Bioresource Technology 101 (2010) 2623–2628

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Bioresource Technology

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Anaerobic digested dairy manure as a nutrient supplement for cultivationof oil-rich green microalgae Chlorella sp.

Liang Wang, Yecong Li, Paul Chen, Min Min, Yifeng Chen, Jun Zhu, Roger R. Ruan *

Department of Bioproducts and Biosystems Engineering, University of Minnesota, 1390 Eckles Avenue, St. Paul, MN 55108, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 July 2009Received in revised form 19 October 2009Accepted 21 October 2009Available online 24 November 2009

Keywords:Anaerobic digested dairy manureAlgaeNutrients removalLipid contentChlorella

0960-8524/$ - see front matter Published by Elsevierdoi:10.1016/j.biortech.2009.10.062

* Corresponding author. Tel.: +1 612 625 1710; faxE-mail addresses: wangx739@umn.edu (L. Wang

Ruan).

The present study was to investigate the effectiveness of using digested dairy manure as a nutrientsupplement for cultivation of oil-rich green microalgae Chlorella sp. Different dilution multiples of10, 15, 20, and 25 were applied to the digested manure and algal growth was compared in regardto growth rate, nutrient removal efficiency, and final algal fatty acids content and composition. Slowergrowth rates were observed with less diluted manure samples with higher turbidities in the initial cul-tivation days. A reverse linear relationship (R2 = 0.982) was found between the average specific growthrate of the beginning 7 days and the initial turbidities. Algae removed ammonia, total nitrogen, totalphosphorus, and COD by 100%, 75.7–82.5%, 62.5–74.7%, and 27.4–38.4%, respectively, in differentlydiluted dairy manure. COD in digested dairy manure, beside CO2, proved to be another carbon sourcefor mixotrophic Chlorella. Fatty acid profiles derived from triacylglyceride (TAG), phospholipid and freefatty acids showed that octadecadienoic acid (C18:2) and hexadecanoic acid (C16:0) were the twomost abundant fatty acids in the algae. The total fatty acid content of the dry weight increased from9.00% to 13.7% along with the increasing dilution multiples. Based on the results from this study, aprocess combining anaerobic digestion and algae cultivation can be proposed as an effective way toconvert high strength dairy manure into profitable byproducts as well as to reduce contaminationsto environment.

Published by Elsevier Ltd.

1. Introduction

Land disposal methods have traditionally been used to manageanimal manures (de Godos et al., 2009). However, the potential ofexporting tremendous nitrogen and phosphorus into water bodiesmakes it not sustainable. Conventional biological treatments suchas activated sludge sequential-batch processes (Zhang et al.,2006; Wang et al., 2009) provide potential approaches for a cor-rect waste management, but the associated high energy inputs re-tard their widespread implementation in rural areas (de Godoset al., 2009). Algae based wastewater treatment processes havebeen gaining tremendous attentions since 1960s (Golueke andOswald, 1962; Lau et al., 1995; Tam and Wong, 2000) becausethey could potentially offer many advantages over the commonactivated sludge process that requires high energy input for aer-ation and high cost for subsequent sludge processing. The successof an algae based system relies on the ability of the algal cells toassimilate organic carbon (heterotrophic growth) (Burrell et al.,1984) as well as inorganic nutrients such as nitrogen and phos-

Ltd.

: +1 612 624 3005.), ruanx001@umn.edu (R.R.

phorus (Lau et al., 1995) from the wastewater for their growthwithout an aerobic environment being created and maintained.The obtained algal biomass contain high ratios of hydrocarbonsin the form of extractable lipids such as free fatty acids, triacyl-glyceride (TAG), phospholipid and glycolipid, and are ideal feed-stocks for biodiesel production. Manure grown algae can also beused as soil conditioner (Wilkie and Mulbry, 2002), animal feedsupplement (Barlow et al., 1975) and fermentation substrate (Ue-noa et al., 1998). With all the above-mentioned merits, co-locat-ing algae production with animal waste treatment could bemade environmentally effective as well as economically viablein a not distant future.

Recovery of nutrients from swine manure wastewaters by mic-roalgae has been extensively investigated by a number of research-ers and a lot of experience has been gained in pilot-scaleoperations (Barlow et al., 1975; Fallowfield and Garrett, 1985; deGodos et al., 2009) with algal productivities varying from 7 to33 g/m2/day and simultaneous reductions of COD, nitrogen andphosphorus. The experience gained for dairy manure is not asmuch as that for swine manure but still can be found in several lit-eratures (Lincoln et al., 1996; Woertz, 2007; Mulbry et al., 2008).From an outdoor algal turf scrubber (ATS) raceway system, Mulbryet al. (2008) concluded that projected annual operation costs were

2624 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628

well below the costs cited for upgrading existing water treatmentplants in sensitive watersheds, indicating the algal technology fordairy manure treatment very appealing from the environmentalstandpoint.

The above studies mainly focused on the algae productivity andnutrient recovery efficiency, and few have discussed the algalgrowth type (heterotrophic or autotrophic) in a high organicstrength wastewater like diary manure, nor did concern aboutthe lipid content and fatty acid profile of the dairy manure grownalgae. The objective of this study was to test the effectiveness ofcultivating a wildly isolated green alga, Chlorella, sp. in diluted di-gested diary manures sampled from a local dairy farm using batch-wise experiments. Carbon usage related to the growth type, therelationship between growth rates and turbidities, as well as the fi-nal algal fatty acid content and composition will be discussed indepth.

2. Methods

2.1. Algae strain and culture condition

Algae strain was a wild-type Chlorella sp. isolated from localfreshwater. It was preserved in Tris–Acetate–Phosphorus (TAP)(Harris, 1989) media containing the following chemicals: NH4Cl(400 mg/L), MgSO4�7H2O (100 mg/L), CaCl2�2H2O (50 mg/L),K2HPO4 (108 mg/L), KH2PO4 (56 mg/L), Tris (hydroxymethyl) ami-nomethane (2420 mg/L), glacial acetic acid (1 mL/L), and trace ele-ments solution (1 mL/L), consisting of Na2EDTA 50 (g/L),ZnSO4�7H2O (22 g/L), CaCl2�2H2O (0.05 g/L), H3BO3 (11.4 g/L),MnCl2�4H2O (5.06 g/L), FeSO4�7H2O (4.99 g/L), CoCl2�6H2O (1.61 g/L), CuSO4�5H2O (1.57 g/L), (NH4)6Mo7O24�4H2O (1.10 g/L), KOH(16 g/L). Algae were inoculated at 10% (v/v) in 250 mL Erlenmeyerflasks containing 100 mL liquid medium. The culture flasks wereincubated under stationary condition at 25 ± 2 �C and200 lmol�m�2s�1 continuous cool-white fluorescent light illumi-nation on a shaker with 150 rpm rotation speed. All the experi-ments were carried out in duplicate and average values werereported.

2.2. Characteristics of dairy manure

Undigested and digested dairy manures were collected fromHaubenschild Farm, Princeton, Minnesota, where a plug-flowanaerobic digester has been in operation since 1999. The charac-teristics of the dairy manure are shown in Table 1. Ammonium(NH4

+–N), total nitrogen (TN), total phosphorus (TP) and chemicaloxygen demand (COD) were determined for both undigested anddigested dairy manures following the Hach DR 5000 Spectropho-tometer Manual (Hach, 2008). Total solids (TS) and total volatilesolids (TVS) were performed following the standard methods(APHA, 2005). The possible volatile fatty acids or organic sub-stances in undigested and digested diary manures were deter-mined using gas chromatography–mass spectrometry (GC–MS)(Agilent 7890–5975C, USA). The GC was equipped with a flame

Table 1Characteristics of the manure before and after digestion.

Parameters Before digestion After digestion

NH3–N (mg N/L) 1782 2232TKN (mg N/L) 3305 3456TP (mg PO4/L) 266 249.7COD (mg/L) 38230 23760TS 9.70% 6.60%TVS 8.00% 5.10%

ionization detector and a DB-5-MS capillary column. The oventemperature was 80 �C, held for 5 min, raised to 290 �C at a rateof 4 �C/min, and held at 290 �C for 5 min, while the injector anddetector temperature were set at 250 �C and 230 �C, respectively.The carrier gas (helium) was controlled at 1.2 mL/min. Chromato-graphic data were recorded and integrated using Agilent data anal-ysis software. The compounds were identified in the NIST MassSpectral Database and quantified by comparing the peak area with1000 mg/L acetic acid as an external standard (Sigma–Aldrich,MO).

The digested dairy manure was used as the feedstock for grow-ing algae. Four dilution multiples (10, 15, 20, 25) were applied tothe digested manure before inoculation. Diluted manure liquidsamples were filtered through glass microfiber filters (934-AH,Whatman, USA) to remove large particles and indigenous bacteria.Turbidities were determined before and after algal cultivation foreach sample using a nephelometer (Model 2100N Turbidimeter,Hach, USA). Results are reported in Nephelometric Ratio TurbidityUnits (NTRU’s).

2.3. Determination of algal growth

Optical density (OD) of the inoculated manure at 680 nm wasmeasured daily as the algal density indicator using a spectropho-tometer (Genesys 5, Spectronic Instruments, UK). A linear relation-ship between OD and dry weight (DW, g/L) was determined for thisstrain:

Dry weightðg=LÞ ¼ 0:3477OD680 � 0:0068; R2 ¼ 0:997

The growth rate (GR, d�1) was calculated by fitting the OD forthe first 5 days of culture to an exponential function:

GR ¼ ðln ODt � ln OD0Þ=t

where OD0 is the optical density at initial day, ODt is the opticaldensity for day t and t is the time between the two measurements.

2.4. Fatty acid analysis

Algae cells were harvested by centrifugation and then dried by afreeze dryer (Savant Instruments Inc., USA) before being analyzedfor lipid content. Fatty acid content and composition analysiswas performed by following two consecutive steps including prep-aration of fatty acid methyl ester (FAME) and GC–MS analysis.FAME was prepared by a one-step extraction–transesterificationmethod as described by (Indarti et al., 2005), after suitable modifi-cation. Dried algae samples (about 100 mg) were weighed intoclean, 25 mL screw-top glass bottles, to which 10 mL mixture ofmethanol, concentrated sulfuric acid, and chloroform(4.25:0.75:5) was added. Transesterification was carried out in90 �C water bath (Cole-Parmer, USA) for 90 min. Upon completionof the reaction, the chloroform layer containing FAME was care-fully collected and subject to GC–MS analysis. The GC wasequipped with a flame ionization detector and a DB-5-MS capillarycolumn. The oven temperature was 80 �C, held for 5 min, raised to290 �C at a rate of 4 �C/min, and held at 290 �C for 5 min, while theinjector and detector temperature were set at 250 �C and 230 �C,respectively. The carrier gas (helium) was controlled at 1.2 mL/min. Chromatographic data were recorded and integrated usingAgilent data analysis software. The compounds were identified inthe NIST Mass Spectral Database and quantified by comparingthe peak area with that of the external standard (C18:2) (Sigma–Aldrich, MO).

L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628 2625

2.5. Physico-chemical analysis

Liquid samples for nutrient consumption analysis were col-lected on the first day and the last day of the experiment. Thewhole experiment ended at the 21st day when all the 4 growthcurves started to level off. Samples were centrifuged at 5000 rpmfor 15 min and supernatants were collected for analyses of ammo-nium (NH4

+–N), total nitrogen (TN), total phosphorus (TP) andchemical oxygen demand (COD). Measurements of NH4

+, TN, TPand COD were performed following the Hach DR 5000 Spectropho-tometer Manual (Hach, 2008). Removal rates for those parameterswere calculated by dividing the difference between the first dayand final day concentrations by the first day concentration, andthen multiplied by 100. The carbon, hydrogen and nitrogen contentof dried algae samples were measured using an elementary ana-lyzer (CE-440, Exeter Analytical Inc., USA).

3. Results and discussion

3.1. Dairy manure characteristics before and after digestion

The Haubenschild Farms digester is a covered 350,000-gallonconcrete tank installed in the ground, the temperature of whichwas controlled 95–105 �F by suspended heating pipes. The digesterwas designed with a maximum treating capacity of 1000 cows anda retention time of 20 days. Table 1 shows the characteristics of themanure before and after digestion. The total nitrogen and totalphosphorus did not change much since anaerobic digesters areknown to reduce negligible amounts of nutrients (Lusk, 1998),while ammonium increased obviously, due to the anaerobic bio-conversion of proteins contained in manure into amino acids andthen to ammonia (Cheng and Liu, 2002). The organic substancesin the undigested diary manure detected by GC–MS were aceticacid and propionic acid, accounting for 31.3% and 27.5% of the totalundigested COD, respectively. Isopropoxycarbamic acid ethyl esterwas the only organic substance detected in the digested sample,which was about 18.8% of the total digested COD. Anaerobic diges-tion reduced COD, TS and TVS by 30–40%, which was comparableto the typical performance level of anaerobic digesters treatingdairy manure (Martin, 2003; Demirer and Chen, 2004). Therefore,critical nutrients were still retained in the dairy manure afteranaerobic digestion, making it a good supplement for growingalgae.

3.2. Algal growth and nutrient removal efficiencies

Algal growth as indicated by optical density measured at680 nm in 10�, 15�, 20� and 25� diluted digested dairy manure

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16 18 20 22Days of Cultivation

Opt

ical

Den

sity

at 6

80 n

m

10*dilution15*dilution20*dilution25*dilution

Fig. 1. Growth curves of Chlorella sp. in digested dairy manure with differentdilution multiples.

were shown in Fig. 1. This wild-isolated Chlorella sp. could survivein all of the diluted dairy manure samples and no obvious lagphases were observed. Comparing between the serial dilutions, itis noticed that algae grew faster in 20� and 25� diluted samplesin the first 7 days. After the 7th day, the growth curve of algae in25� diluted sample started to level off due to earlier exhaustionof less amount of nutrients. The algae in 10� diluted samplespicked up their growth rate in the latter part of the cultivation per-iod. The average specific growth rates under the four conditions inthe first 7 days were 0.282, 0.350, 0.407 and 0.409 d�1, respec-tively. These growth rates were comparable to that grown in thecommercial Bristol medium (0.3644 d�1) under axenic conditions(Lau et al., 1994).

Algal growth is directly affected by the availability of nutrients(El-Nabarawy and Welter, 1984), light (Sorokin and Krauss, 1958),the stability of pH (Azov and Shelef, 1987) and temperature (Talbotand de la Noiie, 1993) and the initial inoculation density (Lau et al.,1995). It is expected that the higher the initial inoculum density,the better the growth and the higher the nutrient removal effi-ciency (Lau et al., 1995) while problems such as self-shading, accu-mulation of auto-inhibitors (Darley, 1982) are not present. Lauet al. (1995) studied the effect of initial algal inoculum sizes onthe nutrient removal efficiency for the primary settled sewageand found that the superconcentrated culture of Chlorella vulgaris(with an initial inoculum of 1 � 107 cells m1�1) did not exhibitany self-shading limitation of growth and nutrient removal. Theinoculation size of present study was about 1.2 � 107 cells m1�1,which was considered to be an optimal level for most efficientnutrient reduction (Lau et al., 1995) compared with inoculationsizes of 106 (medium) and 105 (low) cells m1�1.

The relationship between initial 7-day average specific growthrates and original sample turbidities was plotted in Fig. 2. The ini-tial slower growth rate in less diluted manure sample correlatedwell with their higher turbidities (R2 = 0.982), indicating that tur-bidity is an important parameter determining the suitability of awastewater for algae growth. Turbidity, caused by suspended mat-ter or impurities that interfere with the clarity of the water, ex-presses the extent of the light being scattered and absorbedrather than transmitted in straight lines through a water sample.The impurities responsible for turbidity may include clay, silt, fi-nely divided inorganic and organic matter, soluble colored organiccompounds, and plankton and other microscopic organisms (USEP-A, 2009). Since the manure was diluted and filtered before testing,the high turbidities of 10� and 15� diluted samples should be lar-gely attributed to some finely divided inorganic and organic matterinstead of particles and microorganisms. The 20� and 25� dilutedmanures with turbidities of 95 and 75 NTRU, respectively, did notshow much difference in the growth rate, suggesting that the light

y = -789.32x + 393.83R2=0.982

020406080

100120140160180200

0.25 0.3 0.35 0.4

Specific growth rate (1/d)

Turb

idity

(NTR

U)

Fig. 2. Correlation between sample turbidities and average algal specific growthrates in the first 7 cultivation days.

Table 2Turbidities and removal rates for different samples.

Parameters 10*dilution 15*dilution 20*dilution 25*dilution

Turbidity before treatment (NRTU) 180 120 95 75Turbidity after treatment (NRTU) 57.5 45 31 24.5Removal rate 68.0% 62.5% 67.4% 67.3%

2626 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628

penetration would be affected only when the turbidity went be-yond a certain level. As time moved on, algae removed those par-ticles accounted for turbidity (Table 2), which was anotherreason, why the growth in less diluted samples caught up in thelatter part of the cultivation, beside the aspect that continued algalgrowth could also be supported by the higher nutrient concentra-tions in less diluted samples.

3.3. Nutrient removal efficiencies

For all eukaryotic algae, the only forms of inorganic nitrogenthat are directly assimilable are nitrate (NO�3 ), nitrite (NO�2 ), andammonium (NHþ4 ) (Barsanti and Gualtieri, 2006).

Most of the nitrogen in the digested dairy manure was in theform of ammonium nitrogen, and therefore was readily availableto algae. Results from this experiment showed that ammoniumwas completely removed within the 21-day growth period for allof the four diluted manure samples (Fig. 3a) regardless how highthe initial concentration (81–178 mg/L) was, which is in agreementwith results reported by De la Noüe and Bassères (1989). Thereforethe possible toxicity or inhibition of high levels of NH4 to the mic-

0

20

40

60

80

100

120

140

160

180

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220

10*dilution 15*dilution 20*dilution 25*dilution

10*dilution 15*dilution 20*dilution 25*dilution

NH

3-N

con

cent

ratio

ns (m

gN/L

)

NH3-N_initial

NH3-N_final

100%removal

100%removal

100%removal

100%removal

0

50

100

150

200

250

300

TKN

con

cent

ratio

ns (m

g N

/L) TKN_initial

TKN_final

82.5%removal

75.7%removal

78.3%removal

77.6%removal

(a)

(c)

Fig. 3. (a) Initial and final ammonium concentrations and removal rates; (b) initial and firemoval rates and (d) initial and final COD concentrations and removal rates.

roalgae (Abeliovich and Azov, 1976) was not present for this strainin tested samples, proving the strain to be ammoniacal-N tolerant.

Total nitrogen was greatly reduced by 75.7–82.5% but not com-pletely removed (Fig. 3b), indicating there were still some organiccompounds that could not be converted to ammonium nitrogenand assimilated by algae. A significant reduction (62.5–74.7%) oftotal phosphorus was also found for all of the four samples(Fig. 3c), which was also observed by Woertz (2007) who achievedaround 76% phosphate reduction when treating digested dairymanure with algae but starting at a lower concentration of7.7 mg PO4/L. CODs in the manure were utilized by algae as oneof carbon sources to some extent (27.4–38.4%) but not as efficientas nitrogen and phosphorus (Fig. 3d). The experiment was per-formed in axenic condition and therefore the reduction of CODcould only be attributed to consumption by algae. In photoautotro-phic culture of microalgae, CO2 is the sole carbon source and can befixed into organic compounds such as protein, sugars and lipids(Hirataa and Hayashitania, 1998). However, it is not the case forsome species of Chlorella, facultative algae, whose metabolic path-way can alter with supply of organic substrates such as organicacids or glucose (Eny, 1951). Endo et al. (1977) reported that the

10*dilution 15*dilution 20*dilution 25*dilution

10*dilution 15*dilution 20*dilution 25*dilution

0

5

10

15

20

25

30

35

Tota

l pho

spho

rus

conc

entra

tions

(mg

PO4/

L)

TP_initialTP_final

70.1%removal

62.5%removal

71.6%removal 74.7%

removal

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200

400

600

800

1000

1200

1400

1600

CO

D c

once

ntra

tions

(mg/

L)

COD_initial

COD_final

38.4%removal 27.4%

removal34.3%removal

27.9%removal

(b)

(d)

nal TKN concentrations and removal rates; (c) initial and final TP concentrations and

Table 3Results from elementary analysis of dried algae samples grown under the fourdilution multiples.

10*dilution 15*dilution 20*dilution 25*dilution

C/N 2.64 3.16 3.3 3.81C% 41.6 42.8 44.5 45.6H% 6.42 6.63 6.78 6.92(C + H)% 48.02 49.43 51.28 52.52

Table 4Calculated ratios of carbon, nitrogen and phosphorus removed by algae (thedifference of initial and final concentration in the sample was the amount removed).

Quotient Values

COD/TKN 2.48 2.24 2.99 2.21aC/N 0.93 0.84 1.12 0.83COD/TP 23.6 20.0 22.5 17.0TKN/TP 9.54 8.95 7.51 7.68

a C in C/N is the carbon equivalence of COD (O2) consumed.

Table 5Lipids in Chlorella vulgaris grown on both organic and inorganic medium (MURAKAMIet al., 1997).

Lipid Percentage (%) of dry weight

Organicmedia

Inorganicmedia

Neutral lipid (waxester 80% and TAG 20%) 2.8 1.88Phospholipid 8.6 6.2Glycolipid 5.7 5.7Trans-hexadecanoic acid 1.6 2.3

L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628 2627

growth rate was approximately the same as the sum of growthrates in the autotrophic and heterotrophic cultures when the cellswere cultured under mixotrophic condition (in light with acetate).Definite growth due to the presence of the organic acids was alsoobserved by Eny (1949), indicating that they are used by Chlorellaas a source of carbon. Organic acids are known to exist as interme-diates in the pathway of carbohydrate metabolism of most animalsand bacteria (Eny, 1951). The synthesis of storage materials such ascarbohydrate and protein may result either from photosynthesis orfrom oxidative assimilation of an organic substrate (Myers andCramer, 1947). Respiration of the storage materials provides en-

Table 6Fatty acid profiles derived from triacylglycerol, phospholipid and free fatty acids in algae.

aFatty acid 10*dilution (%)

Total fatty acid/dry weight 9.00Saturated fatty acids (% of total fatty acids) 27.114:0 0.1015:0 0.6016:0 20.617:0 3.4018:0 2.4020:0Monounsaturated fatty acids (% of total fatty acids) 19.116:1 6.6018:1 12.50Polyunsaturated fatty acids (% of total fatty acids) 53.816:2 10.416:3 6.0018:2 27.218:3 10.2

a Fatty acids are defined by the number of carbon atoms followed by the numb(C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0); (2) MUFA, monounsaturated fatty acidC18:3).

ergy for cell division and proliferation (Adams and Early, 2004).It is no doubt that the growth rate can be accelerated significantlyif some organic carbon sources can be used directly by microalgaein the oxidative assimilation process for storage material produc-tion. As the result from elemental analysis suggested that the algaecells had C/N ratios varying between 2.64 and 3.81 (Table 3), com-pared with the C/N ratios (0.83–1.12) consumed in the dairy man-ure supplemented growth media by algae in Table 4, it isconcluded that algae used the organic carbon in the digested dairymanure only as part of their carbon source while CO2 was alsoassimilated through the process of autotrophic photosynthesis.

3.4. Algal fatty acids content and composition

Based on Murakami et al. (1997), lipids in C. vulgaris grown onboth organic and inorganic media included neutral lipid, phospho-lipid, glycolipid, and Trans-hexadecanoic acid. The percentages ofthese lipids in their algae samples (dry weight base) are shownin Table 5. The percentage of TAG of the total lipid generally varieswith species and physiological state of the culture, ranging be-tween 38% and 65% (Tambiev et al., 1989), while lipid of some spe-cies consists mainly of esters of sterols and hydrocarbons (to 60%)(Tambiev et al., 1989). Biodiesel, by definition, is the methyl esterproduced from transesterification of TAG in plant oils or animalfats. When taking a close look at the lipid composition in Chlorellacells, it is noticed that methyl esters derived from free fatty acidsand phospholipids also fall into the biodiesel category. Thus, theone-step extraction–transesterification process used in this studymay provide a direct measurement of an algal species/strain’s po-tential as a biodiesel feedstock. The FAMEs obtained in this studyshould be derived from several lipid species including free fattyacids, TAGs, and phospholipid in the algal bodies.

Fatty acids are primary metabolites of acetyl CoA pathway,which is genetically determined, evolutionarily very old, andtherefore conservative (Petkov and Garcia, 2007). For green algaChlorella, the fatty acid composition of 14:0, 16:0, 16:1, 16:2,16:3, 18:0, 18:1, 18:2, a-18:3 is confirmed under all kinds of con-ditions such as photoautotrophic and heterotrophic cultivation,nitrogen starvation, and outdoor in a photobioreactor (Petkovand Garcia, 2007). Table 6 shows the fatty acid profiles derivedfrom TAG, phospholipid and free fatty acids in algae cultivatedon diluted digested dairy manure samples in our study. Exceptfor those mentioned by Petkov and Garcia (2007), 15:0, 17:0 and20:0 fatty acids were also observed in small quantities in our algae

15*dilution (%) 20*dilution (%) 25*dilution (%)

11.0 13.6 13.728.3 30.7 33.00.30 0.30 0.400.40 0.30 0.4022.2 24.3 26.03.00 2.60 2.202.10 3.00 3.800.30 0.20 0.2022.2 29.9 27.710.80 9.10 7.5011.40 20.80 20.2049.4 39.2 39.18.60 6.80 5.70

30.6 32.4 33.410.2

er of double bonds after the colon, classified to (1) SFA, saturated fatty acidss (C16:1 + C18:1); (3) PUFA, polyunsaturated fatty acids (C16:2 + C16:3 + C18:2 +

2628 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628

samples. Octadecadienoic acid (C18:2) was the most abundantfatty acid in algae bodies under all conditions, ranging from27.2% to 33.4% of the total fatty acids. Hexadecanoic acid (C16:0)was the second abundant fatty acid, ranging from 20.6% to 26.0%.Both acids increased in their contents when cultivated under morediluted samples, indicating their accumulation as storage materialscould be enhanced when subject to low/scare nutrient environ-ment. The total fatty acid contents increased from 9.00% to 13.7%of dry weight with increasing dilution multiples. Taking the celldry weight density (1.57, 1.47, 1.71, 1.48 g/L, respectively) into ac-count, the final total fatty acids concentrations were 0.141, 0.162,0.233, and 0.203 g/L, respectively, for 10�, 15�, 20�, 25� dilutedsamples. The saturated fatty acids increased from 27.1% to 33.0%of total fatty acids while the polyunsaturated fatty acids decreasedfrom 53.8% to 39.1% of total fatty acids with dilution rates from 10to 25. The monounsaturated fatty acids did not show a similartrend but generally were accumulated more in more dilutedsamples.

4. Conclusions

Vast amounts of animal manures produced from concentratedanimal feeding operations present opportunities for economicgains if proper utilization strategies are employed (Demirer andChen, 2004). Anaerobic digestion has been widely employed topartially treat dairy manure and generate on-site energy. A processof combining anaerobic digestion further with algae cultivation canoffer many environmental benefits with the production of abun-dant algal biomass to serve as a biofuel feedstock, a fertilizer oran animal feed and thus provide a valuable solution to the refrac-tory dairy wastes.

Acknowledgements

The authors are grateful to Richard Huelskamp for supplyingmanure samples and Blanca C. Martinez for providing help in thelabs. The study was supported in part by grants from the Universityof Minnesota Initiative for Renewable Energy and the Environment(IREE) and the Center for Biorefining.

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