Applied Energy 128 (2014) 103–110

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Applied Energy

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Biodiesel production from algae cultivated in winter with artificialwastewater through pH regulation by acetic acid

http://dx.doi.org/10.1016/j.apenergy.2014.04.0390306-2619/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +86 2087057760; fax: +86 2087057677.E-mail address: wangzm@ms.giec.ac.cn (Z. Wang).

Liandong Zhu a,b, Erkki Hiltunen a,b, Qing Shu c, Weizheng Zhou c, Zhaohua Li d, Zhongming Wang c,⇑a Faculty of Technology, University of Vaasa, FI-65101 Vaasa, Finlandb Vaasa Energy Institute, FI-65101 Vaasa, Finlandc Guangzhou Institute of Energy Conversion, Key Laboratory of Renewable Energy and Gas Hydrate, Chinese Academy of Sciences, Guangzhou 510640, PR Chinad Faculty of Resources and Environment, Hubei University, Wuhan 430062, PR China

h i g h l i g h t s

�Wintering cultivation of algae inartificial wastewater wasinvestigated.� pH regulation could improve the

nutrient removal and biomassproductivity.� High lipid content and productivity

were obtained by regulating pH.� Biodiesel yield in the pH-regulated

group was 19.44% of dry weight.� It is efficient to grow C. zofingiensis in

winter by pH regulation using aceticacid.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 May 2013Received in revised form 9 April 2014Accepted 11 April 2014

Keywords:Chlorella zofingiensisPilot scaleBiodieselpH regulationArtificial wastewaterNet energy ratio

a b s t r a c t

Algae have been considered as a promising biodiesel feedstock. One of the major factors affectinglarge-scale algae technology application is poor wintering cultivation performance. In this study, anintegrated approach is investigated combining freshwater microalgae Chlorella zofingiensis winteringcultivation in pilot-scale photobioreactors with artificial wastewater treatment. Mixotrophic culture withthe addition of acetic acid (pH-regulation group) and autotrophic culture (control group) were designed,and the characteristics of algal growth, lipid and biodiesel production, and nitrogen and phosphateremoval were examined. The results showed that, by using acetic acid three times per day to regulatepH at between 6.8 and 7.2, the total nitrogen (TN) and total phosphate (TP) removal could beincreased from 45.2% to 73.5% and from 92.2% to 100%, respectively. Higher biomass productivity of66.94 mg L�1 day�1 with specific growth rate of 0.260 day�1 was achieved in the pH-regulation group.The lipid content was much higher when using acetic acid to regulate pH, and the relative lipid produc-tivity reached 37.48 mg L�1 day�1. The biodiesel yield in the pH-regulated group was 19.44% of dryweight, with 16–18 carbons as the most abundant composition for fatty acid methyl esters. The findingsof the study prove that pH adjustment using acetic acid is efficient in cultivating C. zofingiensis inwastewater in winter for biodiesel production and nutrient reduction.

� 2014 Elsevier Ltd. All rights reserved.

104 L. Zhu et al. / Applied Energy 128 (2014) 103–110

1. Introduction

Today there are increasing concerns over global climate changeand the energy crisis, caused in part by increasing global energydemand and depleting fossil resources [1]. It is widely acceptedthat the fossil-based economy is getting closer and closer to theend of its lifecycle. As a result, it is very important to be able toforesee and prevent any shortage in future energy supply, and toprovide access to renewable and sustainable energies for the mar-ketplace. In response to the challenges outlined above, biologicalresearch for energy feedstock production is a promising scientificdirection to solve the global energy issues in an effort to worktoward sustainable development. Of all the renewable energytypes, bioenergy is considered to have the highest potential to sat-isfy the energy needs of modern society for both developed anddeveloping countries, and will hopefully become one of the mainworld energy sources. Microalgae have been described as one ofthe most promising feedstock for biofuel production [2,3]. Cur-rently biodiesel is mainly obtained from oil-rich seeds, but thisprocess has not been considered as a means to replace fossil fuelsdue to feedstock limitation [4]. Compared to first- and second-gen-eration biofuel feedstock, microalgae have many advantages, suchas higher oil content, higher rate of photosynthesis, the ability togrow in wastewater as well as water produced from oil and gasextraction, higher biomass productivities than oil-seed crops, andno need to compromise for the production of food, fodder andother products derived from crops [5–9].

Microalgae production in wastewaters represents one of thebest measures toward the reduction of excessive nutrients, withthe advantage of a faster uptake of nutrients such as nitrogenand phosphorus. Microalgae cultivated in wastewaters can con-sume significantly less fresh water and improve water quality[10]. This is because no or limited chemical fertilizers, which areeasily dissolved in rainwater or runoff, need to be applied to thesystem [6]. Arbib et al. [11] investigated long-term outdoor opera-tion of a tubular airlift pilot photobioreactor as tertiary treatmentof urban wastewater using microalgae Scenedesmus obliquus, andachieved average removal of TN and TP of 89.68% and 86.71%,respectively. The maximum removal of chemical oxygen demand(COD) was 73.18% when Arthrospira platensis was cultivated inolive-oil mill wastewater, while phosphorus and nitrates in someruns were completely removed [12]. Zhu et al. [7] cultivated Chlo-rella zofingiensis in piggery wastewater under indoor conditions forten days and achieved COD, TN and TP removal of more than 78%,82% and 95%, respectively.

However, most previous studies have been carried out undersuitable or optimal conditions, for example with appropriateztemperature and illumination control, sufficient carbon sourcesupplemented with CO2, and so forth [13–16]. Quinn et al. [17]introduced a model of microalgae growth derived from indus-trial-scale outdoor photobioreactor growth data, and mapped outthe near-term feasible cultivation locations and correspondingproductivity potentials for the USA. The potential for year-roundcultivation of algae is limited, especially in regions with four evi-dently distinct seasons. Growing Nannochloropsis oculata outdoorsin closed vertical flat photobioreactors, total lipids ranged from11% of dry weight in winter to 30% of dry weight in autumn, andthe amount of monounsaturated fatty acids in algal cells witnessedan increase during autumn [18]. Slegers et al. [19] carried out sce-nario studies to investigate the effect of decision variables on pho-tobioreactor performance in The Netherlands, France and Algeria,and found that geographical location is an important factor toinfluence the internal light profile and thus the productivity. Thus,the cultivation performance of microalgae under outdoor and coldclimate conditions is poor, showing that this apparently promising

approach is region-dependent. This will hinder the scale-up andcommercial application of algae cultivation for biodiesel produc-tion. Thus, how to improve the performance of microalgal cultiva-tion and nutrient removal in winter is an urgent issue that needs tobe solved if sustainable development is to be achieved for the algalindustry.

In this study, an integrated approach is presented combiningthe wintering cultivation of freshwater microalgae Chlorella zofin-giensis in pilot-scale photobioreactors with artificial wastewatertreatment. The integration is especially important since the micro-algal biofuel applications appear to be strongly economicallyconvenient only in conjunction with wastewater treatment [20].Two treatments (pH-regulation group and control group) weredesigned. In the pH-regulation group a certain amount of aceticacid was used to adjust the pH values of the culture. To summarize,the objectives of this study were: (1) to investigate the winteringperformance of C. zofingiensis cultivated in pH-regulated artificialwastewater; (2) to reveal the nutrient removal abilities in winter;and (3) to specify the productivities of biomass, lipids and biodieselunder winter conditions.

2. Materials and methods

2.1. Microalgae strain and pre-culture conditions

Chlorella zofingiensis was obtained from a lab in the GuangzhouInstitute of Energy Conversion of the Chinese Academy of Sciences.And it was grown in a BG11 medium [21], consisting of: (1) the fol-lowing solid chemicals: NaNO3 (1.5 g L�1), K2HPO4�3H2O(40 mg L�1), Na2CO3 (20 mg L�1), CaCl2�2H2O (36 mg L�1), MgSO4-

�7H2O (75 mg L�1) and FeCl3�6H2O (3.15 mg L�1) and (2) the fol-lowing ingredients: citric acid (6 mg L�1) and 1 mL per liter oftrace elements solution consisting of MnCl2�4H2O (1.81 mg L�1),H3BO3 (2.86 mg L�1), Na2MoO4�2H2O (0.39 mg L�1), CuSO4�5H2O(0.08 mg L�1), Co(NO3)2�6H2O (0.05 mg L�1), ZnSO4�7H2O(0.22 mg L�1) and concentrated H2SO4 (1 mL). The initial pH ofthe medium was adjusted to 6.8. The seed culture was grown ina 1.37 L tubular bubble column photobioreactor (tbcPBR), whichhas a height of 70 cm, an internal diameter of 5 cm and a thicknessof 1.5 mm. The culture with 800 mL of the medium, was cultivatedunder indoor conditions with a surrounding temperature of25 ± 1 �C, aerated with compressed air with 5–6% CO2 and contin-uously illuminated with cool white fluorescent lamps on one sideof the photobioreactor (light intensity of 230 ± 20 lmol m–2 s�1).

2.2. Culture of C. zofingiensis with artificial wastewater

Experiments were carried out in southern China, where typicalsubtropical monsoon climate conditions are found. The tubularbubble column photobioreactor (tbcPBR) at pilot scale was con-structed using Plexiglass for the treatment of artificial wastewaterin conjunction with microalgal growth. Each tbcPBR had a height of172 cm, external diameter of 27.2 cm and thickness of 5 mm. Thus,the volume was 40 L. The artificial wastewater was prepared usinglocal rainwater to dissolve urea [CO(NH2)2] (29 mg L�1), NaNO3

(1.5 g L�1), KH2PO4 (60 mg L�1), MgSO4 (60 mg L�1), and FeSO4-

�7H2O (1 mg L�1). The characteristics and features of the artificialwastewater are summarized in Table 1.

A volume of 33 L of artificial wastewater was introduced intoeach tbcPBR. Subsequently, the artificial wastewater was pre-treated with 10% NaClO at a ratio of 0.1 mL per 1 L substrate from8 pm on 29th December 2012 until the sunrise in the next day,after which the liquid was exposed to sunlight in the daytime of30th December 2012 (light intensity of 835 ± 450 lmol m–2 s�1).Two treatments (pH-regulation group and control group) were

Table 1Characteristics of the artificial wastewater used in the experiments.

Parameter Concentration

pH 6.4TN (mg N L�1) 277.0TP (mg PO4

–3 – P L�1) 43.0Na (mg L�1) 406.4K (mg L�1) 20.3Mg (mg L�1) 13.0Si (mg L�1) 6.3Ca (mg L�1) 8.5Fe (mg L�1) 0.202Cu (mg L�1) 0.037Al (mg L�1) 0.094

L. Zhu et al. / Applied Energy 128 (2014) 103–110 105

designed. From the 2nd day of cultivation onwards, a certainamount of acetic acid (HAc) with a density of 1.0492 kg L�1 wasapplied three times per day to keep the pH values of the pH-regu-lation cultures between 6.8 and 7.2. The experiment began at 9 amon 31st December 2012. Before the introduction of algae seeds, thepH values of the media were adjusted according to pre-determinedvalues. Afterward, a volume of 850 mL of seed microalgae suspen-sion with an optical density (OD680) of 11.000 was introduced intoeach tbcPBR. Air was continuously introduced into the base by asparger attached at the bottom. The range of average dailysurrounding air temperature was from 6.0 to 17.2 �C, and the rangeof average daily sunlight intensity was from 79 to 935 lmol m–

2 s�1 (9 am to 5 pm during the day). All the experiments were car-ried out in duplicate. The experiment lasted for 15 days until 9 amon 15th January 2013.

2.3. Analytical procedures

2.3.1. Sampling and nutrients analysisAt the end of this experiment a volume of 3 mL microalgae was

sampled from each PBR for TN and TP removal analysis. Thesamples were first centrifuged at 1811.16g for 10 min. Then, thesupernatants were filtered using a 0.45 lm nylon membrane filter,after which the filtrates were appropriately diluted. Finally, TN andTP of the filtrates were analyzed following the Hach DR 2700 Spec-trophotometer Manual [22]. The percentage removal was obtainedusing the following formula:

Percentage removal W% ¼ 100%� ðC0 � CiÞ=C0 ð1Þ

where C0 and Ci represent the mean values of nutrient concentra-tion at initial time t0 and time ti, respectively.

2.3.2. Determination of microalgae growthThe optical density of C. zofingiensis at 680 nm was determined

every day using a spectrophotometer (Jingke 722N, Shanghai,China). A linear relationship between the dried biomass and theOD680 was pre-determined, as shown below:

Dry weight ðg L�1Þ ¼ 0:2432� OD680; R2 ¼ 0:9926 ð2Þ

The specific growth rate (l) in the exponential phase of algalgrowth was measured. The expression followed Eq. (3) [7,23,24]below:

l ðday�1Þ ¼ lnðN2=N1Þ=ðt2 � t1Þ ð3Þ

where N1 and N2 represent dry biomass (g L�1) at time t1 and t2,respectively. The biomass productivity (P) was determined usingthe following expression:

P ¼ ðDWi � DW0Þ=ðti � t0Þ ð4Þ

where DWi and DW0 represent dry biomass (g L�1) at time ti and t0

(initial time), respectively.

Microalgae cells from each tbcPBR were collected after C. zofin-giensis grew for 15 days. The samples were centrifuged at 5032.08gfor 15 min. Supernatants were decanted and cell solids werewashed carefully with distilled water, after which the cell solidswere freeze-dried at –80 �C. The dried microalgae biomass wassealed in empty containers for the analysis of lipid extractionand fatty acids methyl esters (FAME).

2.3.3. Lipid extractionThe quantification of the amount of total lipid content was

measured according to a modified method by Bigogno et al. [25].100–150 mg of freeze-dried samples were weighed and thenextracted with 2 mL of methanol containing 10% dimethyl sulfox-ide (DMSO). The extraction took place in a water bath shaker at45 �C for 45 min. The mixture was centrifuged at 1811.16g for10 min, after which the supernatant was collected. The leftoverwas re-extracted twice following the same procedures as men-tioned above. Then the leftover was extracted using a 4 mL mixtureof hexane and ether (1:1, v/v). The extraction took place in a waterbath shaker at 45 �C for 60 min. The mixture was centrifuged at1811.16g for 10 min, after which the supernatant was collected.The leftover was re-extracted twice following the same proceduresas mentioned above. All the supernatants were incorporated and6 mL of distilled water was added to the combined extracts. Thus,the ratio of methanol with 10% DMSO, diethyl ether, hexane anddistilled water was 1:1:1:1 (v/v/v/v). The organic phases containinglipids were collected and combined into a pre-weighed glass tube.Afterward, the samples were evaporated to dryness under the pro-tection of nitrogen. Then the lipids were freeze-dried at –80 �C for24 h. Finally, the lipids were measured gravimetrically as a % of thedry weight.

2.3.4. FAME content analysisA one-step extraction-transesterification method by Indarti

et al. [26] was applied to analyze the content of the FAME aftersuitable modifications. 20 mg of freeze-dried samples of C. zofingi-ensis were weighed. Transesterification was performed using2.5 mL of methanol with 2 wt.% of concentrated sulfuric acid(H2SO4) as the catalyst. The transesterification took place in awater bath shaker at 80 �C for 2.5 h. When the reaction was com-pleted, the mixtures were cooled down to room temperature. Then,1 mL of distilled water and 2 mL of hexane were added into themixture to form two phases. The upper phase, which containedFAME, was transferred to a clean, 10 mL bottle and dried withwater-free Na2SO4. At this point the sample was prepared wellfor the analysis of the FAME composition, which was carried outby a gas chromatograph with a FID detector (Shimadzu GC-2010,Japan). The temperatures of the injector and detector were set at300 and 280 �C, respectively. The temperature gradient wasprogrammed from 130 to 180 �C with an increase of 10 �C/min fol-lowed by a rise to 210 �C with 2 �C/min and then the temperaturewas fixed at 210 �C for 3 min. The compounds detected were iden-tified and quantified using the NIST Mass Spectral Search Program.

All the experiments were carried out in duplicate and averagevalues were reported. Results were performed with EXCEL [27]and SPSS 11.5 for Windows [28] and analysis of variance (ANOVA)was determined wherever applicable.

3. Results and discussion

3.1. Microalgal growth

The growth characteristics of C. zofingiensis in artificial waste-water in winter within 15 days were investigated, as shown inFig. 1. Amazingly, C. zofingiensis in both cultures could survive well

Fig. 2. The fluctuation of irradiance and air temperature of each day (9:00 am–5:00 pm) during C. zofingiensis cultivation (means ± sd).

106 L. Zhu et al. / Applied Energy 128 (2014) 103–110

under outdoor conditions (Figs. 2 and 3), although a slight decreasein biomass weight was observed in the beginning. These curves inFig. 1 illustrate the lag phase, exponential phase and stationaryphase during algal growth. A visible lysis phase is missing becausethe cultivation period was not long enough in this study. The lagphase in this study was much longer than that found by Zhuet al. [29], who cultivated C. zofingiensis in piggery wastewaterunder indoor conditions with temperature at around 25 �C. How-ever, after acclimation to a cold environment for about 4 days C.zofingiensis in pH-regulated culture successfully stepped into theexponential stage, while algae in the control group witnessed theincreasingly exponential phase from Day 6 onwards. The pH-regu-lated culture enjoyed a rapidly exponential phase of 9 days (fromDay 4 to Day 13), which was 3 days longer than the control group(from Day 6 to Day 12).

As illustrated in Fig. 2, under outdoor conditions the change ofweather, temperature and light intensity was inconsistent. During15 day experiments under uncontrolled environment, the outdoorair temperature and light intensity (on the basis of single measure-ment) varied from 5.7 to 20.0 �C, and from 6 to 1810 lmol m–2 s�1,respectively. The specific growth rate l of C. zofingiensis cultivatedin pH-regulated culture and the control group was 0.260 and0.208 day�1, with a doubling time of 2.67 and 3.34 days, respec-tively (Table 2). The l in this study was lower than the 0.337 day�1

achieved by Zhu et al. [7], who cultivated C. zofingiensis outdoorswhere the air temperature varied from 21.5 to 34.5 �C. Anotherstudy has shown high values of l of 0.994 and 0.447 day�1 witha doubling time of 0.697 and 1.54 days, in spring and autumn,respectively [30]. The authors [30] produced C. zofingiensis in 60 Lflat-plate photobioreactors. The final biomass productivity of pH-regulated culture arrived at 66.94 mg L�1 day�1, which was around3.6 times higher than that of the control group (18.48 mg L�1 -day�1). The biomass productivity of the pH-regulated culture inthis study was advantageously higher than that in the researchby Min et al. [31], who achieved 34.6 mg L�1 day�1 biomass whenthey cultivated Chlorella sp. in a pilot-scale photobioreactor usingcentrate wastewater. The pH-regulation group combined higherbiomass productivity with higher specific growth rate, since HAccould act as a carbon source for algal growth [32]. In addition,HAc could adjust the pH values to the suitable range, which wasverified to benefit the growth of algae [33,34].

3.2. Nutrient removal

The TN and TP removals in the pH-regulation group and controlgroup for the 15 day wintering cultivation are exhibited in Fig. 4.

Fig. 1. Growth curves for C. zofingiensis grown in artificial wastewater in winterwithin 15 days (means ± sd). HAc+ and HAc� accordingly represent the cultureswith and without the addition of acetic acid in all cases in this paper.

As can be seen from Fig. 4, TN removals in the pH-regulationgroup and control group reached 73.49% and 45.19%, respectively.The microalgal cell required nitrogen for protein, nucleic acid andphospholipid synthesis. The pH-regulation group witnessed muchhigher biomass productivity than the control group (Fig. 1 andTable 2). That is why TN removal in the pH-regulation group wasevidently higher (1.9-fold) than in control group. Phosphateremoval in the present study was achieved at a relatively highlevel; removal in other studies has been lower (70.3%) for Chlorellapyrenoidosa [35]. TP removals in the pH-regulation group wereachieved at 100%, and the consumed phosphorus was mainly usedand assimilated for the synthesis of C. zofingiensis. The pH value inthe control group was obviously increased from 6.4 to 10.9, result-ing from the photosynthesis of microalgae and the reduction of theorganic pollutant. The increase of pH in cultures could help con-tribute to the precipitation of phosphorus and the increase of phos-phate adsorption on microalgal cells [36]. Thus, although thecontrol group had low biomass productivity, a decrease of phos-phorus levels occurred. Significant differences (P < 0.01) in TNand TP removal were found between the treatments with pH reg-ulation and without pH control.

Meanwhile, bacterial consumption could also be another con-tributor to degrade TN and TP by microbial processes [37,38].The air used in this study was not sterilized, for example, with amembrane filter. In addition, the tbcPBR used in this study is asemi-closed system with a removable cap on the top. Thus, the cul-ture system contains bacteria via air exchange and introduction,which could also contribute to the degradability of pollutants[38,39]. Several previous studies have reported that the interactionof algae and other microorganisms, such as bacteria, in the culturesystem could contribute to wastewater nutrient removal[35,38,40]. In a microalgal–bacterial culture system, microalgaecan consume the CO2 that is produced by bacteria through respira-tion and in turn provide O2, which is essential for bacteria to bio-degrade organic pollutants [29]. Microalgae can enhancebacterial activity via the excretion of certain extracellular com-pounds, while bacterial growth can improve microalgal metabo-lism by releasing growth-promoting factors, by decreasing O2

concentration in the culture, or by decomposing larger substancesfor algal cells to efficiently assimilate [29,38].

3.3. Lipid content and productivity

The lipid content of C. zofingiensis grown in artificial wastewaterin winter over 15 days is shown in Fig. 5. High lipid content wasachieved in this study. Low-light conditions (Fig. 2) can preventthe release of reactive oxygen species to avoid damage to mem-brane lipids [41]. One previous study showed that lipids, especiallytriacylglycerides, were usually synthesized under low light and

Fig. 3. Cultivation of C. zofingiensis in pilot-scale tbcPBRs using artificial wastewater. The left two tbcPBRs belong to the pH-regulated group.

Table 2Growth parameters of C. zofingiensis grown in artificial wastewater in winter within 15 days (means ± sd).

Treatment Specific growth rate l (day�1) Doubling time (days) Biomass increase (g L�1) Biomass productivity (mg L�1 day�1)

HAc+ 0.260 ± 0.012 2.67 ± 0.13 1.004 ± 0.002 66.94 ± 0.14HAc� 0.208 ± 0.022 3.34 ± 0.36 0.277 ± 0.023 18.48 ± 1.56

Fig. 4. N and P removals by C. zofingiensis grown in artificial wastewater in winterwithin 15 days (means ± sd). Fig. 5. Lipid content and productivity of C. zofingiensis grown in artificial waste-

water in winter with 15 days (means ± sd).

L. Zhu et al. / Applied Energy 128 (2014) 103–110 107

temperature conditions or photo-oxidative stress [42]. In addition,it has been found that lower temperature can lead to an increase ofstructural lipids to facilitate cell membrane fluidity [18]. The pH-regulation culture (55.98%) witnessed much higher lipid contentthan the control group (30.31%). This indicated that regulatingpH to an optimal level during microalgae cultivation could increasethe lipid content. Significant differences (P < 0.01) in lipid contentwere found between pH-regulation and without-pH-controlgroups. Similar results had also been observed by Han et al. [34],who found that the lipid content of C. pyrenoidosa was 1.15-foldhigher when controlling pH at 7 with CO2, compared to withoutpH control. Previous studies show that many microalgae accumu-late lipids as storage materials, and their accumulation can bestimulated under environment stress, such as nutrient deficiency[29,43,44]. In the later period of this experiment TP deficiency orlimitation occurring in the pH-regulation culture could helpenhance lipid synthesis and at the same time inhibit protein syn-thesis [44]. Another study demonstrated that lipid storage in algaecould be increased by P starvation [45].

The lipid productivities of C. zofingiensis in two groups werecalculated as shown in Fig. 5. The lipid productivity in the pH-reg-ulated group (37.48 mg L�1 day�1) was 6.7-fold higher than incontrol group (5.60 mg L�1 day�1). The results in this study werelower than the 82.5 mg L�1 day�1 of lipid productivity obtainedby Han et al. [34], who controlled pH at 7 with CO2 when growingC. pyrenoidosa indoors. The data they achieved was 2.64-foldhigher than without pH control. In this study the differences inthe lipid productivity of C. zofingiensis between the two treatmentsresulted from the differences in their biomass productivities andlipid contents.

3.4. FAME composition and biodiesel productivity

Fatty acid methyl esters are the chemical ingredients of biodie-sel. In this present study, the FAME compositions of C. zofingiensismainly consisted of C18:3 (octadecatrienoic acid methyl ester) andC18:2 (octadecadienoic acid methyl ester) in the followingsequence C18:3 > C18:2, as shown in Fig. 6. This observation is in

Table 3Biodiesel yields and productivity of C. zofingiensis grown in artificial wastewater inwinter within 15 days.

Treatment Biodiesel yields (% of dw) Biodiesel productivity(mg L�1 day�1)

HAc+ 19.44 13.01HAc� 11.18 2.07

108 L. Zhu et al. / Applied Energy 128 (2014) 103–110

contrast to the findings of Zhu et al. [7], who achieved the threemain proportions of FAME compositions in the relationshipC18:2 > C16:0 > C18:3, when cultivating C. zofingiensis indoorsand outdoors in autumn.

As can be seen from Fig. 6, C. zofingiensis in this study had ashorter carbon chain for fatty acids, mainly containing 16–18 car-bons, which was ideal for biodiesel conversion [29]. The C16:0(palmitic acid methyl ester), C16:1 (palmitoleic acid methyl ester),C18:0 (stearic acid methyl ester), C18:2 and C18:3 of C. zofingiensisin the pH-regulated and control cultures represented the majorportion of fatty acid methyl esters compositions, accounting for atotal of 89.67% and 86.67%, respectively. Previous studies havereported that culturing conditions, growth period, and environ-mental situations can greatly affect lipid content and fatty acidcomposition [7,46]. The unsaturated fatty acid methyl esters inthe pH-regulated and control cultures were predominant in theFAME profile, accordingly accounting for 79% and 89%. These val-ues were much higher than those achieved during indoor cultiva-tion of C. zofingiensis [7,29,47]. Temperature can affect the fattyacid composition of microalgae by changing fatty acid metabolismin algal cells [48], and the degree of unsaturation in fatty acidsusually has a negative correlation with temperature [18,49]. It isworthy of note that the pour point of biodiesel can be reduced asthe percentage of unsaturated fatty acid methyl esters increases[7,29]. This property can benefit countries with cold climates likeNordic nations by reducing the pour points of biodiesel [7,24].

The quantification of biodiesel yields from C. zofingiensis isshown in Table 3. It can be found that only around one third of lip-ids can be transferred into biodiesel, since not all lipids can be con-verted to FAME, for example, chlorophyll, glycolipid andphospholipid [29,47]. The FAME yields with 19.44% of dry weightwere achieved in the pH-regulation group. The biodiesel productiv-ity of the pH-regulated culture was 11.18 mg L�1 day�1, around5.4 times higher than that of the control group.

3.5. Efficiency analysis

During 15 day cultivation outdoors, the total consumption ofHAc in the pH-regulation culture was 68.25 mL (Table 4), whichis equal to 71.61 g. There was a very positive and significant linearrelationship (P < 0.01) between the daily HAc usage and the dailydry weight concentration. The regression equation of the linearrelationship is y = 7.8405x + 0.9379, R2 = 0.9721, where the lettery represents the daily HAc usage, while the letter x means the dailydry weight concentration of C. zofingiensis. The biomass productiv-ity of C. zofingiensis in the pH-regulated culture was 0.47 g/g HAc.De Swaaf et al. [50] used HAc to produce microalgae biomass via

Fig. 6. Fatty acids methyl ester profile of C. zofingiensis grown in artificialwastewater in winter within 15 days.

a fed-batch operation under heterotrophic conditions, and theyachieved a biomass productivity of less than 0.14 g/g HAc, whichis much lower than that observed from this study. When cultivat-ing C. zofingiensis in bench-scale outdoor ponds in winter for6 days, Huo et al. [51] found that the HAc regulation group wit-nessed much higher lipid content (31.8%) than the CO2 regulationgroup (17.9%).

The net energy ratio (NER) for biomass production was definedas the ratio of the energy produced as biomass over the totalenergy consumption. To sustain an energy generation system,NER must be greater than 1, and as high as possible [43]. In thisstudy the NER calculation is based on the estimation that the drybiomass productivity over a 3 month winter period is 1 ton. Theresults are shown in Table 5. As can be seen from the table, theNER value in the pH-regulation group was around 3 times higherthan that in the control group. However, the winter pilot-scaleoperation in this study is not economically feasible since the NERis much less than 1. Similarly, Jorquera et al. [52] reported thatthe NER for biomass production was 0.20, when producing Nanno-chloropsis sp. in tubular photobioreactors. Rodolfi et al. [43]reported that the NER for algae production in second-generationflat-panel photobioreactors was barely above 1, since the energycosts for mixing and harvesting were high.

The potential energy balance of microalgae production varieswidely, depending on the microalgae species, cultivation season,cultivation system, energy savings realized by the co-products,and so on [54]. The high-energy inputs, which include wastewaterfilling, air pumping, culture mixing for suspension, and biomassharvesting, dewatering and drying, potentially result in a negativeNER for the process. In this study, the biomass productivity underwinter conditions was low, and thus the energy content recoveredfrom biomass was limited, causing a low NER value. On the otherhand, the NER of C. zofingiensis cultivation in this study is unex-pectedly lower than 1, due to the high costs in air mixing and bio-mass harvest. However, several studies suggest that the outdoorcultivation with substantially high light intensity and high temper-ature can generate more biomass and lipids compared to low lightand low temperature conditions [18,55,56]. For example, Olofssonet al. [18] grew Nannochloropsis oculata outdoors, and obtained 2.3and 1.1 g L�1 biomass in autumn and winter, respectively. Theycame to a conclusion that summer and autumn were the favorableseasons for microalgae cultivation to produce biodiesel in thesouthern Portugal (subtropical areas). Similarly, in this study out-door cultivation was carried out in southern China, where typicalsubtropical monsoon climate conditions are found. Therefore, itis expected that substantially high biodiesel productivity mightbe achieved from microalgae cultivated in warm climates withwastewater through pH regulation by acetic acid.

Although pH regulation of culture can enhance biodiesel pro-duction (Table 3), the relative NER value was still lower than 1(Table 5), reflecting a poor economic performance. The economicfeasibility of commercial microalgae production for biofuel appli-cation has been analyzed in numerous studies [8,57,58], whereall the authors point out that economics are currently the mainbarrier to produce microalgae on a large scale for biofuel applica-tion. Nonetheless, that our energy supply must be transitionedfrom fossil fuels to renewable energies is inevitable, and any effort

Table 4The daily usage of HAc in C. zofingiensis wintering cultivation in artificial wastewater.

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Dry weight (g L�1) 0.06 0.06 0.08 0.10 0.13 0.16 0.21 0.36 0.48 0.64 0.76 0.89 0.98 0.99 1.00Usage (mL) 0.00 1.00 1.50 2.25 2.00 2.50 3.25 4.00 5.25 6.00 7.00 8.00 8.50 8.50 8.50

Table 5NER analysis for C. zofingiensis biomass production with artificial wastewater in winter.a

Parameter HAc� HAc+

Cell density (g L�1) 0.277 1.004Biomass productivity (mg L�1 day�1) 18.48 66.94Cultivation time per run (d) 15.0 15.0tbcPBR volume required for biomass production of 1 ton (m3) 601.3 166.0Wastewater needed (m3) 3610 996Total energy consumption for biomass production (MJ)b 358,082 96,494Calorific values of Chlorella (MJ kg�1)c 24 24Energy produced as biomass (MJ) 23,500 23,500NER for biomass production 0.07 0.24

a In this study the NER calculation is based on the estimation that the dry biomass productivity over a 3 month winter period is 1 ton.b Estimated values, including wastewater filling, air pumping, harvest and drying.c 24 MJ kg�1 is used according to Illman et al. [53].

Fig. 7. Proposed algal biorefinery framework for the production of high-value products and biofuels. Harvest water, nutrients from algal residues or leftovers, and CO2 fromanaerobic digestion can be recycled into algae production system.

L. Zhu et al. / Applied Energy 128 (2014) 103–110 109

to fix the above barriers is important and worthwhile. Microalgalbiorefinery is a promising approach to improve the economics ofmicroalgal biodiesel production (Fig. 7). During algae cultivation,cultivation strategies such as nutrient starvation can be designedto induce the accumulation of some targeted constituents likelipids, proteins, pigments, nutraceuticals and other commerciallysignificant materials. Before algal biodiesel conversion, high-valueproducts, such as fine chemicals, animal feed, human nutrition,protein, pharmaceuticals and cosmetics, can be extracted. Inaddition, the appropriate reuse of algal residues should berecommended.

4. Conclusion

This study showed that C. zofingiensis was a valuable candidatefor outdoor production in winter conditions when regulating pHwith HAc. Higher biomass productivity of 66.94 mg L�1 day�1 withspecific growth rate of 0.260 day�1 was achieved in the pH-regula-tion group since acetic acid could also act as a carbon source foralgal growth. TN removal in the pH-regulation group reached73.49%, which was 1.63-fold higher than that in the control group.TP removal in the pH-regulation group (100%) was a little higherthan in the control group (92.21%). Much higher lipid contentwas observed in the pH-regulated group, and the lipid productivitywas 37.48 mg L�1 day�1, which was 6.7-fold higher than that in the

control group. The FAME yields of C. zofingiensis grown in the pH-regulation group and control group were 19.44% and 13.01%,respectively. Fatty acids obtained were suitable for the productionof biodiesel with good quality.

Acknowledgement

This work was funded by the Fortum Foundation of Finland, theSouth Ostrobothnia Regional Fund of the Finnish Cultural Founda-tion and the National Basic Research Program of China (Grant No.2011CB200905). The authors are indebted to the following peoplefor their assistance, input and advice (alphabetical order): Lei Qin,Pengzhong Feng, Shuhao Huo, Zhongbin Xu. The authors wouldalso like to thank the three anonymous reviewers for their helpfulcomments and suggestions that greatly improved the manuscript.

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