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Bioconversion of crude glycerol to glycolipids in Ustilago maydis
Bioresource Technology 102 (2011) 3927–3933
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Bioconversion of crude glycerol to glycolipids in Ustilago maydis Yanbin Liu, Chong Mei John Koh, Lianghui Ji ⇑ Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore
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Article history: Received 28 July 2010 Received in revised form 22 November 2010 Accepted 23 November 2010 Available online 28 November 2010 Keywords: Biosurfactant Crude glycerol Glycolipids Fed-batch fermentation Ustilago maydis
a b s t r a c t Ustilago maydis is known to produce glycolipid-type biosurfactants. Here, we show that U. maydis is able to efficiently convert biodiesel-derived crude glycerol to glycolipids. We have optimized the medium composition and environmental factors for bioconversion of crude glycerol to glycolipids. The synthetic medium (MinCG) contains 50 g L 1 crude glycerol and 20.3 mg L 1 ammonium citrate as the carbon and nitrogen sources, respectively. The supplementation of trace amount of amino acids, Group-B vitamins and precursors of glycolipids, mannose and erythritol, also improved the final yield. At pH 4.0 and 30 °C, 32.1 g L 1 total glycolipids was produced in a 8.2-day fed-batch bioprocess. Methanol at 2% or above severely inhibited cell growth and production of glycolipids. Our results suggest that U. maydis is an excellent host for the bioconversion of crude glycerol to value-added products. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Crude glycerol is a by-product of the biodiesel industry, accounting for approximately 10% (v/v) of the oil transesterification reaction (Wang et al., 2009). It is sometimes regarded as a waste product because of the cost associated with its disposal (Yazdani and Gonzalez, 2007). To date, utilization of glycerol, particularly crude glycerol, remains limited although there has been a number of reports on the conversion of glycerol to value-added products through chemical methods (Johnson and Taconi, 2007) or biological methods (Athalye et al., 2009; Papanikolaou and Aggelis, 2002). The end products include 1,3-propanediol, dihydroxyacetone, ethanol, succinate, propionic acid, glyceric acid, citric acid, hydroxypyruvic acids, polyhydroxyalcanoate, pigments and biosurfactants (da Silva et al., 2009). In recent years, remarkable exploration on the utilization and conversion of crude glycerol are on the way with improving yields (Ethier et al., 2010; Moon et al., 2010; Sabourin-Provost and Hallenbeck, 2009). Biosurfactants have been a subject of notable interest in recent years owing to their low toxicity, biodegradability and structural diversity. A small number of microorganisms have been reported to produce biosurfactants, which have diverse industrial applications, such as enhanced oil recovery, crude oil drilling, lubrication, surfactant-aided bioremediation, health care and food (Cameotra and Makkar, 2004). Glycolipids contain a carbohydrate (monosaccharide, disaccharide or oligosaccharide) and one or more lipophilic moieties consisting of saturated, unsaturated and/or hydroxylated ⇑ Corresponding author. Tel.: +65 6872 7483; fax: +65 6872 7007. E-mail address: [email protected] (L. Ji). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.115
fatty acids or fatty alcohols. The amphiphilic nature of the glycolipids make them very promising as a biosurfactant (Morita et al., 2009). Certain strains of Ustilago maydis were reported to secrete large amounts of glycolipid type biosurfactants, mainly mannosylerythritol lipids (MEL) and ustilagic acid (UA) under nitrogen-limiting conditions (Hewald et al., 2005). Both MEL and UA have excellent surface-active properties. Although glycolipids can be also produced by some species in Pseudomonas, Candida, Rhodococcus and Pseudozyma genera, U. maydis is by far the best-characterized species. With the wealth of genetic resources, genomic information, and genetic and molecular tools (see review by (Brefort et al., 2009)), U. maydis is a superior host for the bioengineering of improved strains for bioconversion of glycerol and other renewable resources. Recently, refined glycerol has been successfully applied to the production of rhamnolipid and MEL-type biosurfactants (Morita et al., 2007; Rahman et al., 2002). It has also been reported that Candida bombicola is able to produce 60 g L 1 sophorolipid from a biodiesel co-product stream (BCS), which is composed of 40% glycerol and 34% hexane-solubles (Ashby et al., 2005). Here, we present a simple method for conversion of crude glycerol to UA and MEL in U. maydis.
2. Methods 2.1. Source of crude glycerol and U. maydis strains Crude glycerol was obtained from Biofuel Research Pte. Ltd. (Singapore). The sample was derived from alkali-catalyzed
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transesterification of waste cooking oil with methanol. To analyze the composition of crude glycerol, the sample was diluted 4-fold with water and passed through a 0.2 lm filter (Pyle et al., 2008). Glycerol and methanol contents were determined by HPLC according to the methods described below. The soap content was estimated by adjusting the pH of crude glycerol to 1.0 using HCl and centrifuged at 4000g for 20 min. Soap content is the dark-red top layer, which was carefully removed and weighed (Liang et al., 2010). Ash content was determined by heating the sample at 550 °C overnight in a furnace (Fenton, UK) before the ash weight was determined (Pyle et al., 2008). Viscosity was measured with an Anton Paar rheometer (Physica MCR 301, Germany) utilizing a D-CP50 cone (50 mm in diameter) at 25 °C. The cone angle and distance between cone and plate was 1o and 0.5 mm, respectively. U. maydis L8 strain (a1 b1) used was as described (Ji et al., 2010). Strains were stored at 4 °C and renewed every 4 weeks. 2.2. Media and culture conditions U. maydis strains were cultured at 30 °C in 250 ml baffled shake flasks on an orbital shaker with an agitation speed of 250 rpm. YPD (10 g L 1 yeast extract, 20 g L 1 peptone and 20 g L 1 glucose) was purchased from Sigma (USA). The composition of YPG medium is similar to YPD except glucose is replaced with glycerol. Basic fermentation medium MinCG (minimum crude glycerol medium) contained 2.05 g K2HPO4, 1.45 g KH2PO4, 0.6 g MgSO4, 0.3 g NaCl, 10 mg CaCl2, 1 mg FeSO4, 0.5 mg ZnSO4, 0.5 mg CuSO4, 0.5 mg H3BO4, 0.5 mg MnSO4, 0.5 mg NaMoO4 (per liter), and indicated concentration of crude glycerol and nitrogen source. The medium pH was adjusted to 4.0. In medium shifting experiments, seed culture in exponential growth stage was harvested by centrifugation, washed with sterile water and resuspended in fermentation medium at a 1:10 ratio.
The concentration of ammonium in fermentation medium was quantified using the salicylate method (Nimura, 1973; Verdouw et al., 1978). Glycerol and methanol were quantified by HPLC. Crude glycerol or fermentation samples were filtered through a 0.2 lm membrane and run through a 300 7.0 mm Aminex 87H column (Bio-Rad) at a constant flow rate of 0.7 ml min 1 using 5 mM sulfuric acid as the mobile phase. The column was maintained at 50 °C, and glycerol and methanol were detected with a Refractive Index Detector (Shimadzu, Japan). Concentrations of glycerol and methanol were determined using calibration curves built with the respective standard chemicals (Sigma, USA). For analysis of glycolipids, small-scale extraction of glycolipids was performed essentially as previously reported with some modifications (Hewald et al., 2005). Cell suspension was mixed with half volume of ethyl acetate, centrifuged, and the upper phase was concentrated for 10 min in a SpeedVac concentrator and dissolved in methanol. Large-scale extraction and purification of MEL were performed as described (Rau et al., 2005). For purification of UA, the total glycolipid extract was washed with PBS buffer and 40% (w/v) methanol sequentially. UA was recovered by centrifugation, and the pellet was dried under vacuum and resuspended in methanol. Glycolipid profiling was performed by thin-layer chromatography (TLC) on silica plates (Silica gel 60, Merck) with a solvent system consisting of chloroform–methanol–water (65:25:4, v/v). Silica plates were thoroughly air-dried at room temperature and Table 1 Composition of crude glycerol after autoclavinga.
2.3. Fed-batch fermentation For fed-batch fermentation, a single colony of U. maydis was inoculated in 2.5 mL YPD medium, which has a carbon to nitrogen ratio (C:N ratio) of 7.5. The saturated seed culture was inoculated (2.5% inoculation rate) in YPD medium and cultured till it reached 5–10 OD600 units, at which the cells were harvested by centrifugation, washed with sterile water and inoculated to 1.5 L MinCG medium containing 50 g L 1 crude glycerol (41.7 g L 1 glycerol), 20.3 mg L 1 ammonium citrate and 10 mg L 1 each of alanine, aspartic acid and vitamin B (C:N ratio = 81.5). Fed-batch fermentation was carried out in a 2 L BiostatÒ B plus bioreactor (Sartorius Stedium, Germany). Fermentation was initiated when the residual glycerol dropped below 15 g L 1, at which the culture should have reached the exponential phase. A mixture containing 460 g L 1 crude glycerol and 132.2 mg L 1 ammonium citrate (C:N ratio = 5000) was fed at 3 mL h 1. Dissolved oxygen level (pO2) and airflow was maintained constantly at 30% and 1.0 vvm, respectively.
Concentration (g L
Glycerol Methanol Soap Water Ash Others Total
1024.32 ± 2.46 NDb 15.70 ± 2.50 142.33 ± 6.02 32.98 ± 0.16 12.65 ± 2.09 1228.05 ± 1.98
1
)
% (w/w) 83.41 ± 0.20 ND 1.28 ± 0.23 11.59 ± 0.34 2.69 ± 0.01 1.03 ± 0.17r 100 ± 0.16
a Data are means of three independent repeats ± standard deviations. b Not detected.
Table 2 Total fatty acid contents (percent of total fatty acid, TFA) in feedstocks and fermentation products. Fatty acid
C4:0 C5:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C16:2 C17:0 C18:0 C18:1 C18:2 C20:0 C22:0 Others Total
2.4. Effects of methanol Methanol was supplemented at various concentrations (2%, 5% and 10%, v/v) to basic fermentation medium containing 50 g L 1 of various carbon sources (glucose, pure glycerol or crude glycerol) and 3.39 g L 1 ammonium citrate. 2.5. Quantification methods Cell biomass (dry cell weight) was determined by drying the cell pellet in a 105 °C oven until constant weight was reached.
Composition
a b
Crude glycerol
–a – – – – – – – 10.68 0.61 – – 4.62 26.91 20.59 1.90 1.20 33.48 100
Glycolipids Glucose
Glycerol
Crude glycerol
3.16 1.83 5.25 7.30 6.54 11.50 2.63 12.00 11.10 17.27 0.45 – 4.64 3.29 4.92 – – 8.14 100
2.19 1.61 5.27 4.30 2.16 5.12 12.57 2.56 26.13 12.66 5.33 0.44 3.88 4.28 5.09 – – 6.41 100
2.43 – 22.39 (4.16) 3.98 (1.83) 1.84 6.06 13.19 3.73 25.14 (4.32) – – – 5.28 3.79 6.48 – – 5.68 100
(1.27)b (2.53)
(1.44) (3.28)
(0.98) (1.10)
(3.29) (1.83)
Not detected. Numbers shown in brackets are the compositions of b-hydroxylated fatty acids.
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sugar-containing compounds were visualized by spraying the plates with a mixture of glacial acetic acid–sulfuric acid–p-anisaldehyde (100:2:1, v/v) and heated at 150 °C for 5 min. For HPLC analysis, purified glycolipid samples were diluted 5-fold and filtered through a 0.2 lm membrane. HPLC separation was performed by loading a 2 lL sample into a 150 4.6 mm C18 silica column (Waters, USA), and run at a constant flow rate of 1 mL min 1 using methanol plus 0.05% formic acid as the mobile phase. Glycolipids were detected using an Evaporative Light Scattering Detector (ELSD) (Shimadzu, Japan). The column temperature was maintained at 45 °C and the ELSD was set at 40 °C. For quantification, hexane-purified glycolipids from U. maydis L8 strain were used as the standard and the concentration of glycolipids was the average of three repeats.
were analyzed by GC–MS, an Agilent 7890 GC coupled to a mass spectrometer (HP 5975C, Agilent). The system was equipped with a 27 m Rtx-5 MS capillary column (Agilent J&W, 0.25 mm i.d., 0.25 lm film thickness). Data collection and analyzing were performed as described previously (Hartig, 2008).
2.6. Fatty acid analysis
3.1. Characterization of crude glycerol
Fatty acid profiles of crude glycerol, glycolipids and cell biomass were determined after esterification to fatty acid methyl esters (FAME) using methanolic HCl (Sigma) as described previously (Chi et al., 2007; Ulberth and Henninger, 1992). The FAME samples
It has been reported previously that compositions of crude glycerol samples varied widely among oil feedstocks used and biodiesel production processes (Dasari, 2007; Pyle et al., 2008). In this study, crude glycerol derived from waste cooking oil showed a dark
2.7. Light microscopy Cells were observed under a Nikon Eclipse 80i microscope equipped with CFI Plan Apochromat objectives (Nikon, Japan) and images were acquired with a Nikon DS camera and Nikon ACT-2U software. 3. Results and discussion
Fig. 1. Production of glycolipids from glycerol and crude glycerol containing media. (A) Cell biomass yields. (B) The residual carbon sources. (C) Glycolipids yields. Small-scale fermentation was performed at 30 °C on a shaking platform (250 rpm) in triplicates in 250 ml conical flasks using MinCG media supplemented with 0.5 g L 1 NH4NO3 and 50 g L 1 of one the carbon sources indicated. (D) Cell morphology after a 12-day fermentation with glycerol and crude glycerol as the sole carbon source. The scale bar (10 lm) is shown.
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brown color with viscosity of 137 Centipoise at 25 °C. The crude glycerol sample used in this study has been adjusted to pH 6.0 and autoclaved in order to dissimilate the residual methanol (Athalye et al., 2009; Thompson and He, 2006). The treated crude glycerol contained 83.41% (w/w) glycerol, 11.59% water, 1.28% soap and 2.69% ash (Table 1). Methanol was undetectable after autoclaving (121 °C, 15 min) although it was present at 1.10% (w/w) in the original sample, similar to what has been reported previously (Chi et al., 2007; Pyle et al., 2008). Palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2) were the main fatty acids in the crude glycerol sample (Table 2). 3.2. Strains and culture conditions for bioconversion of crude glycerol to glycolipids U. maydis L8 strain was cultured in a series of synthetic media to assess glycolipid production. TLC analysis showed that it was possible to produce both UA and MEL when a minimal synthetic medium (MinG) containing 50 g L 1 of refined glycerol as the sole carbon source in the culture medium (data not shown). Surprisingly, cells grew significantly better (Fig. 1A) and consumed more glycerol (Fig. 1B) when crude glycerol was used as the sole carbon source. Importantly, total amount of glycolipids produced was comparable to that obtained with glucose as the sole carbon source (Fig. 1C). In addition, there was no discernible difference in cell morphology when cultured in the two glycerol sources (Fig. 1D). These results suggest that crude glycerol is a potential carbon source for production of glycolipids in U. maydis.
ranging between 24 °C and 35 °C in MinCG, containing 50 g L 1 crude glycerol as the sole carbon source and 0.5 g L 1 NH4NO3. The highest amount of glycolipids was produced at 30 °C after 6 days (Fig. 2A). The glycolipid yield varied greatly at various pH, with the highest observed at pH 4.0 (Fig. 2B). In addition, dissolved oxygen levels in the medium were investigated by culturing cells with an agitation speed between 100 and 300 rpm. Marginal improvement in the production of glycolipids was observed when media aeration was performed between 200 and 300 rpm (data not shown). Hence, production of glycolipids was done in MinCG medium (pH 4.0) at 30 °C with an agitation speed of 200 rpm in the subsequent experiments. 3.4. Optimization of culture medium To determine the optimal concentration of carbon source, MinCG medium was supplemented with various volume of crude
3.3. Effects of culture temperature and medium pH To optimize the conditions for bioconversion from crude glycerol to glycolipids, U. maydis was cultured at various temperature
Fig. 2. Effects of fermentation temperature and medium pH. MinCG medium with 50 g L 1 crude glycerol and 0.5 g L 1 NH4NO3 was used at various temperature (A) or medium pH (B). The fermentation cultures were sampled on the 5th day and quantified by HPLC. Results are average of three repeats.
Fig. 3. Effects of crude glycerol concentration, nitrogen sources and the concentration of ammonium citrate. (A) Effects of crude glycerol concentrations. MinCG media with different concentrations of crude glycerol and 0.5 g L 1 NH4NO3 were used for fermentation. The fermentation cultures were sampled on the 5th day and analyzed for dry cell weight and total glycolipid yield. (B) Effects of nitrogen sources. All nitrogen sources were kept at 10 mM. (C) Effects of ammonium citrate concentrations. MinCG media with 50 g L 1 crude glycerol and various concentrations of ammonium citrate were used for fermentation. The fermentation cultures were sampled on the 5th day. All results are average of triplicates.
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glycerol. Both glycolipid yield and cell biomass increased steadily from 10 to 50 g L 1 of crude glycerol. Further increase in the amount of crude glycerol affected the glycolipid yield negatively, even though cell biomass continued to increase in the whole range of carbon source feeding tested. The best result was obtained with 50 g L 1 crude glycerol, yielding 6.7 g L 1 total glycolipids (Fig. 3A). To determine the effects of nitrogen sources, eight inorganic and three organic nitrogen sources were tested, with each concentration kept as 10 mM. The results showed that organic nitrogen sources favored the accumulation of cell mass but had a negative effect on the accumulation of glycolipids. On the other hand, inorganic nitrogen sources, such as ammonium nitrate, ammonium diphosphate and ammonium citrate enhanced the yield of glycolipids. The addition of 10 mM ammonium citrate gave the best result, yielding 4.8 g L 1 total glycolipids in a 5-day culture (Fig. 3B). Thus, ammonium citrate was selected as the sole nitrogen source. To determine the optimal concentration of ammonium citrate required for fermentation, shaking flasks cultures were performed in MinCG media supplemented with 1–200 mM of ammonium citrate. During a 5-day fermentation, raising its concentration from 15 to 20 mM significantly increased the yield of glycolipids (Fig. 3C). As expected, the production of glycolipids was highly dependent on the level of nitrogen source. When ammonium citrate was added beyond 50 mM, glycolipid production was essentially terminated while cell mass accumulation was not negatively affected until the concentration reached over 100 mM. Collectively, the optimal fermentation medium for the production of glycolipids contains 50 g L 1 of glycerol and 1.6 g L 1 ammonium citrate with C:N ratio of 27. Subsequently, the effects of trace elements and oligosaccharides were assessed. Twenty basic amino acids and two enzyme cofactors (NAD+ and Vitamin B1+B6) were supplemented to the MinCG medium individually at 10 mg L 1 for amino acids and enzyme cofactors. Aspartic acid, alanine and B-group vitamins displayed
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substantial enhancing effects on the production of glycolipids (Fig. 4A). Interestingly, although cell mass accumulation was not affected, asparagine and lysine had an inhibiting effect on glycerol utilization (Fig. 4A and B). Supplementation of NAD+ did not improve glycolipid yield. Therefore, aspartic acid, alanine and B-group vitamins are recommended for the medium. In the last step of the optimization, precursors of glycolipids, i.e., mannose and erythritol, were supplemented to MinCG medium separately. Although supplementation in low concentration (0.1 g L 1) of either precursor inhibited the production of glycolipids, the yield of UA and MEL was found to increase steadily from 1 to 20 g L 1 (Fig. 4C). Notably, glycerol consumption was concomitantly increased (Fig. 4D). These results suggest that supplementation of mannose and erythritol significantly improved the production of glycolipids in U. maydis cells when crude glycerol was used as the main carbon source. However, neither is recommended for the medium due to the high cost of both precursors.
3.5. Fatty acid profiles in glycolipids Glycolipids produced using glucose as the sole carbon source produced fatty acid profile that was similar to that reported previously (Spoeckner et al., 1999) (Table 2). In general, fatty acid compositions of total glycolipids were comparable among the three different carbon sources used (glucose, pure glycerol and crude glycerol), except that palmitic acid (C16:0) was about 2.5-fold higher in media with pure or crude glycerol. Palmitomeic acid (C16:1) was absent when crude glycerol was used, and compared with glucose, its level was reduced substantially (26.6%) when pure glycerol was used (Table 2). In addition, hexanoic acid (C6:0) level increased more than 4-fold with crude glycerol as the sole carbon source (Table 2). These results suggest that the production of palmitic acid was enhanced when glycerol is used as a
Fig. 4. Effects of other supplements. MinCG media with 50 g L 1 crude glycerol, 20.3 mg L 1 ammonium citrate and various other supplements were used for fermentation. The fermentation cultures were sampled on the 5th day. Results are average of triplicates. Effects of all 20 amino acids, vitamin B1+B6 and NAD+ on glycolipids and biomass production are shown in (A) and residual glycerol in (B). All nutrients were supplemented at 0.1 mg L 1. The blank columns indicate the media that lead to relatively higher glycolipid production. Effects of sugar supplements are shown in (C) and (D).
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(Pyle et al., 2008) and Crypthecodinium cohnii (de Swaaf et al., 2003). To determine the effects of methanol residue present in crude glycerol on U. maydis cell growth and production of glycolipids, different concentrations of methanol (0%, 2%, 5% and 10%, v/v) were supplemented to the fermentation media containing glucose, glycerol or autoclaved crude glycerol as the sole carbon source. The addition of 2% methanol severely inhibited cell growth and glycolipid production with a corresponding drop in carbon source utilization. At 5%, cell growth and glycolipid production were abolished (Fig. 5). These results demonstrate that high concentration of methanol (>2%) is detrimental to cell growth and production of glycolipids, irrespective of the main carbon source used in U. maydis. However, as methanol in the crude glycerol used is present at just about 1.10% (0.05% in fermentation media with 50 g L 1 crude glycerol), any negative effects are likely to be small. 3.7. Fed-batch fermentation
Fig. 5. Effects of methanol in fermentation medium. (A) Cell biomass. (B) Residual carbon sources in the medium. (C) Glycolipids yield. Results are average of three independent repeats.
carbon source, and certain components in crude glycerol may strongly enhance the production of short-chain hexanoic acid. 3.6. Effects of residual methanol on production of glycolipids Methanol may negatively or positively affect cell growth and docosahexaenoic acid production in Schizochytrium limacinum
To confirm the effectiveness of the medium composition and culture conditions developed, fed-batch fermentation was performed. In the fermentation medium that had a C:N ratio of 27, mannose and erythritol were not added. The cells began to produce glycolipids after nitrogen was consumed, causing the C:N ratio to increase to 110. The glycolipid yield peaked on the 8th day of fed batch fermentation (175 h in total). At this time point, about 32.1 g L 1 total glycolipids were produced (Fig. 6). This is approximately double what was previously reported (16.3 g L 1), which used much more costly refined glycerol in combination with mannose supplementation (Morita et al., 2007). As UA is much more toxic than MEL to the fungal cells (Haskins and Thorn, 1951; Teichmann et al., 2010), we believe that the biggest hurdle in our process may lie in the toxicity of UA during fermentation. Therefore, it is reasonable to assume that a higher level of glycolipids could be produced when UA-deficient mutants, e.g. Dcyp1 (Hewald et al., 2006), is used as the biocatalyst. In recent years, good progress has been made in the understanding of glycolipid biosynthesis pathway in U. maydis (Hewald et al., 2006; Teichmann et al., 2007). It is expected that our glycolipid production process can be further improved by genetic engineering. For example, over-expression of rua1 promotes UA synthesis even in the presence of a rich nitrogen source (Teichmann et al., 2010). Similarly, over-expression of precursors for glycolipid biosynthetic genes, for example, mannose-6-phosphate isomerase, phosphomannomutase and mannose-1-phosphate guanylyltransferase, might mimic the
Fig. 6. Fed-batch fermentation. U. maydis L8 strain was cultured in a 2-L fermentor in MinCG medium containing 50 g L 1 crude glycerol (41.7 g L 1 glycerol), 20.3 mg L 1 ammonium citrate, 10 mg L 1 each of alanine, aspartic acid and B-group vitamins, for 175 h. The arrow indicates the time point (80 h) where fed-batch fermentation was initiated by medium with 460 g L 1 crude glycerol and 132.2 mg L 1 ammonium citrate.
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effect of precursor supplementation. Perhaps, the most interesting genes to explore are those that improve stress tolerance, such as those encoding proteins involved in the unfolded protein response (Travers et al., 2000) and cell wall integrity (Scrimale et al., 2009). 4. Conclusion In this study, we have shown that U. maydis is able to efficiently utilize and convert crude glycerol to glycolipid-type biosurfactant in a relatively simple synthetic medium using crude glycerol as the sole carbon source. Through further strain improvements, we expect ample room for further improvement in glycolipids yield. Also, engineered strains of U. maydis may be a good biocatalyst for conversion of crude glycerol to other value-added products. Acknowledgements This work is financially supported by the Singapore Economic Development Board and Temasek Trust. We thank Dr. Yunping Bu, Temasek Life Sciences Laboratory (Singapore), and Madam Lai Kwai Wong, Department of Chemistry, National University of Singapore (Singapore), for the GC and GC–MS analyzes. References Ashby, R., Nunez, A., Solaiman, D., Foglia, T., 2005. Sophorolipid biosynthesis from a biodiesel co-product stream. J. Amer. Oil. Chem. Soc. 82 (9), 625–630. Athalye, S.K., Garcia, R.A., Wen, Z., 2009. Use of biodiesel-derived crude glycerol for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare. J. Agric. Food Chem. 57 (7), 2739–2744. Brefort, T., Doehlemann, G., Mendoza-Mendoza, A., Reissmann, S., Djamei, A., Kahmann, R., 2009. Ustilago maydis as a Pathogen. Annu. Rev. Phytopathol. 47, 423–445. Cameotra, S.S., Makkar, R.S., 2004. Recent applications of biosurfactants as biological and immunological molecules. Curr. Opin. Microbiol. 7 (3), 262–266. Chi, Z., Pyle, D., Wen, Z., Frear, C., Chen, S., 2007. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochem. 42 (11), 1537–1545. da Silva, G.P., Mack, M., Contiero, J., 2009. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol. Adv. 27 (1), 30–39. Dasari, M. 2007. Crude glycerol potential described. In: Feedstuffs, vol. 15, p. 16. de Swaaf, M.E., Pronk, J.T., Sijtsma, L., 2003. Fed-batch cultivation of the docosahexaenoic-acid-producing marine alga Crypthecodinium cohnii on ethanol. Appl. Microbiol. Biotechnol. 61 (1), 40–43. Ethier, S., Woisard, K., Vaughan, D., Wen, Z., 2010. Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Bioresour. Technol. 102 (1), 88–93. Hartig, C., 2008. Rapid identification of fatty acid methyl esters using a multidimensional gas chromatography–mass spectrometry database. J. Chromatogr. A 1177 (1), 159–169. Haskins, R., Thorn, J., 1951. Biochemistry of the ustilaginales: VII. Antibiotic activity of ustilagic acid. Can. J. Bot. 29 (6), 585–592. Hewald, S., Josephs, K., Bölker, M., 2005. Genetic analysis of biosurfactant production in Ustilago maydis. Appl. Environ. Microbiol. 71 (6), 3033–3040. Hewald, S., Linne, U., Scherer, M., Marahiel, M.A., Kämper, J., Bölker, M., 2006. Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis. Appl. Environ. Microbiol. 72 (8), 5469–5477.
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Ji, L., Jiang, Z.D., Liu, Y., Koh, C.M., Zhang, L.H., 2010. A simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells. Fungal Genet. Biol. 47 (4), 279–287. Johnson, D., Taconi, K., 2007. The glycerin glut: options for the value-added conversion of crude glycerol resulting from biodiesel production. Environ. Prog. 26 (4), 338–348. Liang, Y., Sarkany, N., Cui, Y., Blackburn, J.W., 2010. Batch stage study of lipid production from crude glycerol derived from yellow grease or animal fats through microalgal fermentation. Bioresour. Technol. 101 (17), 6745–6750. Moon, C., Ahn, J.H., Kim, S.W., Sang, B.I., Um, Y., 2010. Effect of biodiesel-derived raw glycerol on 1,3-propanediol production by different microorganisms. Appl. Biochem. Biotechnol. 161 (1–8), 502–510. Morita, T., Fukuoka, T., Imura, T., Kitamoto, D., 2009. Production of glycolipid biosurfactants by basidiomycetous yeasts. Biotechnol. Appl. Biochem. 53 (Pt 1), 39–49. Morita, T., Konishi, M., Fukuoka, T., Imura, T., Kitamoto, D., 2007. Microbial conversion of glycerol into glycolipid biosurfactants, mannosylerythritol lipids, by a basidiomycete yeast, Pseudozyma antarctica JCM 10317(T). J. Biosci. Bioeng. 104 (1), 78–81. Nimura, Y., 1973. A direct estimation of microgram amounts of ammonia in water without salt-error. Bull. Jpn. Soc. Sci. Fish. 39 (12), 1315–1324. Papanikolaou, S., Aggelis, G., 2002. Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture. Bioresour. Technol. 82 (1), 43–49. Pyle, D.J., Garcia, R.A., 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 (11), 3933– 3939. Rahman, K.S., Rahman, T.J., McClean, S., Marchant, R., Banat, I.M., 2002. Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials. Biotechnol. Prog. 18 (6), 1277–1281. Rau, U., Nguyen, A.H., Roeperc, H., Kochc, H., Langa, S., 2005. Downstream processing of mannosylerythritol lipids produced by Pseudozyma aphidis. Eur. J. Lipid Sci. Technol. 107 (6), 373–380. Sabourin-Provost, G., Hallenbeck, P.C., 2009. High yield conversion of a crude glycerol fraction from biodiesel production to hydrogen by photofermentation. Bioresour. Technol. 100 (14), 3513–3517. Scrimale, T., Didone, L., de Mesy Bentley, K.L., Krysan, D.J., 2009. The unfolded protein response is induced by the cell wall integrity mitogen-activated protein kinase signaling cascade and is required for cell wall integrity in Saccharomyces cerevisiae. Mol. Biol. Cell 20 (1), 164–175. Spoeckner, S., Wray, V., Nimtz, M., Lang, S., 1999. Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources. Appl. Microbiol. Biotechnol. 51 (1), 33–39. Teichmann, B., Linne, U., Hewald, S., Marahiel, M.A., Bölker, M., 2007. A biosynthetic gene cluster for a secreted cellobiose lipid with antifungal activity from Ustilago maydis. Mol. Microbiol. 66 (2), 525–533. Teichmann, B., Liu, L., Schink, K.O., Bölker, M., 2010. Activation of the ustilagic acid biosynthesis gene cluster in Ustilago maydis by the C2H2 zinc finger transcription factor Rua1. Appl. Environ. Microbiol. 76 (8), 2633–2640. Thompson, J., He, B., 2006. Characterization of crude glycerol from biodiesel production from multiple feedstocks. Appl. Eng. Agric. 22 (2), 261–265. Travers, K.J., Patil, C.K., Wodicka, L., Lockhart, D.J., Weissman, J.S., Walter, P., 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101 (3), 249– 258. Ulberth, F., Henninger, M., 1992. One-step extraction/methylation method for determining the fatty acid composition of processed foods. J. Amer. Oil. Chem. Soc. 69 (2), 174–177. Verdouw, H., Van Echteld, C.J.A., Dekkers, E.M.J., 1978. Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12 (6), 399– 402. Wang, X., Dou, P., Zhao, P., Zhao, C., Ding, Y., Xu, P., 2009. Immobilization of lipases onto magnetic Fe(3)O(4) nanoparticles for application in biodiesel production. Chem. Sus. Chem. 2 (10), 947–950. Yazdani, S.S., Gonzalez, R., 2007. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr. Opin. Biotechnol. 18 (3), 213– 219.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Bioconversion of crude glycerol to glycolipids in Ustilago maydis Yanbin Liu, Chong Mei John Koh, Lianghui Ji ⇑ Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore
a r t i c l e
i n f o
Article history: Received 28 July 2010 Received in revised form 22 November 2010 Accepted 23 November 2010 Available online 28 November 2010 Keywords: Biosurfactant Crude glycerol Glycolipids Fed-batch fermentation Ustilago maydis
a b s t r a c t Ustilago maydis is known to produce glycolipid-type biosurfactants. Here, we show that U. maydis is able to efficiently convert biodiesel-derived crude glycerol to glycolipids. We have optimized the medium composition and environmental factors for bioconversion of crude glycerol to glycolipids. The synthetic medium (MinCG) contains 50 g L 1 crude glycerol and 20.3 mg L 1 ammonium citrate as the carbon and nitrogen sources, respectively. The supplementation of trace amount of amino acids, Group-B vitamins and precursors of glycolipids, mannose and erythritol, also improved the final yield. At pH 4.0 and 30 °C, 32.1 g L 1 total glycolipids was produced in a 8.2-day fed-batch bioprocess. Methanol at 2% or above severely inhibited cell growth and production of glycolipids. Our results suggest that U. maydis is an excellent host for the bioconversion of crude glycerol to value-added products. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Crude glycerol is a by-product of the biodiesel industry, accounting for approximately 10% (v/v) of the oil transesterification reaction (Wang et al., 2009). It is sometimes regarded as a waste product because of the cost associated with its disposal (Yazdani and Gonzalez, 2007). To date, utilization of glycerol, particularly crude glycerol, remains limited although there has been a number of reports on the conversion of glycerol to value-added products through chemical methods (Johnson and Taconi, 2007) or biological methods (Athalye et al., 2009; Papanikolaou and Aggelis, 2002). The end products include 1,3-propanediol, dihydroxyacetone, ethanol, succinate, propionic acid, glyceric acid, citric acid, hydroxypyruvic acids, polyhydroxyalcanoate, pigments and biosurfactants (da Silva et al., 2009). In recent years, remarkable exploration on the utilization and conversion of crude glycerol are on the way with improving yields (Ethier et al., 2010; Moon et al., 2010; Sabourin-Provost and Hallenbeck, 2009). Biosurfactants have been a subject of notable interest in recent years owing to their low toxicity, biodegradability and structural diversity. A small number of microorganisms have been reported to produce biosurfactants, which have diverse industrial applications, such as enhanced oil recovery, crude oil drilling, lubrication, surfactant-aided bioremediation, health care and food (Cameotra and Makkar, 2004). Glycolipids contain a carbohydrate (monosaccharide, disaccharide or oligosaccharide) and one or more lipophilic moieties consisting of saturated, unsaturated and/or hydroxylated ⇑ Corresponding author. Tel.: +65 6872 7483; fax: +65 6872 7007. E-mail address: [email protected] (L. Ji). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.115
fatty acids or fatty alcohols. The amphiphilic nature of the glycolipids make them very promising as a biosurfactant (Morita et al., 2009). Certain strains of Ustilago maydis were reported to secrete large amounts of glycolipid type biosurfactants, mainly mannosylerythritol lipids (MEL) and ustilagic acid (UA) under nitrogen-limiting conditions (Hewald et al., 2005). Both MEL and UA have excellent surface-active properties. Although glycolipids can be also produced by some species in Pseudomonas, Candida, Rhodococcus and Pseudozyma genera, U. maydis is by far the best-characterized species. With the wealth of genetic resources, genomic information, and genetic and molecular tools (see review by (Brefort et al., 2009)), U. maydis is a superior host for the bioengineering of improved strains for bioconversion of glycerol and other renewable resources. Recently, refined glycerol has been successfully applied to the production of rhamnolipid and MEL-type biosurfactants (Morita et al., 2007; Rahman et al., 2002). It has also been reported that Candida bombicola is able to produce 60 g L 1 sophorolipid from a biodiesel co-product stream (BCS), which is composed of 40% glycerol and 34% hexane-solubles (Ashby et al., 2005). Here, we present a simple method for conversion of crude glycerol to UA and MEL in U. maydis.
2. Methods 2.1. Source of crude glycerol and U. maydis strains Crude glycerol was obtained from Biofuel Research Pte. Ltd. (Singapore). The sample was derived from alkali-catalyzed
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transesterification of waste cooking oil with methanol. To analyze the composition of crude glycerol, the sample was diluted 4-fold with water and passed through a 0.2 lm filter (Pyle et al., 2008). Glycerol and methanol contents were determined by HPLC according to the methods described below. The soap content was estimated by adjusting the pH of crude glycerol to 1.0 using HCl and centrifuged at 4000g for 20 min. Soap content is the dark-red top layer, which was carefully removed and weighed (Liang et al., 2010). Ash content was determined by heating the sample at 550 °C overnight in a furnace (Fenton, UK) before the ash weight was determined (Pyle et al., 2008). Viscosity was measured with an Anton Paar rheometer (Physica MCR 301, Germany) utilizing a D-CP50 cone (50 mm in diameter) at 25 °C. The cone angle and distance between cone and plate was 1o and 0.5 mm, respectively. U. maydis L8 strain (a1 b1) used was as described (Ji et al., 2010). Strains were stored at 4 °C and renewed every 4 weeks. 2.2. Media and culture conditions U. maydis strains were cultured at 30 °C in 250 ml baffled shake flasks on an orbital shaker with an agitation speed of 250 rpm. YPD (10 g L 1 yeast extract, 20 g L 1 peptone and 20 g L 1 glucose) was purchased from Sigma (USA). The composition of YPG medium is similar to YPD except glucose is replaced with glycerol. Basic fermentation medium MinCG (minimum crude glycerol medium) contained 2.05 g K2HPO4, 1.45 g KH2PO4, 0.6 g MgSO4, 0.3 g NaCl, 10 mg CaCl2, 1 mg FeSO4, 0.5 mg ZnSO4, 0.5 mg CuSO4, 0.5 mg H3BO4, 0.5 mg MnSO4, 0.5 mg NaMoO4 (per liter), and indicated concentration of crude glycerol and nitrogen source. The medium pH was adjusted to 4.0. In medium shifting experiments, seed culture in exponential growth stage was harvested by centrifugation, washed with sterile water and resuspended in fermentation medium at a 1:10 ratio.
The concentration of ammonium in fermentation medium was quantified using the salicylate method (Nimura, 1973; Verdouw et al., 1978). Glycerol and methanol were quantified by HPLC. Crude glycerol or fermentation samples were filtered through a 0.2 lm membrane and run through a 300 7.0 mm Aminex 87H column (Bio-Rad) at a constant flow rate of 0.7 ml min 1 using 5 mM sulfuric acid as the mobile phase. The column was maintained at 50 °C, and glycerol and methanol were detected with a Refractive Index Detector (Shimadzu, Japan). Concentrations of glycerol and methanol were determined using calibration curves built with the respective standard chemicals (Sigma, USA). For analysis of glycolipids, small-scale extraction of glycolipids was performed essentially as previously reported with some modifications (Hewald et al., 2005). Cell suspension was mixed with half volume of ethyl acetate, centrifuged, and the upper phase was concentrated for 10 min in a SpeedVac concentrator and dissolved in methanol. Large-scale extraction and purification of MEL were performed as described (Rau et al., 2005). For purification of UA, the total glycolipid extract was washed with PBS buffer and 40% (w/v) methanol sequentially. UA was recovered by centrifugation, and the pellet was dried under vacuum and resuspended in methanol. Glycolipid profiling was performed by thin-layer chromatography (TLC) on silica plates (Silica gel 60, Merck) with a solvent system consisting of chloroform–methanol–water (65:25:4, v/v). Silica plates were thoroughly air-dried at room temperature and Table 1 Composition of crude glycerol after autoclavinga.
2.3. Fed-batch fermentation For fed-batch fermentation, a single colony of U. maydis was inoculated in 2.5 mL YPD medium, which has a carbon to nitrogen ratio (C:N ratio) of 7.5. The saturated seed culture was inoculated (2.5% inoculation rate) in YPD medium and cultured till it reached 5–10 OD600 units, at which the cells were harvested by centrifugation, washed with sterile water and inoculated to 1.5 L MinCG medium containing 50 g L 1 crude glycerol (41.7 g L 1 glycerol), 20.3 mg L 1 ammonium citrate and 10 mg L 1 each of alanine, aspartic acid and vitamin B (C:N ratio = 81.5). Fed-batch fermentation was carried out in a 2 L BiostatÒ B plus bioreactor (Sartorius Stedium, Germany). Fermentation was initiated when the residual glycerol dropped below 15 g L 1, at which the culture should have reached the exponential phase. A mixture containing 460 g L 1 crude glycerol and 132.2 mg L 1 ammonium citrate (C:N ratio = 5000) was fed at 3 mL h 1. Dissolved oxygen level (pO2) and airflow was maintained constantly at 30% and 1.0 vvm, respectively.
Concentration (g L
Glycerol Methanol Soap Water Ash Others Total
1024.32 ± 2.46 NDb 15.70 ± 2.50 142.33 ± 6.02 32.98 ± 0.16 12.65 ± 2.09 1228.05 ± 1.98
1
)
% (w/w) 83.41 ± 0.20 ND 1.28 ± 0.23 11.59 ± 0.34 2.69 ± 0.01 1.03 ± 0.17r 100 ± 0.16
a Data are means of three independent repeats ± standard deviations. b Not detected.
Table 2 Total fatty acid contents (percent of total fatty acid, TFA) in feedstocks and fermentation products. Fatty acid
C4:0 C5:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C16:2 C17:0 C18:0 C18:1 C18:2 C20:0 C22:0 Others Total
2.4. Effects of methanol Methanol was supplemented at various concentrations (2%, 5% and 10%, v/v) to basic fermentation medium containing 50 g L 1 of various carbon sources (glucose, pure glycerol or crude glycerol) and 3.39 g L 1 ammonium citrate. 2.5. Quantification methods Cell biomass (dry cell weight) was determined by drying the cell pellet in a 105 °C oven until constant weight was reached.
Composition
a b
Crude glycerol
–a – – – – – – – 10.68 0.61 – – 4.62 26.91 20.59 1.90 1.20 33.48 100
Glycolipids Glucose
Glycerol
Crude glycerol
3.16 1.83 5.25 7.30 6.54 11.50 2.63 12.00 11.10 17.27 0.45 – 4.64 3.29 4.92 – – 8.14 100
2.19 1.61 5.27 4.30 2.16 5.12 12.57 2.56 26.13 12.66 5.33 0.44 3.88 4.28 5.09 – – 6.41 100
2.43 – 22.39 (4.16) 3.98 (1.83) 1.84 6.06 13.19 3.73 25.14 (4.32) – – – 5.28 3.79 6.48 – – 5.68 100
(1.27)b (2.53)
(1.44) (3.28)
(0.98) (1.10)
(3.29) (1.83)
Not detected. Numbers shown in brackets are the compositions of b-hydroxylated fatty acids.
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sugar-containing compounds were visualized by spraying the plates with a mixture of glacial acetic acid–sulfuric acid–p-anisaldehyde (100:2:1, v/v) and heated at 150 °C for 5 min. For HPLC analysis, purified glycolipid samples were diluted 5-fold and filtered through a 0.2 lm membrane. HPLC separation was performed by loading a 2 lL sample into a 150 4.6 mm C18 silica column (Waters, USA), and run at a constant flow rate of 1 mL min 1 using methanol plus 0.05% formic acid as the mobile phase. Glycolipids were detected using an Evaporative Light Scattering Detector (ELSD) (Shimadzu, Japan). The column temperature was maintained at 45 °C and the ELSD was set at 40 °C. For quantification, hexane-purified glycolipids from U. maydis L8 strain were used as the standard and the concentration of glycolipids was the average of three repeats.
were analyzed by GC–MS, an Agilent 7890 GC coupled to a mass spectrometer (HP 5975C, Agilent). The system was equipped with a 27 m Rtx-5 MS capillary column (Agilent J&W, 0.25 mm i.d., 0.25 lm film thickness). Data collection and analyzing were performed as described previously (Hartig, 2008).
2.6. Fatty acid analysis
3.1. Characterization of crude glycerol
Fatty acid profiles of crude glycerol, glycolipids and cell biomass were determined after esterification to fatty acid methyl esters (FAME) using methanolic HCl (Sigma) as described previously (Chi et al., 2007; Ulberth and Henninger, 1992). The FAME samples
It has been reported previously that compositions of crude glycerol samples varied widely among oil feedstocks used and biodiesel production processes (Dasari, 2007; Pyle et al., 2008). In this study, crude glycerol derived from waste cooking oil showed a dark
2.7. Light microscopy Cells were observed under a Nikon Eclipse 80i microscope equipped with CFI Plan Apochromat objectives (Nikon, Japan) and images were acquired with a Nikon DS camera and Nikon ACT-2U software. 3. Results and discussion
Fig. 1. Production of glycolipids from glycerol and crude glycerol containing media. (A) Cell biomass yields. (B) The residual carbon sources. (C) Glycolipids yields. Small-scale fermentation was performed at 30 °C on a shaking platform (250 rpm) in triplicates in 250 ml conical flasks using MinCG media supplemented with 0.5 g L 1 NH4NO3 and 50 g L 1 of one the carbon sources indicated. (D) Cell morphology after a 12-day fermentation with glycerol and crude glycerol as the sole carbon source. The scale bar (10 lm) is shown.
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brown color with viscosity of 137 Centipoise at 25 °C. The crude glycerol sample used in this study has been adjusted to pH 6.0 and autoclaved in order to dissimilate the residual methanol (Athalye et al., 2009; Thompson and He, 2006). The treated crude glycerol contained 83.41% (w/w) glycerol, 11.59% water, 1.28% soap and 2.69% ash (Table 1). Methanol was undetectable after autoclaving (121 °C, 15 min) although it was present at 1.10% (w/w) in the original sample, similar to what has been reported previously (Chi et al., 2007; Pyle et al., 2008). Palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2) were the main fatty acids in the crude glycerol sample (Table 2). 3.2. Strains and culture conditions for bioconversion of crude glycerol to glycolipids U. maydis L8 strain was cultured in a series of synthetic media to assess glycolipid production. TLC analysis showed that it was possible to produce both UA and MEL when a minimal synthetic medium (MinG) containing 50 g L 1 of refined glycerol as the sole carbon source in the culture medium (data not shown). Surprisingly, cells grew significantly better (Fig. 1A) and consumed more glycerol (Fig. 1B) when crude glycerol was used as the sole carbon source. Importantly, total amount of glycolipids produced was comparable to that obtained with glucose as the sole carbon source (Fig. 1C). In addition, there was no discernible difference in cell morphology when cultured in the two glycerol sources (Fig. 1D). These results suggest that crude glycerol is a potential carbon source for production of glycolipids in U. maydis.
ranging between 24 °C and 35 °C in MinCG, containing 50 g L 1 crude glycerol as the sole carbon source and 0.5 g L 1 NH4NO3. The highest amount of glycolipids was produced at 30 °C after 6 days (Fig. 2A). The glycolipid yield varied greatly at various pH, with the highest observed at pH 4.0 (Fig. 2B). In addition, dissolved oxygen levels in the medium were investigated by culturing cells with an agitation speed between 100 and 300 rpm. Marginal improvement in the production of glycolipids was observed when media aeration was performed between 200 and 300 rpm (data not shown). Hence, production of glycolipids was done in MinCG medium (pH 4.0) at 30 °C with an agitation speed of 200 rpm in the subsequent experiments. 3.4. Optimization of culture medium To determine the optimal concentration of carbon source, MinCG medium was supplemented with various volume of crude
3.3. Effects of culture temperature and medium pH To optimize the conditions for bioconversion from crude glycerol to glycolipids, U. maydis was cultured at various temperature
Fig. 2. Effects of fermentation temperature and medium pH. MinCG medium with 50 g L 1 crude glycerol and 0.5 g L 1 NH4NO3 was used at various temperature (A) or medium pH (B). The fermentation cultures were sampled on the 5th day and quantified by HPLC. Results are average of three repeats.
Fig. 3. Effects of crude glycerol concentration, nitrogen sources and the concentration of ammonium citrate. (A) Effects of crude glycerol concentrations. MinCG media with different concentrations of crude glycerol and 0.5 g L 1 NH4NO3 were used for fermentation. The fermentation cultures were sampled on the 5th day and analyzed for dry cell weight and total glycolipid yield. (B) Effects of nitrogen sources. All nitrogen sources were kept at 10 mM. (C) Effects of ammonium citrate concentrations. MinCG media with 50 g L 1 crude glycerol and various concentrations of ammonium citrate were used for fermentation. The fermentation cultures were sampled on the 5th day. All results are average of triplicates.
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glycerol. Both glycolipid yield and cell biomass increased steadily from 10 to 50 g L 1 of crude glycerol. Further increase in the amount of crude glycerol affected the glycolipid yield negatively, even though cell biomass continued to increase in the whole range of carbon source feeding tested. The best result was obtained with 50 g L 1 crude glycerol, yielding 6.7 g L 1 total glycolipids (Fig. 3A). To determine the effects of nitrogen sources, eight inorganic and three organic nitrogen sources were tested, with each concentration kept as 10 mM. The results showed that organic nitrogen sources favored the accumulation of cell mass but had a negative effect on the accumulation of glycolipids. On the other hand, inorganic nitrogen sources, such as ammonium nitrate, ammonium diphosphate and ammonium citrate enhanced the yield of glycolipids. The addition of 10 mM ammonium citrate gave the best result, yielding 4.8 g L 1 total glycolipids in a 5-day culture (Fig. 3B). Thus, ammonium citrate was selected as the sole nitrogen source. To determine the optimal concentration of ammonium citrate required for fermentation, shaking flasks cultures were performed in MinCG media supplemented with 1–200 mM of ammonium citrate. During a 5-day fermentation, raising its concentration from 15 to 20 mM significantly increased the yield of glycolipids (Fig. 3C). As expected, the production of glycolipids was highly dependent on the level of nitrogen source. When ammonium citrate was added beyond 50 mM, glycolipid production was essentially terminated while cell mass accumulation was not negatively affected until the concentration reached over 100 mM. Collectively, the optimal fermentation medium for the production of glycolipids contains 50 g L 1 of glycerol and 1.6 g L 1 ammonium citrate with C:N ratio of 27. Subsequently, the effects of trace elements and oligosaccharides were assessed. Twenty basic amino acids and two enzyme cofactors (NAD+ and Vitamin B1+B6) were supplemented to the MinCG medium individually at 10 mg L 1 for amino acids and enzyme cofactors. Aspartic acid, alanine and B-group vitamins displayed
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substantial enhancing effects on the production of glycolipids (Fig. 4A). Interestingly, although cell mass accumulation was not affected, asparagine and lysine had an inhibiting effect on glycerol utilization (Fig. 4A and B). Supplementation of NAD+ did not improve glycolipid yield. Therefore, aspartic acid, alanine and B-group vitamins are recommended for the medium. In the last step of the optimization, precursors of glycolipids, i.e., mannose and erythritol, were supplemented to MinCG medium separately. Although supplementation in low concentration (0.1 g L 1) of either precursor inhibited the production of glycolipids, the yield of UA and MEL was found to increase steadily from 1 to 20 g L 1 (Fig. 4C). Notably, glycerol consumption was concomitantly increased (Fig. 4D). These results suggest that supplementation of mannose and erythritol significantly improved the production of glycolipids in U. maydis cells when crude glycerol was used as the main carbon source. However, neither is recommended for the medium due to the high cost of both precursors.
3.5. Fatty acid profiles in glycolipids Glycolipids produced using glucose as the sole carbon source produced fatty acid profile that was similar to that reported previously (Spoeckner et al., 1999) (Table 2). In general, fatty acid compositions of total glycolipids were comparable among the three different carbon sources used (glucose, pure glycerol and crude glycerol), except that palmitic acid (C16:0) was about 2.5-fold higher in media with pure or crude glycerol. Palmitomeic acid (C16:1) was absent when crude glycerol was used, and compared with glucose, its level was reduced substantially (26.6%) when pure glycerol was used (Table 2). In addition, hexanoic acid (C6:0) level increased more than 4-fold with crude glycerol as the sole carbon source (Table 2). These results suggest that the production of palmitic acid was enhanced when glycerol is used as a
Fig. 4. Effects of other supplements. MinCG media with 50 g L 1 crude glycerol, 20.3 mg L 1 ammonium citrate and various other supplements were used for fermentation. The fermentation cultures were sampled on the 5th day. Results are average of triplicates. Effects of all 20 amino acids, vitamin B1+B6 and NAD+ on glycolipids and biomass production are shown in (A) and residual glycerol in (B). All nutrients were supplemented at 0.1 mg L 1. The blank columns indicate the media that lead to relatively higher glycolipid production. Effects of sugar supplements are shown in (C) and (D).
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(Pyle et al., 2008) and Crypthecodinium cohnii (de Swaaf et al., 2003). To determine the effects of methanol residue present in crude glycerol on U. maydis cell growth and production of glycolipids, different concentrations of methanol (0%, 2%, 5% and 10%, v/v) were supplemented to the fermentation media containing glucose, glycerol or autoclaved crude glycerol as the sole carbon source. The addition of 2% methanol severely inhibited cell growth and glycolipid production with a corresponding drop in carbon source utilization. At 5%, cell growth and glycolipid production were abolished (Fig. 5). These results demonstrate that high concentration of methanol (>2%) is detrimental to cell growth and production of glycolipids, irrespective of the main carbon source used in U. maydis. However, as methanol in the crude glycerol used is present at just about 1.10% (0.05% in fermentation media with 50 g L 1 crude glycerol), any negative effects are likely to be small. 3.7. Fed-batch fermentation
Fig. 5. Effects of methanol in fermentation medium. (A) Cell biomass. (B) Residual carbon sources in the medium. (C) Glycolipids yield. Results are average of three independent repeats.
carbon source, and certain components in crude glycerol may strongly enhance the production of short-chain hexanoic acid. 3.6. Effects of residual methanol on production of glycolipids Methanol may negatively or positively affect cell growth and docosahexaenoic acid production in Schizochytrium limacinum
To confirm the effectiveness of the medium composition and culture conditions developed, fed-batch fermentation was performed. In the fermentation medium that had a C:N ratio of 27, mannose and erythritol were not added. The cells began to produce glycolipids after nitrogen was consumed, causing the C:N ratio to increase to 110. The glycolipid yield peaked on the 8th day of fed batch fermentation (175 h in total). At this time point, about 32.1 g L 1 total glycolipids were produced (Fig. 6). This is approximately double what was previously reported (16.3 g L 1), which used much more costly refined glycerol in combination with mannose supplementation (Morita et al., 2007). As UA is much more toxic than MEL to the fungal cells (Haskins and Thorn, 1951; Teichmann et al., 2010), we believe that the biggest hurdle in our process may lie in the toxicity of UA during fermentation. Therefore, it is reasonable to assume that a higher level of glycolipids could be produced when UA-deficient mutants, e.g. Dcyp1 (Hewald et al., 2006), is used as the biocatalyst. In recent years, good progress has been made in the understanding of glycolipid biosynthesis pathway in U. maydis (Hewald et al., 2006; Teichmann et al., 2007). It is expected that our glycolipid production process can be further improved by genetic engineering. For example, over-expression of rua1 promotes UA synthesis even in the presence of a rich nitrogen source (Teichmann et al., 2010). Similarly, over-expression of precursors for glycolipid biosynthetic genes, for example, mannose-6-phosphate isomerase, phosphomannomutase and mannose-1-phosphate guanylyltransferase, might mimic the
Fig. 6. Fed-batch fermentation. U. maydis L8 strain was cultured in a 2-L fermentor in MinCG medium containing 50 g L 1 crude glycerol (41.7 g L 1 glycerol), 20.3 mg L 1 ammonium citrate, 10 mg L 1 each of alanine, aspartic acid and B-group vitamins, for 175 h. The arrow indicates the time point (80 h) where fed-batch fermentation was initiated by medium with 460 g L 1 crude glycerol and 132.2 mg L 1 ammonium citrate.
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effect of precursor supplementation. Perhaps, the most interesting genes to explore are those that improve stress tolerance, such as those encoding proteins involved in the unfolded protein response (Travers et al., 2000) and cell wall integrity (Scrimale et al., 2009). 4. Conclusion In this study, we have shown that U. maydis is able to efficiently utilize and convert crude glycerol to glycolipid-type biosurfactant in a relatively simple synthetic medium using crude glycerol as the sole carbon source. Through further strain improvements, we expect ample room for further improvement in glycolipids yield. Also, engineered strains of U. maydis may be a good biocatalyst for conversion of crude glycerol to other value-added products. Acknowledgements This work is financially supported by the Singapore Economic Development Board and Temasek Trust. We thank Dr. Yunping Bu, Temasek Life Sciences Laboratory (Singapore), and Madam Lai Kwai Wong, Department of Chemistry, National University of Singapore (Singapore), for the GC and GC–MS analyzes. References Ashby, R., Nunez, A., Solaiman, D., Foglia, T., 2005. Sophorolipid biosynthesis from a biodiesel co-product stream. J. Amer. Oil. Chem. Soc. 82 (9), 625–630. Athalye, S.K., Garcia, R.A., Wen, Z., 2009. Use of biodiesel-derived crude glycerol for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare. J. Agric. Food Chem. 57 (7), 2739–2744. Brefort, T., Doehlemann, G., Mendoza-Mendoza, A., Reissmann, S., Djamei, A., Kahmann, R., 2009. Ustilago maydis as a Pathogen. Annu. Rev. Phytopathol. 47, 423–445. Cameotra, S.S., Makkar, R.S., 2004. Recent applications of biosurfactants as biological and immunological molecules. Curr. Opin. Microbiol. 7 (3), 262–266. Chi, Z., Pyle, D., Wen, Z., Frear, C., Chen, S., 2007. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochem. 42 (11), 1537–1545. da Silva, G.P., Mack, M., Contiero, J., 2009. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol. Adv. 27 (1), 30–39. Dasari, M. 2007. Crude glycerol potential described. In: Feedstuffs, vol. 15, p. 16. de Swaaf, M.E., Pronk, J.T., Sijtsma, L., 2003. Fed-batch cultivation of the docosahexaenoic-acid-producing marine alga Crypthecodinium cohnii on ethanol. Appl. Microbiol. Biotechnol. 61 (1), 40–43. Ethier, S., Woisard, K., Vaughan, D., Wen, Z., 2010. Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Bioresour. Technol. 102 (1), 88–93. Hartig, C., 2008. Rapid identification of fatty acid methyl esters using a multidimensional gas chromatography–mass spectrometry database. J. Chromatogr. A 1177 (1), 159–169. Haskins, R., Thorn, J., 1951. Biochemistry of the ustilaginales: VII. Antibiotic activity of ustilagic acid. Can. J. Bot. 29 (6), 585–592. Hewald, S., Josephs, K., Bölker, M., 2005. Genetic analysis of biosurfactant production in Ustilago maydis. Appl. Environ. Microbiol. 71 (6), 3033–3040. Hewald, S., Linne, U., Scherer, M., Marahiel, M.A., Kämper, J., Bölker, M., 2006. Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis. Appl. Environ. Microbiol. 72 (8), 5469–5477.
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