Seraphim Papanikolaou, Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece. E-mail: email@example.com
To study the biotechnological production of lipids containing rich amounts of the medically and nutritionally important γ-linolenic acid (GLA), during cultivation of the Zygomycetes Thamnidium elegans, on mixtures of glucose and xylose, abundant sugars of lignocellulosic biomass.
Methods and Results
Glucose and xylose were utilized as carbon sources, solely or in mixtures, under nitrogen-limited conditions, in batch-flask or bioreactor cultures. On glucose, T. elegans produced 31·9 g l−1 of biomass containing 15·0 g l−1 lipid with significantly high GLA content (1014 mg l−1). Xylose was proved to be an adequate substrate for growth and lipid production. Additionally, xylitol secretion occurred when xylose was utilized as carbon source, solely or in mixtures with glucose. Batch-bioreactor trials on glucose yielded satisfactory lipid production, with rapid substrate consumption rates. Analysis of intracellular lipids showed that the highest GLA content was observed in early stationary growth phase, while the phospholipid fraction was the most unsaturated fraction of T. elegans.
Thamnidium elegans represents a promising fungus for the successful valorization of sugar-based lignocellulosic residues into microbial lipids of high nutritional and pharmaceutical interest.
Significance and Impact of the Study
Xylitol production and cultivation in bioreactor trials is reported for the first time for T. elegans, while cultivation on xylose-based media resulted in high GLA production by this fungus.
Oleaginous micro-organisms are known for their ability to accumulate in their cells or mycelia significant amounts of lipids, addressed as single cell oils (SCOs) (Ratledge 2004; Papanikolaou and Aggelis 2011a,b; Papanikolaou 2012). The principal micro-organisms that are regarded as oleaginous belong to a relatively limited number of yeasts and a higher number of moulds (Ratledge 1991; Ratledge and Wynn 2002). Especially, various oleaginous Zygomycetes have the potential to produce high-quality fats and lipids, rich in polyunsaturated fatty acids (PUFAs) of high added value. Amongst them, γ-linolenic acid (GLA) receives much attention due to its nutritional importance and its selective pharmaceutical and anticancer properties (Sajbidor et al. 1988; Čertik and Shimizu 1999; Kavadia et al. 2001; Papanikolaou et al. 2007; Fakas et al. 2008a,b; Chatzifragkou et al. 2010). On the other hand, microbial lipids are viewed as an alternative starting material for biodiesel production (the so-called ‘2nd generation biodiesel’), due to the fact that their fatty acid (FA) composition resembles to that of vegetable oils (Li et al. 2007; Papanikolaou and Aggelis 2011a,b; Zhao and Hu 2011; Ruan et al. 2012; Xing et al. 2012). For the accomplishment – realization of the above-mentioned possibility, the need of low-cost sugar-based or similarly metabolized (e.g. biodiesel-derived glycerol) raw materials that can be utilized as carbon sources by oleaginous micro-organisms still represents a challenging aspect (Zhao and Hu 2011; Koutinas and Papanikolaou 2011; Papanikolaou 2012).
Lignocellulosic materials represent the largest and the most attractive biomass resources worldwide that can serve as cheap feedstock of monosaccharides in a variety of microbial fermentations. Lignocellulosic residues from wood, grass, agricultural – forestry wastes and municipal solid wastes are particularly abundant in nature and have a potential for bioconversion (Peters 2007). Glucose and xylose found in several ratios are the principal sugars of lignocellulosic biomass, being produced via chemical and/or enzymatic hydrolytic treatment processes (Lee et al. 1999; Sánchez 2009; Koutinas and Papanikolaou 2011). Therefore, the capability of oleaginous micro-organisms to utilize both C-5 and C-6 sugars as carbon source is highly desired to increase the efficiency of lipid production from lignocellulosic materials (Ruan et al. 2012).
Aim of the present work was to study the biochemical behaviour and ability of a Zygomycetes strain, namely Thamnidium elegans, to accumulate SCO rich in GLA during its cultivation on glucose and xylose, either used as individual substrates or used in several mixtures. It should be mentioned that only few studies can be found in the international literature, investigating the physiological response of the aforementioned micro-organism cultivated in submerged fermentations (Čertik et al. 1997; Papanikolaou et al. 2010; Vamvakaki et al. 2010; Chatzifragkou et al. 2011; Bellou et al. 2012), as most of them employ solid state fermentations (see i.e. Stredansky et al. 2000; Conti et al. 2001; Čertik et al. 2006; Kumar Salar et al. 2012). Additionally, this study was focused on monitoring the FA composition of intracellular lipid and lipid fractions, as well as GLA distribution among them, while cultivation of T. elegans in a bioreactor was pursued for the first time. Insights concerning kinetic evolutions and production of value-added metabolic compounds of the micro-organism are also provided and comprehensively discussed.
Materials and methods
Micro-organisms and culture conditions
Thamnidium elegans CCF-1465 was used in this study. The strain was maintained on potato dextrose agar (PDA; Plasmatec, Dorset, UK) at 4 ± 1°C and subcultivated every month to maintain its viability. The salt composition of the employed growth medium was as follows (in g l−1): KH2PO4, 7·0; Na2HPO4, 2·5; MgSO4·7H2O, 1·5; FeCl3·6H2O, 0·15; ZnSO4·7H2O, 0·02; MnSO4·H2O, 0·06; CaCl2·2H2O, 0·15. Yeast extract and (NH4)2SO4 were used as nitrogen sources at concentrations of 4·0 and 2·0 g l−1, respectively. The pH of the medium was 6·0 ± 0·1 after autoclaving. Glucose and xylose (analytical grade; Sigma) were used either as the sole carbon source at 100 g l−1 or in mixtures in the following foregone proportions (in g l−1): glucose–xylose 50–50; glucose–xylose 75–25 and glucose–xylose 25–75.
Experiments were conducted in 250-ml conical flasks, containing 50 ± 1 ml of growth medium, sterilized at 121°C/20 min and inoculated with 1 ml of spore suspension (around 105–107 CFU). All cultures were incubated in an orbital shaker (New Brunswick Scientific Co., Edison, NJ, USA) at an agitation rate of 180 rev min−1 and incubation temperature of 28 ± 1°C. In all experiments, it was desirable to maintain a medium pH in a value >5·2, therefore an appropriate volume of KOH (5 mol l−1) was periodically and aseptically added into the flasks when needed. Moreover, experimental work was carried out in a 3-l bioreactor (New Brunswick Scientific Co.) with a working volume of 1·5-l. Glucose was used as the sole carbon source at initial concentration of 100 g l−1, while the nitrogen sources used were yeast extract and ammonium sulfate, in concentrations as reported earlier. The initial pH of the medium was 6·0 ± 0·1 and maintained at the desirable value by automatic addition of 5 mol l−1 NaOH, through a peristaltic pump. The bioreactor contained 1·5-l of growth medium, sterilized and inoculated with 2% (v/v) of fungal spore suspension. The agitation rate of the bioreactor was 500–550 rev min−1, air supply was maintained at a constant rate of 1·0 vvm and growth temperature at 28°C.
Determination of dry mycelial biomass (X, g l−1) was performed by collecting the pellets and mycelia by filtration through a 0·09-mm stainless steel sieve and washed twice with distilled water. Biomass (X, g l−1) was determined by dry matter (90 ± 5°C/24 h). Determination of glucose, xylose and produced xylitol in the various glucose–xylose mixtures was conducted by means of HPLC analysis in a Waters 600E device (Waters Corporation, Milford, MA, USA), with an Aminex HPX-87H (300 mm × 7·8 mm; Bio-Rad, Los Angeles, CA, USA) column coupled to a differential refractometer (RI Waters 410; Waters Corp.). Operating conditions were as follows: sample volume 20 μl; mobile phase 0·005 mol l−1 H2SO4; flow rate 0·6 ml min−1; column temperature 65°C. Dissolved oxygen (DO) concentration in shake-flasks was determined with the aid of a selective electrode as described by Papanikolaou et al. (2009), whereas in the bioreactor, determination was performed on-line by using a selective electrode (Mettler Toledo, Urdorf, Switzerland). In any case, DO concentration was maintained during fermentation >20% (v/v) in the medium, during all growth phases. Finally, inorganic ammonium ion (NH4+) concentration was determined by a selective electrode (51927-00; Hach, Loveland, CO, USA).
Total cellular lipid (L, g l−1) was extracted from the dried mycelia with a mixture of chloroform/methanol 2 : 1 (v/v) and weighted after evaporation of the solvent in a rotary evaporator (R-144 apparatus; Büchi Labortechnik, Flawil, Switzerland). Lipids were converted to their corresponding fatty acid methyl-esters (FAMEs) in a two-step reaction with methanolic sodium and hydrochloric methanol (Papanikolaou et al. 2011). GC analysis was carried out on a Fisons 8060 device (Fisons Instruments, Milano, Italy), equipped with a Chrompack column (60 m × 0·32 mm; Chrompack Nederland BV, Middelburg, the Netherlands) and a FID detector (Fisons Instruments); helium was the carrier gas (2 ml min−1). The analysis was isothermally run at 200°C with the injection at 240°C and detector at 250°C. FAMEs were identified by reference to standards. Furthermore, in some trials, cellular lipids were fractionated into their lipid fractions. In brief, a known weight of extracted lipid (around 100 mg) was dissolved in chloroform (1–2 ml) and was fractionated by using a column (25 × 100 mm) of silicic acid, activated by heating overnight at 110°C (Fakas et al. 2006, 2008a). Successive applications of chloroform (100 ml), acetone (100 ml) and methanol (100 ml) produced fractions containing neutral lipids (N), glycolipids plus sphingolipids (G + S) and phospholipids (P), respectively (Guo and Ota 2000; Fakas et al. 2008a,b). The weight of each fraction was evaluated after evaporation of the solvent. Fractions were converted to their corresponding FAMEs and analysed with GC as described previously.
To ascertain the reproducibility of the experimental results related with yeast growth, all of the fermentations were carried out in (at least) duplicate experiments, in which different inocula were employed. Likewise, each experimental point of the kinetics presented in the tables and figures related with the cultures of T. elegans is the mean value of at least two determinations. In all cases, standard error was calculated and it was found to be <15%.
Growth of Thamnidium elegans on glucose or xylose employed as the sole carbon source
At first, T. elegans was cultivated in shake-flasks, with glucose or xylose as the sole carbon source at initial concentration of 100 g l−1, in nitrogen-limited media to direct the microbial metabolism towards the synthesis of intracellular lipid (Papanikolaou et al. 2004, 2008; Koutinas and Papanikolaou 2011; Papanikolaou 2012). Quantitative data obtained from the kinetics are presented in Table 1. On glucose-based media, the strain presented appreciable cell growth, with maximum biomass production corresponding to 31·9 g l−1, after 309 h of fermentation. At nearly the same time, substrate exhaustion was observed in the culture medium. Moreover, intracellular oil was typically accumulated after depletion of the nitrogen source from the culture medium. Specifically, remarkable lipid accumulation was carried out by the strain, 15·0 g l−1, with the corresponding lipid in dry weight yield value (YL/X) equal to 47·1% (w/w). The cellular lipids of T. elegans were found to be particularly rich in GLA, reaching the quantity of 1014 mg l−1 of medium. Additionally, when T. elegans was cultivated on xylose-based media, the fungal strain was capable of producing satisfactory amounts of biomass, 21·4 g l−1 (Fig. 1). The micro-organism consumed totally the available carbon source after ~400 h of fermentation, while sufficient amounts of cellular lipids were also produced, up to 8·9 g l−1. At that given time, the corresponding YL/X value was 41·8% (w/w). On the other hand, an interesting finding that was observed in the current investigation was that the cultivation of T. elegans on xylose, favoured the production of xylitol, reaching a maximum concentration of 31·3 g l−1. The secretion of xylitol can account for the relatively lower final concentrations of produced biomass and cellular lipids by the strain during growth on xylose, as compared with the performance of the micro-organism during growth on glucose. Furthermore, as shown in Fig. 1, a small consumption of produced xylitol occurred at the end of the fermentation, a time point in which xylose was almost depleted from the culture medium. This consumption coincided with a slight further increase in the concentration of total cellular lipids produced by the micro-organism.
Table 1. Kinetic data of Thamnidium elegans cultivation on glucose, xylose or mixtures as carbon source, in shake-flask or bioreactor experiments
Growth of Thamnidium elegans on glucose–xylose mixtures
As a next step, T. elegans was tested for its ability to grow and accumulate intracellular oil during its cultivation on glucose–xylose mixtures, under nitrogen-limited conditions. Data obtained from the kinetics are presented in Table 1. Overall, the micro-organism grew particularly well in all glucose–xylose mixtures, with maximum biomass production ranging from 27·4 to 29·5 g l−1, while in all cases total substrate assimilation occurred. In terms of lipid accumulation, T. elegans proved capable of producing significant amounts of SCO, reaching up to 12·6 g l−1, accompanied by sufficient values of lipid in dry weight, in the range of 36·7–45·9% (w/w). As far as GLA production was concerned, the mixture of glucose–xylose 50–50 (in g l−1) proved to be the most promising substrate among others tested for the accumulation of the aforementioned FA, reaching the maximum value of 974·9 mg l−1 about 381 h after inoculation. Furthermore, it should be mentioned that xylitol production took place in all mixtures. However, the amount of accumulated xylitol remained lower than that achieved on the substrate with xylose as the sole carbon source, indicating in accordance with the relevant literature (Winkelhausen and Kuzmanova 1998; Kim et al. 2009), that xylitol production could be significantly altered by the presence of glucose in the fermentation medium. Specifically, as depicted in Fig. 2, xylitol accumulation began only after glucose was depleted from the culture medium, revealing an obstructive role of glucose on glucose–xylose mixtures, in terms of xylitol secretion. Accordingly, it was observed that in mixtures in which xylose was in excess [i.e. glucose–xylose 25–75 (g l−1)], maximum xylitol production was 10·1 g l−1, a value much higher that the one obtained during cultivation of the strain in the other two mixtures, namely glycose–xylose 50–50 and 75–25 (g l−1) (Table 1). Worth mentioning also was the fact that during cultivation on glucose–xylose mixtures, all or part of the secreted xylitol was reconsumed by the strain during the fermentation process, in favour of lipid production (Fig. 2). Moreover, concerning the uptake of individual sugars in the trials with glucose and xylose used as dual substrates, it must be stressed that simultaneous assimilation of both sugars occurred regardless of the initial concentration of the sugars adjusted into the medium, suggesting the absence of diauxic growth of the micro-organism cultivated on binary mixtures of glucose and xylose. As far as the specificity of sugar uptake rates by the fungus is concerned, as noted in Fig. 3, in the case of glucose or xylose utilization as sole carbon sources, glucose was noticeably faster consumed as compared to xylose. On the contrary, during cultivation of the fungus in equal amounts of glucose–xylose mixtures, the specific uptake rates (rS‴, g l−1 h−1) for both sugars were similar (0·13 and 0·11 g l−1 h−1, for glucose and xylose, respectively).
Growth of Thamnidium elegans in bioreactor experiment
Based on the obtained results regarding lipid accumulation, the cultivation of T. elegans on glucose as the sole carbon source gave the most promising results among all substrates utilized. GLA production was remarkable during growth of the strain on glucose, whereas the corresponding lipid in dry mycelial mass value (YL/X) was interesting (47% w/w). Therefore, the forenamed cultivation was chosen to be performed in a 3-l bioreactor. In this case too, the performance of the micro-organism was satisfactory, by producing remarkable amounts of biomass (up to 30·1 g l−1) that contained noticeable lipid quantities (13·9 g l−1, corresponding to an YL/X value of ~46% w/w – see Table 1). As illustrated in Fig. 4, mycelial biomass production in the bioreactor was almost equal with the corresponding shake-flask experiment. However, the duration of the bioprocess was noticeably shorter in the bioreactor, while the accumulated lipid in the bioreactor was slightly lower than that produced in the shake-flask trial with the same carbon source. Therefore, it can be deduced that the preservation of optimized conditions in the bioreactor resulted in direction of microbial metabolism firstly towards biomass production and secondly towards intracellular lipid accumulation. This suggestion can be also enhanced by the fact that the specific substrate uptake rate (rS‴, g l−1 h−1) of glucose was significantly higher during batch-bioreactor trials, than in flask cultures (Fig. 3). Furthermore, it should be mentioned that the GLA content in the bioreactor trial was slightly reduced compared with the corresponding shake-flask experiment, a fact that could be attributed to the relatively lower lipid production of the micro-organism obtained in the bioreactor experiment.
Thamnidium elegans lipid analysis
Analysis of the lipids produced by T. elegans was conducted during course of fermentation time in all trials, to monitor possible alterations of the lipid profile of the micro-organism, in terms of FA composition. The obtained data are presented in Table 2. It can be easily observed that the predominant FA produced was oleic acid (Δ9C18:1), followed by palmitic acid (C16:0), linoleic acid (Δ9,12C18:2) and GLA (Δ6,9,12C18:3) in significant quantities. On the contrary, palmitoleic acid (Δ9C16:1) and stearic acid (C18:0) were detected in lower amounts. GLA (Δ6,9,12C18:3) was found in large contents (up to 18% w/w) in the produced oil, regardless of the substrate employed (Table 2). However, it is worth noticing that the concentrations of GLA were higher at the beginning of growth, but during the stationary phase, where actual lipid accumulation is carried out, GLA percentage was decreased. Finally, after completion of lipid accumulation process, GLA content of intracellular lipid increased again. Furthermore, oleic acid concentrations increased as oil accumulation progressed, while the opposite behaviour was observed for linoleic acid. Accordingly, the unsaturation index (UI) indicated that as the concentrations of GLA and linoleic acid raised, the lipid composition of the micro-organism became more unsaturated. The only exception in this general trend was noted in the mixture of glucose–xylose 50/50 (in g l−1), where the GLA concentration and hence the UI were increased during lipid accumulation process (Table 2). During cultivation of the strain in glucose–xylose mixtures, the percentage of oleic acid was found to be lower, as compared to those obtained during cultivation on either glucose or xylose as the sole carbon source. The opposite trend was observed for linoleic acid.
Table 2. Fatty acid distribution (in%, w/w) of total cellular lipids of Thamnidium elegans at early stationary (ES), stationary (S) and late stationary (LS) growth phase, during growth on glucose- and xylose-based media, solely or in mixtures
Fractionation of accumulated oil of T. elegans into neutral (N), glycolipids plus sphingolipids (G + S) and phospholipids (P), during lipid accumulation phases at all experiments with glucose and xylose, either as sole carbon source or as a mixture, revealed that the fraction of neutral lipids (N) was the major constituent of total lipids (Table 3). It is worth mentioning that the proportion of neutral lipids (N) nearly in all cases increased during the stationary phase (S), and a further increase occurred at late stationary phase (LS). However, the percentage of G + S and P fractions was high at the beginning of growth but declined thereafter. Moreover, analysis of intracellular lipids into their lipid fractions (N, G + S and P) of T. elegans growing in the bioreactor showed that the proportion of neutral lipids (N) was lower compared with the corresponding percentages in shake-flasks experiments. Furthermore, the polar fraction of cellular lipids (G + S and P) in the bioreactor presented increased percentages, which outweighed the polar lipids proportions of all the other experiments.
Table 3. Percentages (in%, w/w) of neutral (N), sphingolipid and glycolipid (G + S) and phospholipid (P) fractions during lipid accumulation phases of Thamnidium elegans cultivated on glucose and xylose-based media, solely or in mixtures
ES, early stationary growth phase; S, stationary growth phase; LS, late stationary growth phase.
Glucose (100 g l−1)
G + S
Xylose (100 g l−1)
G + S
Glucose–Xylose (50–50 g l−1)
G + S
Glucose–Xylose (75–25 g l−1)
G + S
Glucose–Xylose (25–75 g l−1)
G + S
Bioreactor Glucose (100 g l−1)
G + S
FA analysis of the lipid fractions of T. elegans cultivated in all of the substrates showed that the FA Δ9C18:1 was the dominant one followed by the FAs C16:0, Δ9,12C18:2 and GLA (Δ6,9,12C18:3) (Table 4). As previously stated, neutral lipids (N) was the major constituent of total lipids; thus, neutral fraction composition was found alike with FA profile of total lipids. Accordingly, neutral lipids (N) contained high amounts of the FA Δ9C18:1, while the fraction of G + S had the highest C16:0 content. Furthermore, it was observed that fraction of N lipids maintained a somehow constant FA composition with age and the UI remained almost invariable. However, the phospholipids fraction (P) was of great interest as it was particularly enriched in PUFAs and especially in GLA (Table 4). Indeed, the GLA percentage in phospholipids fraction was significant high (almost 20% w/w). As a result, the P fraction was more unsaturated than G + S and N fractions due to the high proportion of GLA and linoleic acid. Finally, it should be mentioned that the phospholipid fraction showed a marked decrease in GLA gradually with age in all experiments accompanied by a distinct decrease in the UI (Table 4). Nevertheless, the GLA content in P fraction during the cultivation of T. elegans in the mixture of glucose–xylose 75 : 25 (g l−1) presented a somehow different profile. The percentage of GLA during the stationary phase was higher (13·3%) than that during the early stationary phase (10·4%). However, as the cultivation progressed, the GLA content in P fraction was obviously reduced.
Table 4. Fatty acid composition (in%, w/w) in lipid fractions of Thamnidium elegans cellular lipids, during growth on glucose and xylose-based media, solely or in mixtures, in the late stationary (LS) growth phase
Lipids produced from microbial sources attract a potential industrial and financial interest due to their specific characteristics (Ratledge 1992, 1994; Čertik and Shimizu 1999; Papanikolaou and Aggelis 2011a,b; Koutinas and Papanikolaou 2011; Zhao and Hu 2011; Papanikolaou 2012). Especially, various oleaginous Zygomycetes have been employed for the biotechnological production of lipids, rich in PUFAs of pharmaceutical and nutritional interest, such as GLA, during their growth on a variety of carbon sources (Čertik et al. 1997; Kavadia et al. 2001; Fakas et al. 2008b, 2009a; Chatzifragkou et al. 2010, 2011). The present study was focused on the potentiality of a Zygomycetes strain, T. elegans, to utilize glucose and xylose, the principle products deriving from hydrolysis of lignocellulosic residues, as carbon sources for the production of intracellular lipids.
Glucose was an ideal substrate for the production of SCO by T. elegans; the rather remarkable amount of 15·0 g l−1 of fat (47·1%, w/w, of lipid in dry mycelial biomass) was obtained in shake-flask experiment with high initial glucose concentration (S0 = 100 g l−1). In this trial, the highest production of GLA was achieved, 1014 mg l−1. Furthermore, the micro-organism grew satisfactorily on xylose and produced fair amounts of microbial lipid, whereas the maximum GLA concentration obtained was 534 mg l−1. Moreover, T. elegans grew sufficiently in all mixture trials, while lipid synthesis was significant, reaching the maximum value of 12·6 g l−1. GLA production was also remarkable as up to 980 mg l−1 were synthesized. Regarding xylose catabolism by yeast and fungal strains, after sugar transport across the cell membrane via facilitative diffusion or active transport, xylose is subjected to a two-step reduction and oxidation (Winkelhausen and Kuzmanova 1998). Firstly, xylose is reduced to xylitol by either NADH- or NADPH-dependent xylose reductase. Then, xylitol is secreted from the cell or oxidized to xylulose by a NAD- or NADP-dependent xylitol dehydrogenase, followed by its phosphorylation to yield in the synthesis of xylulose-5-P. This intermediate will either be directly cleaved into glycerinaldehyde-3-P and acetyl-P (the so-called ‘phosphoketolase reaction]) or will enter into the pentose phosphate pathway (Jeffries 1983; Winkelhausen and Kuzmanova 1998; Sonderegger et al. 2004; Papanikolaou and Aggelis 2011a). In some cases, the existence of glucose in the fermentation medium as cosubstrate has been reported to partially inhibit xylose consumption, while the catabolism of the latter starts after the preferred sugar (i.e., glucose) is depleted (Winkelhausen and Kuzmanova 1998; Stulke and Hillen 1999; Kim et al. 2009). In our case, glucose and xylose were simultaneously consumed regardless of their initial concentrations into the medium, and therefore, no diauxic growth was observed, while xylitol secretion was observed after disappearance of glucose from the fermentation medium. Similarly, the cultivation of another oleaginous micro-organism, Trichosporon cutaneum AS 2.571, on glucose–xylose mixtures in flasks and bioreactor, was not accompanied by diauxic growth, with glucose and xylose being consumed simultaneously (Hu et al. 2011). In contrast, shake-flask cultures of Trichosporon fermentans CICC 1368 on sulfuric acid treated bagasse hydrolysate composed of mixtures of pentoses and hexoses, were accompanied initially by assimilation of hexoses (glucose plus galactose) and thereafter by assimilation of pentoses (xylose plus arabinose) (Huang et al. 2012a). Moreover, the literature indicates that the stoichiometry of glucose (and similar types of hexoses) metabolism has as result that ~1·1 moles of acetyl-CoA are generated from 100 g of glucose catabolized. On the other hand, when xylose is catabolized through the phosphoketolase reaction, ~1·2 moles of acetyl-CoA are generated per 100 g of xylose utilized, while the employment of the pentose phosphate pathway yields ~1·0 mole of acetyl-CoA formed per 100 g of xylose utilized (Ratledge 1988; Papanikolaou and Aggelis 2011a). In the current investigation, comparing the equivalent experiments of initial sugar glucose or xylose concentration at 100 g l−1 (see Table 1), and by taking into consideration that 1 mole of xylitol is generated through 1 mole of catabolized xylose (Sonderegger et al. 2004) (meaning therefore that ~1 g of xylitol is produced by the catabolism of 1 g of xylose), it may be assumed that the lipid yield per glucose consumed for T. elegans was 0·15 g g−1, while the respective one for xylose consumed was ~0·11 g g−1. Thus, it seems that T. elegans metabolized xylose favourably through the pentose phosphate pathway, corroborating the results reported for the fungus Mortierella isabellina and in disagreement with the results for the fungus Cunninhamella echinulata (Fakas et al. 2009a).
Cultivation of T. elegans on xylose and xylose–glucose mixtures resulted in enhanced xylitol production. Yeast strains and especially those belonging to the genus Candida are considered to be the best xylitol producers. Particularly, the strain Candida sp. 559-9 was found to accumulate up to 210 g l−1 xylitol when cultivated on media containing 300 g l−1 xylose (Ikeuchi et al. 1999). Also, a recombinant bacterium, Corynebacterium glutamicum pEKEx2-XYL1 produced up to 34·4 g l−1 of xylitol, during fed-batch cultivation on xylose (Kim et al. 2009). Regarding fungal strains, there is only one report referring to Petromyces albertensis, which accumulated 39·8 g l−1 of xylitol when cultivated for 10 days on 100 g l−1 xylose-based media (Dahiya 1991). These results are in agreement with the biochemical behaviour of T. elegans cultivated on 100 g l−1 xylose, which produced 31·3 g l−1 of xylitol about 240 h (10 days) after inoculation. The above-mentioned polyol was totally or partially reconsumed by the fungus, in favour of lipid production.
Cultivation of T. elegans in a bioreactor experiment gave satisfactory results concerning the produced biomass (30·1 g l−1) and the accumulated lipid (13·9 g l−1) but GLA yield was lower (742·0 mg l−1) compared with the corresponding shake-flask trial (1014 mg l−1). Comparison between shake-flask and bioreactor experiments for T. elegans growing on glucose revealed that cultivation in bioreactor was accompanied by a slightly higher uptake rate of glucose compared with the shake-flask experiments, while final total biomass and total lipid quantities were slightly higher in the shake-flask experiment (see Fig. 4). Thus, both cultivation strategies can be considered satisfactory for investigation and production of SCOs, while as in the present work, other studies indicate slight variations in the kinetic results achieved between shake-flask and bioreactor trials concerning oleaginous Zygomycetes (Kennedy et al. 1994; Chatzifragkou et al. 2010).
The highest quantity of GLA achieved in the current investigation was 1014 mg l−1 that is a rather satisfactory value compared with the literature; concerning other micro-organisms grown in flasks, C. echinulata CCRC 31840 produced 964 mg l−1 of GLA under optimized conditions (Chen and Chang 1996). Fakas et al. (2009a) demonstrated that the strain C. echinulata ATHUM 4411 cultivated on xylose, accumulated 1119 mg l−1 of GLA, while growth on tomato waste hydrolysate yielded 800 mg l−1 (Fakas et al. 2008b). Mortierella isabellina growing on lactose-enriched cheese whey produced GLA to ~300 mg l−1 (Vamvakaki et al. 2010). Cunninhamella echinulata produced ~500 mg l−1 on media based on waste molasses, while significant decolorization-detoxification of the medium was observed together with GLA production (Chatzifragkou et al. 2010). The highest quantity of GLA reported in the literature has been obtained by a mutant of Mortierella ramanniana (strain MM15-1) cultivated on media containing extremely high initial sugar amounts (initial glucose at 300 g l−1), and a concentration of ~5550 mg of GLA per l of culture medium was reported when growth was carried out on a specific type of bioreactor (Hiruta et al. 1997).
Despite their abundance in nature, lignocellulosic sugars (i.e. xylose) have been only recently utilized for SCO production; Ruan et al. (2012) investigated the ability of M. isabellina ATCC 42613 to produce lipids during its cultivation on xylose–glucose mixtures. The strain was found capable of maintaining a constant level of lipid yield and FA composition, reaching 8·8 g l−1 of SCO on xylose-based media. In another study, a Lipomyces starkeyi strain efficiently produced microbial oil during cofermentation of cellobiose/xylose mixtures, yielding a lipid content of 0·52 g g−1 (Gong et al. 2012). Zhang et al. (2011) reported the suitability of lignocellulosic sugars as substrates for SCO production by the oleaginous yeast Rhodotorula glutinis ATCC 15125 (lipid produced of ~2 g l−1). Trichosporon fermentans CICC 1368 was cultivated in shake-flask experiments with mixtures of glucose and xylose (at a ratio of 2/1 w/w) and the production of biomass and SCO was optimized in relation with the initial concentration of sugar added into the medium, the incubation temperature and the inoculum size. Then, the impact of several inhibitors typically found after lignocellulosic chemical hydrolysis upon biomass and SCO production was studied, and the highest quantities of biomass and lipid in dry weight recorded in media without addition of inhibitors were 24 g l−1 and ~62% w/w (Huang et al. 2012b). Also, the addition of some inhibitors (e.g. gallic acid) in small concentrations stimulated the production of biomass in this micro-organism (Huang et al. 2012b). Moreover, in flask cultures of T. cutaneum in mixtures of glucose and xylose, variable quantities of SCO were produced, with xylose being a less favourable substrate compared with glucose (Hu et al. 2011). The yeast Yarrowia lipolytica Po1 g was cultivated on various types of lignocellulosic hydrolysates and total biomass and lipid content quantities of 10·8–11·4 g l−1 and 48–59% w/w have been reported (Tsigie et al. 2011, 2012). Finally, shake-flask nitrogen-limited trials with corn fibre hydrolysate (containing principally glucose and xylose and to lesser extent arabinose and mannose) were performed with the strain M. isabellina M2, and the maximum quantities of biomass and SCO produced were 21·4 and 11·7 g l−1, respectively (Xing et al. 2012). Representative literature results and their comparisons with the current investigation concerning SCO production during growth on pure lignocellulosic sugars and/or lignocellulosic hydrolysates are seen in Table 5.
Table 5. Lipid production by various oleaginous microorganisms on lignocellulosic sugars or xylose-based media
Fungal growth was accompanied by proportional changes in amounts of lipid fractions as well as in their FA composition. Detailed lipid analysis of T. elegans showed specific trends with age which largely reflect the physiological role of individual lipids. More specifically, the fraction of neutral lipids (N) was the major component of total lipids followed by G + S and P fractions. In addition, the increase in neutral lipid content in aged mycelia could be indicative of the accumulation of some lipid storage with time. These results are in agreement with the general accepted energy storage role of neutral lipids, while phospholipids are considered as structural lipids of cell membranes and for this reason they are synthesized during active growth (Fakas et al. 2007, 2009a). These observations probably explain the high percentages of polar lipids (G + S and P) in the bioreactor cultivation due to the significant biomass production and the accordingly decreased lipid accumulation. Work with C. echinulata ATHUM 4411 grown on tomato waste hydrolysate showed that during lipid degradation neutral lipids were preferentially utilized against the other two fractions (G + S and P) (Fakas et al. 2007).
Among lipid fractions of T. elegans phospholipids (P) presented the highest GLA concentration, which reached the value of 20% (w/w) and consequently the UI of P fraction was higher than the corresponding in the other two fractions (N, G + S). According to Fakas et al. (2009b), lipid analysis of the strain T. elegans grown on raw glycerol showed that phospholipids were particularly rich in PUFAs and especially GLA. However, the GLA content in P fraction did not exceed the percentage of 10% (w/w). Furthermore, studies of C. echinulata ATHUM 4411 during its cultivation on tomato waste hydrolysate revealed that P fraction contained significant amounts of GLA, which ranged from 18 to 25% w/w (Fakas et al. 2006). Detail study of P fraction profile in our work indicated that the proportion of GLA in phospholipids decreased with age accompanied by a subsequent decrease in the UI. Also, as indicated by Fakas et al. (2006), the decrease in the GLA amount (expressed as percentage w/w of total lipid) in polar lipids of C. echinulata ATHUM 4411 was accompanied by an increase in the N fraction. Consequently, it can be deduced that this phenomenon occurs due to the fact that lipid accumulation constitutes a secondary metabolic process (Kavadia et al. 2001) and as a result the produced lipid is stored in neutral fraction as triacylglycerol (TAG) (Fakas et al. 2006, 2007, 2009a,b). This means that lipid is accumulated during course of the fermentation and, thus, GLA content in lipid fractions is found to gradually decrease.
Overall, the fungus T. elegans proved to be a promising micro-organism during its cultivation on glucose and xylose substrates, principal sugars of hydrolysed lignocellulosic biomass, yielding significant amounts of intracellular oil, rich in GLA. By taking into account that microbial lipid production still represents an expensive process, the successful bio-transformation of sugar-based lignocellulosic residues as substrates represents an attractive alternative.
The current investigation was partially funded by the project entitled ‘Research on oleaginous micro-organisms and development of new biotechnological processes’ (Acronym: ‘MicroOil’), action ‘ARISTEIA’, in the operational programme ‘Education and Lifelong Learning,’ 2007–2013 (financial support: GSRT – Ministry of Education and Religious Affairs, Culture and Sports; European Union).
Conflict of Interest
The authors have no conflict of interest to declare.