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Bioresource Technology 99 (2008) 3051–3056

Conversion of sewage sludge into lipids by Lipomyces starkeyifor biodiesel production

C. Angerbauer a, M. Siebenhofer b, M. Mittelbach c, G.M. Guebitz a,*

a Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austriab Department of Chemical Engineering and Environmental Technology, Graz University of Technology, Inffeldgasse 25/C/II, 8010 Graz, Austria

c Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, 8010 Graz, Austria

Received 7 February 2007; received in revised form 29 May 2007; accepted 1 June 2007Available online 24 August 2007

Abstract

The potential of accumulation of lipids by Lipomyces starkeyi when grown on sewage sludge was assessed. On a synthetic medium,accumulation of lipids strongly depended on the C/N ratio. The highest content of lipids was measured at a C/N-ratio of 150 with 68%lipids of the dry matter while at a C/N-ratio of 60 only 40% were accumulated. Within a pH range from 5.0 to 7.5 the highest lipid accu-mulation was found at pH 5.0 while the highest yield per litre was pH 6.5. Although sewage sludge had no inhibitory effects on growth oraccumulation on L. starkeyi when added to synthetic medium, there was no significant growth on untreated sewage sludge. However,pretreatment of sludge by alkaline or acid hydrolysis, thermal or ultrasonic treatment lead to accumulation of lipids by L. starkeyi withhighest values of 1 g L�1 obtained with ultrasound pre-treatment. Based on the content of free fatty acids and phosphorus, lipids accu-mulated from sewage sludge could serve as a substrate for the production of biodiesel.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Lipids; Fat accumulating yeast; Lipomyces starkeyi; Biodiesel; Sewage sludge

1. Introduction

In many countries world-wide utilisation of sewagesludge in agriculture is restricted due to the presence oftoxic substances. Thus, in many cases sewage sludge hasto be eliminated using expensive and environmentallyunfriendly ways like the disposal on landfills or incinera-tion. Apart from heavy metals like copper and zinc,organic hazardous substances (xeno-hormones) like linearalkyl-benzene-sulfonates, nonylphenol, hydrocarbons ofmineral oils and chlorine-paraffins which can reach thegroundwater table are a major concern in the utilisationof sewage sludge as a fertiliser in agriculture according toa recent study (Federal Environmental AgencyGermany(BMU), 2004). In addition, there was also a lack on hygie-nic requirements obviously required for a high quality fer-

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.06.045

* Corresponding author. Tel.: +43 316 873 8312; fax: +43 316 873 8815.E-mail address: guebitz@tugraz.at (G.M. Guebitz).

tiliser. Due to these new insights, for example, Germanlaws will enforce incineration of sewage sludge as from01.06.2005 with all its economical disadvantages (e.g. highenergy consumption) (TEXTE des Umweltbundesamtes).

Although anaerobic digestion is commonly used for thestabilisation of sewage sludge reducing the volume of result-ing end products, this process is rather slow and requiredlarge reactors. As an alternative to the anaerobic conversionof sewage sludge to mainly methane (CH4) and carbondioxide (CO2), the carbon moieties contained in sludgecould be converted into lipids by aerobic micro-organisms.These lipids can serve as a raw material for the productionof biodiesel. Biodiesel, a fuel produced by transesterifica-tion of fats with methanol has the potential to replace fossildiesel (Mittelbach and Remschmied, 2004). Recently, inseveral countries laws have been established enforcing a cer-tain content of biofules required in fuels. While sugar (poly-saccharide) based materials have a potential for bioethanolproduction, complex biomass sources such as sewage sludgeare usually used for biogas production.

3052 C. Angerbauer et al. / Bioresource Technology 99 (2008) 3051–3056

Lipids serve as storage materials in some lipid accumu-lating yeasts, e.g. Rhodotorula graminis. It is reported thatyeasts can store up to 70% of lipids in dry matter (Guerzoniet al., 1985). First data in lipid accumulation and the con-ditions for the fermentation have been reported alreadymore than 40 years ago (Mulder et al., 1962). Theseauthors observed that under nitrogen limiting conditionsand the presence of a carbon-source in excess organismsstarted to store lipids. Therefore a high carbon to nitrogen(C/N)-ratio, around 100, is a basic requirement for theaccumulation of lipids. From different yeasts studiedincluding Candida curvata, Trichosporon cutaneum, Rho-

dosporidium toruloides, and L. starkeyi the latter organismseemed to store the largest quantities of lipids (Holdsworthand Ratledge, 1988). Physical factors such as the concen-tration of some ions like Zn2+ and Mn2+ affected lipidaccumulation and to a lesser extent Fe3+, Ca2+, K+ andNHþ4 (Naganuma et al., 1985a,b).

Compared to other lipid accumulating yeasts like C.

curvata D, T. cutaneum and R. toruloides; L. starkeyi

showed only a minimal reutilisation of the stored lipids(Holdsworth et al., 1988). Natural habitat of L. starkeyiis soil and ensilage (Lodder, 1970), where the organismdegrades carbohydrates using extracellular carbohydrolas-es. Both, a-amylase and dextranase from L. starkeyi (Kanget al., 2004; Park et al., 2003; Lee et al., 2003a,b) and thebiodegradation of triazine herbicides (Nishimura et al.,2002) have been subject of recent studies.

Polyunsaturated fatty acids, e.g. arachidonic acid, ofyeast accumulated lipids are of interest as a dietary supple-ment, therefore an understanding of the biosynthetic path-ways involved in the production is desired (Ratledge, 2004;Ratledge and Wynn, 2002). ‘‘Malic enzyme’’ is a keyenzyme in the process of lipid accumulation (Ratledge,2002). Furthermore, Yarrowia lipolytica is studied in thisrelation (Mlickova et al., 2004a,b; Papanikolaou and Agge-lis, 2003; Papanikolaou et al., 2003) since it grows onindustrial fats and in the process accumulating lipids ofhigher quality.

In this study we describe for the first time that lipidsstored by L. starkeyi when grown on sewage sludge havea potential for the production of biodiesel.

2. Methods

2.1. Organism and culture conditions

Lipomyces starkeyi DSM 70295 a lipid accumulatingyeast was grown under aerobic conditions at 30�C andpH 5 with a reciprocal shaker at 120 rpm. The basal med-ium consisted of [gL�1]: KH2PO4 12.5; Na2HPO4 1;(NH4)2SO4 0.5; MgSO4 Æ 7H2O 2.5; CaCl2 Æ 2H2O 0.25;yeast extract 1.9; glucose Æ H2O 40 and 0.625 mL trace-element-solution. All chemicals were dissolved in 400 mLdistilled water in Erlenmeyer flasks. The trace-element-solution contained [gL�1]: FeSO4 Æ 7H2O 16; MnSO4 Æ H2O4; Al2(SO4)3 Æ 18H2O 5.52; CoCl2 Æ 6H2O 2.92; ZnSO4 Æ

7H2O 0.8; Na2MoO4 Æ 2H2O 0.8; CuCl2 Æ 2H2O 0.4;H3BO3 0.2 and KI 1.6 in 5 N hydrochloric acid. All chem-icals were of analytical grade and obtained from Sigma–Aldrich or Merck.

2.2. Sewage sludge

‘‘Raw sewage sludge’’ before anaerobic fermentationand stabilisation was obtained from the communal waste-water treatment plant Graz-Gossendorf, Austria. It con-sisted of one third surplus activated sludge and twothirds of primary sludge. The sludge was homogenised withan Ultraturax-apparatus.

2.3. Sludge medium

The ammonium nitrogen content (NHþ4 –N) of raw sew-age sludge was determined and glucose as carbon-sourcewas added to reach the desired C/N-ratio. To 50 g raw sew-age sludge the required amount of glucose and distilledwater were added to reach a final volume of 400 mL. UsingH3PO4 the pH-value was adjusted to 5.0. This resultingsludge medium was sterilised, inoculated with 5 ml activelygrowing L. starkeyi cultivated as described above and incu-bated for 240 h.

2.4. Dissolved organic carbon (DOC) fraction of sludge

One hundred gram sewage sludge was centrifuged at4066 g decanted the supernatant, washed the residue twicewith 50 mL distilled water and united the supernatants, dis-tilled water was added to a final volume of 200 mL.

2.5. Digests

All heating processes were carried out in a small auto-clave from the CertoClav Sterilizer GmbH, Traun, Austria.Suggestions for the alkaline hydrolysis were from Rusta-mov (2002) and for the acid digest from Choi and Mathews(1996). For digests using ultrasound a BANDELIN Sono-plus HD 70 from BANDELIN electronic, Berlin, Germanywith a maximum power of 70 W was used.

2.5.1. Alkaline digest

To 100 g of raw sewage sludge, 5 g Na2CO3 and 2.5 gKOH were added and heated for 40 min at 125�C.After hydrolysis, the sludge was auburn coloured and thealkaline medium was adjusted with phosphoric acid topH 5, distilled water was added to a final volume of400 mL.

2.5.2. Acid digest

The acid hydrolysis was done similarly using 100 g rawsewage sludge and 18 g phosphoric acid (85%). After heat-ing for 40 min at 125 �C the treated sludge was adjusted topH 5 with sodium hydroxide solution and distilled waterwas added to give a final volume of 400 mL.

Table 1Accumulation of lipids by Lipomyces starkeyi at different C/N-ratios

C/N-ratio 150 60 30 20 15

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2.5.3. Thermal treatment

The raw sewage sludge was heated at 125 �C for 1, 1.5and 2 h. After the procedure, similar to the hydrolysis, apH 5 was adjusted and distilled water to a final volumeof 400 mL was added.

2.5.4. Ultrasonic treatment

The sludge was treated with 40% of the maximum powerof the ultrasonic generator and with ultrasonic pulses at 7 sintervals (3 s break). One hundred gram of raw sewagesludge in plastic bottles were treated for 2, 2.5 and 3 h with-out cooling. Distilled water was added and pH wasadjusted to 5 as described above.

2.6. NHþ4 –N test

The ammonium nitrogen content was determined withan ammonium cell test from Merck (Spectroquant) accord-ing to the instructions manual. The measuring range fromthe tests used was from 0.20 to 8.00 mg L�1 and 4.0 to80.0 mg L�1 NHþ4 –N.

2.7. Lipids determination

The determination of the lipid content was carried outaccording to Moreton (1989). The sample in the determi-nation was fermentation broth. A 1 mL sample taken fromthe incubation mixture was hydrolysed with perchloricacid to release intracellular lipids, followed by a saponifica-tion to release glycerol from lipids (triacylglycerols). Theglycerol content was determined using an enzymatic esti-mation glycerol assay kit from Boehringer Mannheim.Out of the glycerol content the amount of lipids was calcu-lated assuming a standard triacylglycerol containing oleicacid.

2.8. Lipid extraction

The extraction was done according to Thakur et al.(1988). From 400 mL fermentation broth the cells wereharvested for the estimation.

2.9. Dry matter and DOC

The washed cells were dried to constant weight in vialsby treatment in an oven at 105 �C. To estimate the carboncontent in media with sewage sludge used by L. starkeyi theDOC was measured with the Total Organic Carbon Ana-lyzer, TOC-VCPH/CPN from the Shimadzu Corporation(Kyoto, Japan). Together with the NHþ4 –N the C/N-ratiowas calculated.

Accumulatedlipids in drymatter (%)

68 ± 1 40 ± 1 37 ± 1 34 ± 1 34 ± 1

Yield on lipidsin the broth(g L�1)

6.4 ± 0.2 5.9 ± 0.2 4.9 ± 0.2 4.6 ± 0.2 4.1 ± 0.2

2.10. RNA determination

The determination was done according to Liwarska-Bizukoje and Ledakowicz (2003).

2.11. Fatty acid composition

Fatty acid composition was assayed by gas chromato-graphy on an HP 5890 GC apparatus equipped with aflame ionization detector on a J + W DB-5 HT(15.0 m · 320 lm · 0.19 lm) capillary column, accordingto a standard procedure (American Oil Chemists’ Society,1989).

2.12. Determination of sulphur and phosphorus

Sulphur content was determinated with a Mitsubishi TS-100 trace sulphur and nitrogen analyzer according to ENISO 20846, an ultraviolet fluorescence method.

Phosphorus or the equivalent phosphatide content wasdetermined by burning the sample in the presence of zincoxide, followed by the spectrophotometric measurementof phosphorus as a blue phospomolybdic acid complexaccording to A.O.C.S. official method Ca 12–55.

3. Results and discussion

3.1. Lipid storage by L. starkeyi cultivated on basal medium

In a first stage, the influence of the C/N-ratio on accu-mulation of lipids by L. starkeyi was determined compar-ing C/N-ratios of 150, 60, 30, 20 and 15. The results arein shown in Table 1.

The highest content of lipids was measured at a C/N-ration of 150, with 68% lipids of the dry matter. The drymatter in the fermentation broth was 9.5 g L�1, giving alipid-concentration of 6.4 g L�1. In the cultivation testswith a C/N-ratio of 60 only 40% of lipids were accumu-lated, however the dry matter was much higher(14.6 g L�1). From this it follows that the concentrationof lipids was 5.9 g L�1. At the lower C/N-ratios the maxi-mum percentage and lipid-concentration was decreasing.Although the productivity (lipid-concentration) at C/N-ratio 150 and 60 were similar (6.4 g L�1 and 5.9 g L�1),the lipid-concentration of 6.4 g L�1 in the fermentationwith the C/N-ratio 150 was reached after 190 h while inthe cultivation at a C/N-ratio 60 took 220 h. Summarisingthe results it can be concluded that the optimum for accu-mulation of lipids by L. starkeyi was a C/N-ration between60 and 150. Thus, in a subsequent experiment a C/N-ratioof 100 was used which lead to a lipid content of 56.3%equivalent to a concentration of 7.5 g L�1.

Table 2Accumulation of lipids by Lipomyces starkeyi at different pH-values

pH-values 5.0 5.5 6.0 6.5 7.0

Accumulatedlipids in drymatter (%)

56 ± 1 51 ± 1 50 ± 1 51 ± 1 7 ± 1

Yields on lipidsin the broth(gL�1)

7.5 ± 0.2 7.1 ± 0.2 5.6 ± 0.2 7.7 ± 0.2 1.1 ± 0.2

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In a second step the influence of the pH-value on lipidaccumulation was studied using a C/N-ratio of 100. Rawsewage sludge from a treatment plant varies quite stronglywith regard to the pH-value (from 5 to 7.5) and thus a pHrange from 5.0 to 7.5 was chosen for these experiments.The results of these experiments are shown in Table 2.The highest lipid content was found at a pH 5.0 whilethe yield per litre was highest at a pH 6.5. Thus, therewas not a big influence on the accumulation of lipids inthe pH-range 5.5 to 6.5. However, at pH 7.0 the accumula-tion of lipids decreased dramatically.

In the literature different pH-values are reported foroptimal lipid accumulation which seemed to depend onthe carbon sources used. An optimum pH of 4.9 for lipidaccumulation was found when glucose was used as a car-bon source (Naganuma et al., 1985a), while pH 4.0 wasthe optimum when ethanol was used as carbon-source(Yamauchi et al., 1983). Holdsworth and Ratledge (1988)worked with glucose at pH 5.5, because they comparedL. starkeyi with other organisms. However the pH-rangefrom 5.5 to 7.0 is not reported.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

0 1 3 4 5 7 8Time [d]

RN

A [m

g/L]

62

Fig. 1. RNA-concentration over the time in basal medium and sludge-medium. (j) RNA-concentration in basal medium; (}) RNA-concentra-tion in sludge-medium.

3.2. Lipid storage by L. starkeyi cultivated on sewage sludge

In a first stage, the sludge medium was adjusted toC/N-ratios of 100 and 60 by addition of glucose. For theC/N-ratios of 100 and 60, 35.6% and 32.2% lipids wereaccumulated, respectively. For the media with C/N-ratiosof 100 and 60, a part of dry matter due to fibres presentin the sludge of 50.8% and 57.2%, respectively, can beassumed to be constant. Based thereon a lipid content of72.3% and 75.2%, respectively, was calculated for the C/N-ratios of 100 and 60. The yields per litre cultivationbroth gave more comparable results of 6.7 g L�1 for theC/N-ratio 100 and 5.2 g L�1 for the C/N-ratio 60. Theseresults compared with results from basal medium indicatethat there were no inhibitory effects on growth or accumu-lation on L. starkeyi by sewage sludge present in the culti-vation medium.

In a next step the utilisation of carbon moieties presentin sludge for lipid accumulation was studied. The solublefraction of sludge was compared to the basal glucose med-ium following dry matter and DOC during cultivation. Inthe basal medium 95.6% of the DOC was used by L. star-

keyi, while 66.1% of the sludge-DOC was converted.In an experiment using raw sludge the dry matter

remained nearly constant throughout the cultivation per-

iod (11.6 g L�1), while the DOC remained constant ataround 730 mg L�1. The NHþ4 –N varied from 58 to135 mg L�1, indicating a release of NHþ4 from organicnitrogen compounds (proteins) present in sludge. Due tothis fact the C/N-ratio varied accordingly between 6 and12, which is far from being optimal for the lipid accumula-tion. Additionally no significant growth was detected basedon dry matter determination which was verified by follow-ing the RNA concentration (Fig. 1). The initial RNA con-centration of around 4 mgL�1 is due to micro-organismsalready present in the sludge.

The insignificant degradation of fibres (polysaccarides)was somehow unexpected since L. starkeyi is known forthe production of extracellular carbohydrolases (Kanget al., 2004; Park et al., 2003; Lee et al., 2003a,b). In orderto increase the amount of usable DOC and C/N-ratio ofthe medium and to stabilise the NHþ4 -concentration differ-ent pre-treatment methods of sludge were compared.

3.3. Digests for the sewage sludge

Common raw sewage sludge consists of proteins, carbo-hydrates, lipids and fibres. The fibre fraction partially con-sists of bacterial extra-cellular polysaccharides (Brown andLester, 1980) and a hydrolysis of these fibres to sugarscould increase the amount of carbon which can be usedby L. starkeyi. L. starkeyi can only metabolise glycerolbut no lipids. Thus, the aim of the digest was also thehydrolysis of lipids yielding glycerol and free fatty acid.However, the pre-treatment should not lead to degradationof proteins to NHþ4 affecting the C/N-ratio in a negativeway.

All four digesting methods, alkaline and acid hydrolysis,thermal and ultrasonic treatment, gave a higher amount onaccumulated lipids than in fermentations with untreatedraw sewage sludge (Fig. 2). The best results were obtainedwith the sewage sludge pre-treated with ultrasound. Ultra-sound is also a good method for industry. The advantagesare ultrasound is insusceptible to impurities, e.g. littlestones, the experience in lab-use, pilot-plants and industrial

0

0.2

0.4

0.6

0.8

1

1.2

1.4

raw sewagesludge

ultrasonictreatment

thermaltreatment

acid hydrolysis alkalinehydrolysis

different treatments of raw sewage sludge

lipid

s [g

L-1]

Fig. 2. Accumulation of lipids by Lipomyces starkeyi from pre-treatedsewage sludge.

C. Angerbauer et al. / Bioresource Technology 99 (2008) 3051–3056 3055

pilot-plants, only in industrial long-term usage few data areavailable (Mueller et al., 2001).

3.4. Properties of the accumulated lipids

The extracted lipids were solid at room temperature andhad a slightly yellow colour.

In Table 3 the fatty acid composition, the content of freefatty acids, phosphorus and sulphur are listed. For the pro-duction of biodiesel the preparation of fatty acid methylesters has to be carried out in two steps due to the high con-tent of free fatty acids. After pre-esterification with metha-nol transesterification can be carried out under alkalinecatalysis. The fatty acid composition is quite similar topalm oil with a very high content of palmitic acid. Com-pared to palm oil also the content of stearic acid is higher.Because of the total content of saturated fatty acids ofabout 70% excellent burning characteristics like very highcetane number of the fatty acid methyl esters can be esti-mated (Mittelbach and Remschmied, 2004). However, thehigh content of saturated fatty acid acids also leads to poorlow temperature behaviour of the resulting biodiesel, how-ever, blending with mineral diesel could overcome thisproblem. The sulphur and phosphorus content is verylow, so the limits for biodiesel according to EN 14214 with10 mg/kg each can be met easily.

Table 3Chemical composition of the lipids accumulated in Lipomyces starkeyi

grown on sewage sludge. Distribution of fatty acids, content of sulphur,phosphorus and free fatty acids

Distribution of fatty acids Distribution of fatty acids

Myristic acid C14:0 0.90% Linolenic acid C18:3 0.12%Palmitic acid C16:0 55.93% Arachidic acid C20:0 0.48%Palmitoleic acid C16:1 1.85% Gadoleic acid C20:1 0.18%Stearic acid C18:0 13.8% Behenic acid C22:0 0.48%Oleic acid C18:1 25.89% Not identified 0.37%Linoleic acid C18:2 <0.1%

Free fatty acids Phosphorus content Sulphur content12.18% 7 mg/kg 12 mg/kg

Summarising the results lipids accumulated by L. star-

keyi are suitable for the production of biodiesel.

4. Conclusion

The C/N ration is a crucial parameter for the accumula-tion of lipids by L. starkeyi. While high amounts of lipids(around 70% of dry matter) can be accumulated by thisorganism in a synthetic medium, there was no significantgrowth on raw sewage sludge. However, pre-treatment ofsludge especially by ultrasound can make this substrateaccessible to L. starkeyi. Based on the content on free fattyacids and phosphorus, lipids accumulated from sewagesludge could serve as a substrate for the production of bio-diesel. Future investigations should focus on the optimiza-tion of the pre-treatment method of sludge to approachthose lipid accumulation yields obtained in a syntheticmedium.

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