, E.G., Chistyakova, T.I., Mironov, A.A., Kamzolova, S.V., Morgunov, I.G. and Vainshtein, M.B. (2014) Arachidonic acid synthesis from biodiesel-derived waste by Mortierella alpina. Eur J Lipid Sci Technol 116, 429–437. Diamantopoulou, P., Papanikolaou, S., Kapoti, M., Komaitis, M., Aggelis, G. and Philippoussis, A. (2012) Mushroom polysaccharides and lipids synthesized in liquid agitated and static cultures. Part I: Screening various mushroom species. Appl Biochem Biotechnol 167, 536–551. Diamantopoulou, P., Papanikolaou, S., Komaitis, M., Aggelis, G. and Philippoussis, A. (2014) Patterns of major metabolites biosynthesis by different mushroom fungi grown on glucosebased submerged cultures. Bioproc Biosyst Eng 37, 1385–1400. Duarte, S.H., de Andradea, C.C.P., Ghiselli, S. and Maugeri, F. (2013a) Exploration of Brazilian biodiversity and selection of a new oleaginous yeast strain cultivated in raw glycerol. Bioresour Technol 138, 377–318. Duarte, S.H., Ghiselli, S. and Maugeri, F. (2013b) Influence of culture conditions on lipid production by Candida sp. LEB-M3 using glycerol from biodiesel synthesis. Biocatal Agric Biotechnol 2, 339–343. Ethier, S., Woisard, K., Vaughan, D. and Wen, Z. (2011) Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Bioresour Technol 102, 88–93. Fakas, S., Papanikolaou, S., Galiotou-Panayotou, M., Komaitis, M. and Aggelis, G. (2009a) Biochemistry and biotechnology of single cell oil. In New Horizons in Biotechnology ed. Pandey, A., Larroche, C., Soccol, C.R. and Dussard, C.G. pp. 38–60. New Delhi: AsiaTech Publishers Inc. Fakas, S., Papanikolaou, S., Batsos, A., Galiotou-Panayotou, M., Mallouchos, A. and Aggelis, G. (2009b) Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina. Biomass Bioenerg 33, 573–580.


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Fontanille, P., Kumar, V., Christophe, G., Nouaille, R. and Larroche, C. (2012) Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresour Technol 114, 443–449. Galiotou-Panagiotou, M., Kalantzi, O. and Aggelis, G. (1998) Modelling of simultaneous production of polygalactorunase and exopolysaccharide by Aureobasidium pullulans ATHUM 2915. Antonie van Leeuwenhoek 73, 155–162. Hansson, L. and Dostálek, M. (1986) Influence of cultivation conditions on lipid production by Cryptococcus albidus. Appl Microbiol Biotechnol 24, 12–18. Juszczyk, P., Tomaszewska, L., Kita, A. and Rymowicz, W. (2013) Biomass production by novel strains of Yarrowia lipolytica using raw glycerol, derived from biodiesel production. Bioresour Technol 137, 124–131. Kachrimanidou, V., Kopsahelis, N., Chatzifragkou, A., Papanikolaou, S., Yanniotis, S., Kookos, I.K. and Koutinas, A.A. (2013) Utilisation of by-products from sunflower-based biodiesel production processes for the production of fermentation feedstock. Waste Biomass Valor 4, 529–537. Kamzolova, S.V., Fatykhova, A.R., Dedyukhina, E.G., Anastassiadis, S.G., Golovchenko, N.P. and Morgunov, I.G. (2011) Citric acid production by yeast grown on glycerol-containing waste from biodiesel industry. Food Technol Biotechnol 49, 65–74. Kitcha, S. and Cheirslip, B. (2013) Enhancing lipid production from crude glycerol by newly isolated oleaginous yeasts: strain selection, process optimization, and fed-batch strategy. Bioenerg Res 6, 300–310. Li, Y.H., Zhao, Z.B. and Bai, F.W. (2007) High density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microb Technol 41, 312–317. Liang, Y.N., Cui, Y., Trushenski, J. and Blackburn, J.W. (2010a) Converting crude glycerol derived from yellow grease to lipids through yeast fermentation. Bioresour Technol 101, 7581–7586.


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Liang, Y.N., Sarkany, N., Cui, Y. and Blackburn, J.W. (2010b) Batch stage study of lipid production from crude glycerol derived from yellow grease or animal fats through microagal fermentation. Bioresour Technol 101, 6745–6750. Louhasakul, Y. and Cheirsilp, B. (2013) Industrial waste utilization for low-cost production of raw material oil through microbial fermentation. Appl Biochem Biotechnol 169, 110–122. Makri, A., Fakas, S. and Aggelis, G. (2010) Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresour Technol 101, 2351– 2358. Meesters, P.A.E.P., Huijberts, G.N.M. and Eggink, G. (1996) High cell density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Appl Microbiol Biotechnol 45, 575–579. Miller, G. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31, 426–428. Moreton, R.S. (1988) Physiology of lipid accumulating yeasts. In Single Cell Oil ed. Moreton, R.S. pp. 1–32. Essex: Longman Scientific & Technical. Morgunov, I.G., Kamzolova, S.V. and Lunina, J.N. (2013) The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation. Appl Microbiol Biotechnol 97, 7387–7397. Papanikolaou, S. and Aggelis, G. (2009) Biotechnological valorization of biodiesel derived glycerol waste through production of single cell oil and citric acid by Yarrowia lipolytica. Lipid Technol 21, 83–87. Papanikolaou, S. and Aggelis, G. (2011a) Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur J Lipid Sci Technol 113, 1031–1051. Papanikolaou, S. and Aggelis, G. (2011b) Lipids of oleaginous yeasts. Part II: Technology and potential applications. Eur J Lipid Sci Technol 113, 1052–1073.


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Papanikolaou, S., Beopoulos, A., Koletti, A., Thevenieau, F., Koutinas, A.A., Nicaud, J.-M. and Aggelis, G. (2013) Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica. J Biotechnol 168, 303–314. Papanikolaou, S., Chatzifragkou, A., Fakas, S., Galiotou-Panayotou, M., Komaitis, M., Nicaud, J.M. and Aggelis, G. (2009) Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose. Eur J Lipid Sci Technol 111, 1221–1232. Papanikolaou, S., Dimou, A., Fakas, S., Diamantopoulou, P., Philippoussis, A., Galiotou-Panayotou, M. and Aggelis, G. (2011) Biotechnological conversion of waste cooking olive oil into lipid-rich biomass using Aspergillus and Penicillium strains. J Appl Microbiol 110, 1138–1150. Papanikolaou, S., Fakas, S., Fick, M., Chevalot, I., Galiotou-Panayotou, M., Komaitis, M., Marc, I. and Aggelis, G. (2008) Biotechnological valorisation of raw glycerol discharged after bio-diesel (fatty acid methyl-esters) manufacturing process: production of 1,3-propanediol, citric acid and single cell oil. Biomass Bioenerg 32, 60–71. Papanikolaou, S., Muniglia, L., Chevalot, I., Aggelis, G. and Marc, I. (2002) Yarrowia lipolytica as a potential producer of citric acid from raw glycerol. J Appl Microbiol 92, 737–744. Papanikolaou, S., Muniglia, L., Chevalot, I., Aggelis, G. and Marc, I. (2003) Accumulation of a cocoa-butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues. Curr Microbiol 46, 124–130. Papanikolaou, S., Sarantou, S., Komaitis, M. and Aggelis, G. (2004) Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media. J Appl Microbiol 97, 867–875. Petrik, S., Marova, I., Haronikova, A., Kostovova, I. and Breierova, E. (2013) Production of biomass, carotenoid and other lipid metabolites by several red yeast strains cultivated on waste glycerol from biofuel production – a comparative screening study. Ann Microbiol 63, 1537–1551.


Accepted Article

Poli, J.S., da Silva, M.A.N., Siqueira, E.P., Pasa, V.M.D., Rosa, C.A. and Valente, P. (2014) Microbial lipid produced by Yarrowia lipolytica QU21 using industrial waste: a potential feedstock for biodiesel production. Bioresour Technol 161, 320–326. Pyle, D.J., Garcia, R.A. and Wen, Z. (2008) Producing docosahexaenoic acid (DHA)-rich algae from biodiesel-derived crude glycerol: effects of impurities on DHA production and algal biomass composition. J Agric Food Chem 56, 3933–3939. Ratledge, C. (1997) Microbial lipids. In Biotechnology, 2nd edn, ed. Kleinkauf, H. and Dohren, H. pp. 135–197. Weinheim: Wiley-VCH. Ratledge, C. and Wynn, J. (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51, 1–51. Rivaldi, J.D., Sarrouh, B.F., Branco, R.F., de Mancilha, I.M. and da Silva, S.S. (2012) Biotechnological utilization of biodiesel-derived glycerol for the production of ribonucleotides and microbial biomass. Appl Biochem Biotechnol 167, 2054–2067. Rivaldi, J.D., Sarrouh, B.F. and da Silva, S.S. (2009) Development of biotechnological processes using glycerol from biodiesel production. In Current Research Topics in Applied Microbiology and Microbial Biotechnology ed. Mendez-Vilas, A. pp. 429–433. Madrid: World Scientific Publishing Co. Formatex Research Center. Rymowicz, W., Fatykhova, A.R., Kamzolova, S.V., Rywinska, A. and Morgunov, I.G. (2010) Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes. Appl Microbiol Biotechnol 87, 971–979. Rymowicz, W., Rywińska, A. and Gładkowski, W. (2008) Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1. Chem Pap 62, 239–246. Rymowicz, W., Rywińska, A., Zarowska, B. and Juszczyk, P. (2006) Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica. Chem Pap 60, 391–394.


Accepted Article

Ryu, B.G., Kim, J., Kim, K., Choi, Y.E., Han, J.I. and Yang, J.W. (2013) High-cell-density cultivation of oleaginous yeast Cryptococcus curvatus for biodiesel production using organic waste from the brewery industry. Bioresour Technol 135, 357–364. Rywińska, A., Juszczyk, P., Wojtatowicz, M., Robak, M., Lazar, Z., Tomaszewska, L. and Rymowicz, W. (2013a) Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications. Biomass Bioenerg 48, 148–166. Rywińska, A., Juszczyk, P., Wojtatowicz, M. and Rymowicz, W. (2011) Chemostat study of citric acid production from glycerol by Yarrowia lipolytica. J Biotechnol 152, 54–57. Rywińska, A. and Rymowicz, W. (2009) Citric acid production from raw glycerol by Yarrowia lipolytica Wratislavia 1.31. In Microbial Conversions of Raw Glycerol ed. Aggelis, G. pp. 19–30. New York: Nova Science Publishers Inc. Rywińska, A. and Rymowicz, W. (2010) High-yield production of citric acid by Yarrowia lipolytica on glycerol in repeated-batch bioreactors. J Ind Microbiol Biotechnol 37, 431–435. Rywińska, A., Rymowicz, W. and Marcinkiewicz, M. (2010) Valorization of raw glycerol for citric acid production by Yarrowia lipolytica yeast. Electron J Biotechnol 13, no 4. Rywińska, A., Tomaszewska, L. and Rymowicz, W. (2013b) Erythritol biosynthesis by Yarrowia lipolytica yeast under various culture conditions. Afr J Microbiol Res 7, 3511–3516. Saenge, C., Cheirsilp, B., Suksaroge, T.T. and Bourtoom, T. (2011) Potential use of oleaginous red yeast Rhodotorula glutinis for the bioconversion of crude glycerol from biodiesel plant to lipids and carotenoids. Proc Biochem 46, 210–218. Santamauoro, F., Whiffin, F.M., Scott, R.J. and Chuck, C.J. (2014) Low-cost lipid production by an oleaginous yeast cultured in non-sterile conditions using model waste resources. Biotechnol Biofuel 7, 34.


Accepted Article

Sarris, D., Galiotou-Panayotou, M., Koutinas, A.A., Komaitis, M. and Papanikolaou, S. (2011) Citric acid, biomass and cellular lipid production by Yarrowia lipolytica strains cultivated on olive mill wastewater-based media. J Chem Technol Biotechnol 86, 1439–1448. Sestric, R., Munch, G., Cicek, N., Sparling, R. and Levin, D.B. (2014) Growth and neutral lipid synthesis by Yarrowia lipolytica on various carbon substrates under nutrient-sufficient and nutrient-limited conditions. Bioresour Technol 164, 41–46. Sitepu, I.R., Selby, T., Lin, T., Zhu, S. and Boundy-Mills, K. (2014) Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species. J Ind Microbiol Biotechnol 41, 1061–1070. Sitepu, I.R., Sestric, R., Ignatia, L., Levin, D.B., German, J.B., Gillies, L.A., Almada, L.A. and BoundyMills, K.L. (2013) Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species. Bioresour Technol 144, 360–369. Taccari, M., Canonico, L., Comitini, F., Mannazzu, I. and Ciani, M. (2012) Screening of yeasts for growth on crude glycerol and optimization of biomass production. Bioresour Technol 110, 488– 495. Tomaszewska, L., Rywińska, A. and Gładkowski, W. (2012) Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol. J Ind Microbiol Biotechnol 39, 1333– 1343. Uçkun Kiran, E., Trzcinski, A. and Webb, C. (2013) Microbial oil produced from biodiesel byproducts could enhance overall production. Bioresour Technol 129, 650–654. Vieira, J.P., Ienczak, J.L., Rossell, C.E., Pradella, J.G. and Franco, T.T. (2014) Microbial lipid production: screening with yeasts grown on Brazilian molasses. Biotechnol Lett 36, 2433–2442. Wen, Z., Pyle, D.J. and Athalye, S.K. (2009a) Glycerol waste from biodiesel manufacturing. In Microbial Conversions of Raw Glycerol ed. Aggelis, G. pp. 1–7. New York: Nova Science Publishers Inc.


Accepted Article

Wen, Z., Pyle, D.J. and Athalye, S.K. (2009b) Production of omega-3 polyunsaturated fatty acids from biodiesel-derived crude glycerol by microalgal and fungal fermentation. In Microbial Conversions of Raw Glycerol ed. Aggelis, G. pp. 41–63. New York: Nova Science Publishers Inc. Wensel, P., Helms, G., Hiscox, B., Davis, W.C., Kirchhoff, H., Bule, M., Yu, L. and Chen, S. (2014) Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation. Algal Res 4, 2–11. Xu, J., Zhao, X., Wang, W., Du, W. and Liu, D. (2012) Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production. Biochem Eng J 65, 30–36. Yang, X., Jin, G., Gong, Z., Shen, H., Bai, F. and Zhao, Z.K. (2014) Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process. Biochem Eng J 91, 86–91. Yazdani, S.S. and Gonzalez, R. (2007) Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 18, 213–219. Zhao, X., Kong, X., Hua, Y., Feng, B. and Zhao, Z.B. (2008) Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi. Eur J Lipid Sci Technol 110, 405–412. Zhong, J.J. and Tang, Y.-J. (2004) Submerged cultivation of medicinal mushrooms for production of valuable bioactive metabolites. Adv Biochem Eng Biotechnol 87, 25–59. Zikou, E., Chatzifragkou, A., Koutinas, A.A. and Papanikolaou, S. (2013) Evaluating glucose and xylose as cosubstrates for lipid accumulation and γ-linolenic acid biosynthesis of Thamnidium elegans. J Appl Microbiol 114, 1020–1032.


Accepted Article

Table 1. Experimental results originated from kinetics of yeast strains grown on crude glycerol in shakeflask experiments. Representations of biomass (DCW, g l-1), lipid (L, g l-1), glycerol consumed (Glolcons, g l1),

fermentation time (h) and lipid in dry biomass (YL/DCW, % w/w) when the maximum quantity of lipids

in dry cell weight (YL/DCW, % w/w) (a) and the maximum concentration of biomass (DCW, g l-1) (b) respectively, were achieved. Culture conditions: growth on 250 ml flasks at 180 ± 5 rpm, 28 ± 1°C, initial glycerol concentration (Glol0) ~30 g l-1, initial molar ratio C/N~100 moles/moles, initial pH = 6.1 ± 0.1, pH 5.0–6.0, oxygen saturation higher than 40% (v/v) for all growth phases. For the trial performed with Lipomyces starkeyi, DCW produced was subjected to acidification before extraction of total lipids. Each experimental point is the mean value of two independent measurements (SE < 10%) Yeast strain

Time

Glolcons

DCW

L

YL/DCW

(g l-1)

(g l-1)

(g l-1)

(%, w/w)

Cryptococcus curvatus NRLL Y-1511

a, b

195

25.7

11.8

1.48

12.5

Yarrowia lipolytica ACA YC 5029

a

22

5.9

3.1

0.28

9.0

b

95

29.1

5.5

0.21

3.8

a

26

8.5

4.1

0.86

20.9

b

190

31.1

6.1

0.27

4.4

Rhodosporidium toruloides DSM 4444

a, b

168

25.5

8.9

2.22

24.9

Rhodosporidium toruloides NRRL Y-

a, b

92

30.4

11.0

2.83

25.7

a

168

21.0

9.0

2.95

32.7

b

192

22.1

9.3

1.10

11.8

Yarrowia lipolytica ACA YC 5033

27012 Lipomyces starkeyi DSM 70296


Accepted Article

Table 2. Experimental results of kinetics of Rhodosporidium toruloides NRRL Y-27012 grown on crude glycerol in shake-flask experiments. Representation of fermentation time (h) in which biomass (DCW, g l1),

lipid (L, g l-1), glycerol consumed (Glolcons, g l-1), lipid in dry biomass (YL/DCW, % w/w) and yield of lipid

produced per unit of glycerol consumed (YL/Glol, g g-1) were achieved when the maximum quantity of lipids in dry cell weight (YL/DCW, % w/w) was obtained. Culture conditions: growth on 250 ml flasks at 180 ± 5 rpm, 28 ± 1°C, various initial glycerol concentrations employed, initial pH = 6.1 ± 0.1, pH 5.0–6.0, oxygen saturation higher than 20% (v/v) for all growth phases, inoculation volume 10% (v/v). Each experimental point is the mean value of two independent measurements (SE < 10%) Glol0

Time

(g l-1)

Glolcons

DCW

L

YL/DCW

YDCW/Glol

YL/Glol

(g l-1)

(g l-1)

(g l-1)

(%, w/w)

(g g-1)

(g g-1)

≈50

195

47.6

16.7

7.89

47.2

0.35

0.17

≈95

288

79.0

23.8

11.19

47.0

0.30

0.14

≈120

395

115.9

30.1

12.04

40.0

0.26

0.10

≈180

360

79.1

16.2

8.80

54.3

0.20

0.11


Accepted Article

Table 3. Experimental results of kinetics of the yeast Lipomyces starkeyi DSM 70296 grown on crude glycerol in shake-flask experiments. Representation of fermentation time (h) in which biomass (DCW, g l1),

lipid (L, g l-1), glycerol consumed (Glolcons, g l-1), lipid in dry biomass (YL/DCW, % w/w) and yield of lipid

produced per unit of glycerol consumed (YL/Glol, g g-1) were achieved when the maximum quantity of lipids in dry cell weight (YL/DCW, % w/w) was obtained. Culture conditions: growth on 250 ml flasks at 180 ± 5 rpm, 28 ± 1°C, various initial glycerol concentrations employed, initial pH = 6.1 ± 0.1, pH 5.0–6.0, oxygen saturation higher than 20% (v/v) for all growth phases, inoculation volume 10% (v/v). Each experimental point is the mean value of two independent measurements (SE < 10%) Glol0

Time

(g l-1)

Glolcons

DCW

L

YL/DCW

YDCW/Glol

YL/Glol

(g l-1)

(g l-1)

(g l-1)

(%, w/w)

(g g-1)

(g g-1)

≈50

144

46.7

11.2

3.70

33.0

0.24

0.08

≈100

336

105.1

23.3

8.16

35.0

0.22

0.08

≈120

470

115.1

34.4

12.34

35.9

0.30

0.11

≈180

310

91.1

18.2

6.74

37.0

0.20

0.07


Accepted Article

Table 4. Fatty acid composition of cellular lipids of Rhodosporidium toruloides NRRL Y-27012, during growth on media composed of crude glycerol added at various initial glycerol (Glol0) concentrations and constant initial nitrogen, under nitrogen-limited conditions. Early growth phase is that in which incubation time is 24–72 h. Late growth phase is that in which incubation time is c. 100 h (for Glol0 ≈ 30 g l-1) and c. 200–250 h (for the higher Glol0 concentrations). Culture conditions as in Tables 1 and 2 Glol0 (g l-1)

Growth phase

C16:0

C18:0

Δ9C18:1

Δ9,12C18:2

≈30

Early

23.0

10.5

55.5

13.0

Late

31.0

8.0

50.2

10.0

Early

21.8

14.4

53.9

7.1

Late

31.4

13.4

48.7

6.0

Early

23.3

11.1

54.1

5.1

Late

34.1

13.0

50.1

1.9

Early

30.4

13.4

47.0

5.3

Late

35.4

12.2

44.0

2.4

≈50

≈95

≈180


Accepted Article

Table 5. Fatty acid composition of cellular lipids of Lipomyces starkeyi DSM 70296, during growth on media composed of crude glycerol added at various initial glycerol (Glol0) concentrations and constant initial nitrogen, under nitrogen-limited conditions. Early growth phase is that in which incubation time is 24–72 h. Late growth phase is that in which incubation time is c. 150 h (for Glol0≈30 g l-1) and c. 200–250 h (for the higher Glol0 concentrations). Culture conditions as in Tables 1 and 3 Glol0 (g l-1)

Growth phase

C16:0

C18:0

Δ9C18:1

Δ9,12C18:2

≈30

Early

35.8

8.8

46.1

5.3

Late

30.9

7.9

52.1

5.6

Early

32.8

8.4

52.3

2.6

Late

28.0

7.1

57.3

7.7

Early

33.1

8.0

53.1

4.4

Late

26.1

7.0

57.0

9.7

≈50

≈120


Accepted Article

Table 6. Experimental results of microbial strains cultivated on glycerol-based media and producing microbial lipid during growth under various fermentation configurations and their comparisons with the present study

Strain

Culture mode

DCW

YL/DCW

(g l-1)

(%

Reference

w/w)

1. Yeasts Cryptococcus albidus CBS 4517 ¶

Shake flasks

1.4

43.8

Hansson and Dostálek (1986)

Rhodosporidium toruloides CBS 14 ¶

Shake flasks

5.8

34.6

Moreton (1988)

118.0

25.0

Meesters et al. (1996)

8.1

43.0

Papanikolaou and Aggelis (2002)

11.4

29.8

Papanikolaou et al. (2003)

4.7

23.1

Makri et al. (2010)

32.9

52.9

Liang et al. (2010a)

5.5

35.2

Saenge et al. (2011)

22.0

49.0

Cui et al. (2012)

42.2

38.2

Fontanille et al. (2012)

Cryptococcus curvatus ATCC 20509 ¶

Fed-batch bioreactor Single stage

Yarrowia lipolytica ACA-DC 50109 ‡ continuous

Yarrowia lipolytica ACA-DC 50109 ‡ a

Yarrowia lipolytica ACA-DC 50109 ¶

Shake flasks Fed Batch bioreactor Fed Batch

Cryptococcus curvatus ATCC 20509 ‡ bioreactor

Rhodotorula glutinis TISTR 5159

Cryptococcus curvatus ATCC 20509 ‡

Shake flasks Fed Batch bioreactor

Yarrowia lipolytica MUCL 28849 ¶

Fed Batch bioreactor


Fed Batch

Accepted Article

Yarrowia lipolytica MUCL 28849 ¶ b

41.0

34.6

Fontanille et al. (2012)

19.2

47.7

Xu et al. (2012)

26.7

69.5

Xu et al. (2012)

23.0

12.9

Celińska and Grajek (2013)

42.0

30.9

Celińska and Grajek (2013)

bioreactor

Rhodosporidium toruloides AS2.1389 ‡

Rhodosporidium toruloides AS2.1389 ‡

Shake flasks Batch bioreactor Fed Batch

Yarrowia lipolytica A10 ¶ bioreactor Fed Batch

Yarrowia lipolytica NCYC 3825 ¶ c

bioreactor

Candida sp. LEB-M3 ‡

Shake flasks

19.7

50.2

Duarte et al. (2013b)

Kodamaea ohmeri BY4-523 ‡

Shake flasks

10.3

53.3

Kitcha and Cheirslip (2013)

Trichosporanoides spathulata JU4-57 ‡

Shake flasks

17.1

43.4


13.8

56.4


5.5

50.8

Louhasakul and Cheirslip (2013)

Fed Batch

Trichosporanoides spathulata JU4-57 ‡ bioreactor Batch

Yarrowia lipolytica TISTR 5151 ‡ d bioreactor

Yarrowia lipolytica JMY1203 ‡ e

Shake flasks

3.2

30.7


Cryptococcus curvatus ATCC 20509 ¶ f

Shake flasks

50.4

37.7

Ryu et al. (2013)

35.3

46.0

Uçkun Kiran et al. (2013)

Batch

Rhodosporidium toruloides Y4 ¶

bioreactor

Yarrowia lipolytica Q21 ‡

Shake flasks

3.85

22.1

Poli et al. (2014)

Metschnikowia pulcherrima

Shake flasks

7.4

40.0

Santamauro et al. (2014)


Shake flasks

11.6

31.0

Sestric et al. (2014)

Shake flasks

24.9

48.9

Yang et al. (2014)

Schizochytrium limacinum SR21 ¶

Shake flasks

14.4

44.8

Chi et al. (2007)

Schizochytrium limacinum SR21 ‡

Shake flasks

18.0

50.6

Chi et al. (2007)

Mortierella isabellina ATHUM 2935 ‡

Shake flasks

8.5

51.7


Cunninghamella echinulata ATHUM 4411 ‡

Shake flasks

7.8

25.6

Fakas et al. (2009b)

Aspergillus niger LFMB 1 ‡

Shake flasks

5.4

57.4

André et al. (2010)

Aspergillus niger NRRL 364 ‡

Shake flasks

8.2

41.4

André et al. (2010)

Schizochytrium limacinum SR21 ‡

Shake flasks

13.1

73.3

Liang et al. (2010b)

Thamnidium elegans CCF 1465 ‡

Shake flasks

16.3

71.1

Chatzifragou et al. (2011a)

≈11

50.2

Ethier et al. (2011)

7.0

53.1

Bellou et al. (2012)

9.7

32.7


6.9

25.1


4.2

15.4


Accepted Article

Yarrowia lipolytica ATCC 20460 ‡ Rhodosporidium toruloides Y4 ‡ 2. Fungi and micro-algae

Single stage

Schizochytrium limacinum SR21 ‡ continuous

Mortierella ramanniana MUCL 9235 ¶

Shake flasks Batch

Mortierella ramanniana MUCL 9235 ¶ bioreactor

Cunninghamella echinulata ATHUM 4411 ¶

Shake flasks Batch

Cunninghamella echinulata ATHUM 4411 ¶ bioreactor

Mortierella alpina LPM 301 ¶

Shake flasks

28.6

33.4

Dedyukhina et al. (2012)

Mortierella alpina NRRL-A-10995 ¶

Shake flasks

26.7

35.4



Batch

Accepted Article

Schizochytrium sp. S31 ¶

≈40

49.1

Chang et al. (2013)

bioreactor

Mortierella alpina LPM 301 ‡ g

Shake flasks

15.6

33.3


Mortierella alpina NRRL-A-10995 ‡ g

Shake flasks

20.5

31.9


Lipomyces starkeyi DSM 70296 ‡

Shake flasks

34.4

35.9

Present study

Rhodosporidium toruloides NRRL Y-27012 ‡

Shake flasks

30.1

40.0

Present study

‡Utilization of crude glycerol. ¶Utilization of pure glycerol. a: Utilization of blend of glycerol with saturated free-fatty acids. b: Utilization of blend of glycerol with volatile fatty acids. c: Strain genetically modified, over-expressing glycerol dehydratase and a wide spectrum of alcohol oxidoreductases. d: Utilization of blend of glycerol with decanter effluent from palm oil mill. e: Strain genetically modified, lacking in the function of methyl-citrate cycle. f: Utilization of blend of glycerol with spent yeast lysate. g: Utilization of biodiesel waste containing blend of glycerol with saturated and unsaturated free-fatty acids.


Accepted Article

Figure 1 Kinetics of biomass produced (■ , g l-1), citric acid produced (● , g l-1) and intracellular lipids produced per unit of dry weight (○, % w/w) of Yarrowia lipolytica ACA-YC 5033 during growth on crude glycerol, at initial glycerol concentration (Glol0) of c. 30 g l-1, under nitrogen-limited conditions. Culture conditions: growth on 250 ml flasks at 185 rpm, initial pH = 6.0 ± 0.1, pH 5.2–6.0, DOT > 20% (v/v), incubation temperature 28 ± 1°C. Each point is the mean value of two independent measurements (SE < 10%).

, g l-1), lipid produced (○, g l-1), intracellular polysaccharides Figure 2 Kinetics of biomass produced (■ produced (●, g l-1) (a), remaining glycerol (● , g l-1), intracellular polysaccharides produced per unit of dry weight (◊, % w/w) and intracellular lipids produced per unit of dry weight (○, % w/w) of Rhodosporidium toruloides NRRL Y-27012 during growth on crude glycerol, at initial glycerol concentration (Glol0) of c. 120 g l-1, under nitrogen-limited conditions. Culture conditions: growth on 250 ml flasks at 185 rpm, initial pH = 6.0 ± 0.1, pH 5.2–6.0, DOT > 20% (v/v), incubation temperature 28 ± 1°C. Each point is the mean value of two independent measurements (SE < 10%).

, g l-1), lipid produced (○, g l-1), intracellular polysaccharides Figure 3 Kinetics of biomass produced (■ produced (●, g l-1) (a), remaining glycerol (● , g l-1), intracellular polysaccharides produced per unit of dry weight (◊, % w/w) and intracellular lipids produced per unit of dry weight (○, % w/w) of Lipomyces starkeyi DSM 70296 during growth on crude glycerol, at initial glycerol concentration (Glol0) of c. 100 g l-1, under nitrogen-limited conditions. Culture conditions: growth on 250 ml flasks at 185 rpm, initial pH = 6.0 ± 0.1, pH 5.2–6.0, DOT > 20% (v/v), incubation temperature 28 ± 1°C. Each point is the mean value of two independent measurements (SE < 10%).


L/DCW

15

15

-1 -1

(%, w/w)

20

Dry cell weight (DCW, g l ); Citric acid (Cit, g l )

20

10 10

Y

Accepted Article

25

5

5

0

0 0

50

100 Time (h)

Fig. 1


150

200

7

-1

6

25

5

20

4 15

3

10

2

5

1

0

0 400

0

50

100

150

200

250

Time (h) Fig. 2a


300

350

-1

30

Intra-cellular polysaccharides (IPS, g l )

8

-1

Dry cell weight (DCW, g l ); Lipid (L, g l )

Accepted Article

35

140 120 -1

30

100 80

20

60 40

10

20 0

Glycerol (Glol, g l )

(%, w/w) IPS/DCW

(%, w/w); Y L/DCW

40

Y

Accepted Article

160

0

50

100

150

200

250

Time (h) Fig. 2b


300

350

0 400

20

8

15

6

10

4

5

2

0

0 350

-1

-1

Intra-cellular polysaccharides (IPS, g l )

10

-1

Dry cell weight (DCW, g l ); Lipid (L, g l )

Accepted Article

25

0

50

100

150

200

Time (h)

Fig. 3a


250

300

100 80

30 60 20 40 10

20

0

0

50

100

150

200

Time (h) Fig. 3b


250

300

0 350

-1

40

Glycerol (Glol, g l )

(%, w/w) IPS/DCW

(%, w/w); Y L/DCW

120

Y

Accepted Article

50

Biomass and lipid production of Chlorella protothecoides under heterotrophic cultivation on a mixed waste substrate of brewer fermentation and crude glycerol.

Bioprospection of bacteria and yeasts from Atlantic Rainforest soil capable of growing in crude-glycerol residues.

Impurities of crude glycerol and their effect on metabolite production.

Impact of glycerol and nitrogen concentration on Enterobacter A47 growth and exopolysaccharide production.

Microbial lipid production: screening with yeasts grown on Brazilian molasses.

Optimization of prodigiosin production by Serratia marcescens using crude glycerol and enhancing production using gamma radiation.

Enhanced ethanol production by fermentation of Gelidium amansii hydrolysate using a detoxification process and yeasts acclimated to high-salt concentration.

Assessing an effective feeding strategy to optimize crude glycerol utilization as sustainable carbon source for lipid accumulation in oleaginous yeasts.

Effects of temperature and substrate concentration on lipid production by Chlorella vulgaris from enzymatic hydrolysates of lipid-extracted microalgal biomass residues (LMBRs).

Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production.

Production of folate in oat bran fermentation by yeasts isolated from barley and diverse foods.

Biohydrogen production by co-fermentation of crude glycerol and apple pomace hydrolysate using co-culture of Enterobacter aerogenes and Clostridium butyricum.

Strain and process development for poly(3HB-co-3HP) fermentation by engineered Shimwellia blattae from glycerol.

High production of 2,3-butanediol from biodiesel-derived crude glycerol by metabolically engineered Klebsiella oxytoca M1.

Selection of oleaginous yeasts for fatty acid production.

Metabolic Flexibility of Yarrowia lipolytica Growing on Glycerol.

Fermentation and aerobic metabolism of cellodextrins by yeasts.

Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process.

Aeration and fermentation strategies on nisin production.

Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis.

Crude glycerol as feedstock for polyhydroxyalkanoates production by mixed microbial cultures.

Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol.

Two-step sequential liquefaction of lignocellulosic biomass by crude glycerol for the production of polyols and polyurethane foams.

Anaerobic digestion of crude glycerol as sole substrate in mixed reactor.