Applied Energy 88 (2011) 3307–3312

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Applied Energy

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Microalgae Chlorella as a potential bio-energy feedstock

Mayur M. Phukan a, Rahul S. Chutia b, B.K. Konwar a,⇑, R. Kataki b

a Department of Molecular Biology & Biotechnology, School of Science & Technology, Tezpur University, Assam, Napaam 784 028, Indiab Department of Energy, School of Energy, Environment & Natural Resources, Tezpur University, Assam, Napaam 784 028, India

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

Article history:Received 15 September 2010Received in revised form 1 November 2010Accepted 18 November 2010Available online 6 January 2011

Keywords:MicroalgaeBiomassThermogravimetryChlorellaBiofuel

0306-2619/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.apenergy.2010.11.026

⇑ Corresponding author. Tel.: +91 9954449458; faxE-mail address: bkkon@tezu.ernet.in (B.K. Konwar

Microalgae are promising biomass species owing to their fast growth rate and high CO2 fixation ability ascompared to terrestrial plants. Microalgae have long been recognized as potentially good source for bio-fuel production because of their high oil content and rapid biomass production. In this study Chlorella sp.MP-1 biomass was examined for its physical and chemical characteristics using Bomb calorimeter,TGDTA, CHN and FTIR. The proximate composition was calculated using standard ASTM methodology.Chlorella sp. MP-1 biomass shows low ash (5.93%), whereas high energy (18.59 MJ/kg), carbohydrate(19.46%), and lipid (28.82%) content. The algal de-oiled cake was characterized by FTIR spectroscopyand thermogravimetric study at 10 �C/min and 30 �C/min to investigate its feasibility for thermo-chem-ical conversion. The present investigation suggests that within the realm of biomass energy technologiesthe algal biomass can be used as feedstock for bio and thermo-chemical whereas the de-oiled cake forthermo-chemical conversion thereby serving the demand of second generation biofuels.

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1. Introduction

As conventional energy sources across the globe are fast deplet-ing, unless the renewable and non-conventional energy sources aretapped, coupled with prudent use and management of energy,humanity is bound to engender a horrific specter of a global energyvacuum. The quest for renewable energy has geared up and one ofthe facets with great potential for satisfying mankind’s primary en-ergy demand is energy derived from biomass. The present centuryhas witnessed major emphasis on the use of biomass as an alterna-tive to fossil fuels due to its renewable nature and reduced CO2

emissions. Biomass energy sources are amongst the most promis-ing, most hyped and most heavily subsidized renewable energysources. Biomass can be sustainable, environmentally benign andeconomically sound. It can provide heat, power and transportationfuels in an environmentally friendly manner, by reducing greenhouse gas emissions and thus can aid in achieving renewable en-ergy targets.

A major insight into the search operation for new sources ofbiomass energy can be offered by microalgae. The generic termmicroalgae refer to a large group of very diverse photosyntheticmicro-organisms of microscopic dimensions. They are sunlight dri-ven oil factories which convert carbon dioxide into potential biofu-els, feeds, foods and high value bioactives [1–9]. Algae are the mostefficient biological producer of oil on the planet and a versatile

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biomass source and may soon be one of the Earths most importantrenewable fuel crops [10]. Microalgae are a major natural sourcefor an enormous array of valuable compounds, including a diver-sity of pigments, for which these photosynthetic micro-organismsrepresent an almost exclusive biological resource [11]. The poten-tial of microalgae as the most efficient primary producer of bio-mass still requires comprehensive understanding, but there islittle doubt that they will eventually become one of the mostimportant renewable energy sources. The use of microalgae asbio-energy feedstock seems to be promising because:

(1) Biomass doubling times in microalgae during exponentialgrowth are commonly as short as 3.5 h [13].

(2) Due to their simple cellular structure, algae have higherrates of biomass and oil production than conventional crops[12]. Oil content in microalgae can exceed 80% by weight ofdry biomass [13].

(3) Their lipid content could be adjusted through alteringgrowth media composition [14].

(4) Salty or waste water can be used for the culture of microal-gae [15].

(5) Can be harvested batch-wise nearly all-year-around provid-ing a reliable and continuous supply of oil [15].

(6) Atmospheric carbon dioxide is the source of carbon for thegrowth of microalgae [15].

(7) Depending on species microalgae produce many kinds of lip-ids, hydrocarbons and other complex oils [5,16,17].

(8) Algae can produce 30–100 times more energy per hectare ascompared to terrestrial crops [18].

Table 1Taxonomic position of Chlorella [20].

Systematic position Fritsch (1935) Bold and Wyne (1978)

Division ChlorophycophytaClass Chlophyceae ChlorophyceaeOrder Chlorococcales ChlorellalesFamily Chlorellaceae ChlorellaceaeGenus Chlorella Chlorella

Table 2Media composition used for culturing Chlorella sp. MP-1 (g L�1).

Composition Modified Chu 13 BBM BG11 Basal

3308 M.M. Phukan et al. / Applied Energy 88 (2011) 3307–3312

(9) The production of biofuels from algae can be coupled withflue gas CO2 mitigation, waste water treatment and produc-tion of high value chemicals [18].

The potential value of any biomass depends on the chemicaland physical properties of molecules from which it is made. Thiswork is an endeavor to study the biomass properties of Chlorellaspp. as several characteristics affect the performance of biomassfuel including the calorific value, moisture content and physico-chemical properties and to evaluate the potential of the above asfeedstock for the production of biofuel basing on their chemicaland physical characteristics. The study also investigates the feasi-bility of algal de-oiled cake for thermo-chemical conversion.

KNO3 200 – – 100NaNO3 – 250 1500 –K2HPO4 40 74 40 –KH2PO4 – 17.5 – –CaCl2�2H2O 80 24 36 –MgSO4�7H2O 100 73 75 40Na2CO3 – – 20 –NaCl – 25 – –FeSO4 – 5 – –EDTA – 45 –Citric acid 100 – 6 –Ammonium ferric citrate – 6Ferric citrate 10 – – –Ca(NO3)2�4H2O – – – 150b-Na2glyserophosphate – – – 50EDTA-Na2 – – 1 2.71Vitamin B12 – – – 0.0001Biotin – – – 0.0001Thiamine-HCL – – – 0.01H3BO3 – – 2.86 –MnCl2�4H2O – – 1.81 0.108ZnSO4�7H2O – – 0.22 0.066Na2MoO4�2H2O – – 0.39 0.0075CuSO4�5H2O – – 0.08 –Co(NO3)2�6H2O – – 0.05 –FeCl3�6H2O – – – 5.888CoCl2�6H2O – – – 0.012Trisaminomethane – – – 500

2. Description of the species

Chlorella Beijerinck (Gr. Chloros, green; ella, diminutive). Thetaxonomic position of Chlorella is depicted in Table 1. Chlorella isa single celled, spherical non-motile green alga 2.0–10.0 lm indiameter. Chlorella occurs in both fresh and marine water. Somecall Chlorella ubiquitous since it occurs in various different habi-tats. They are generally found in fresh water of ponds and ditches,in moist soil or other damp situations such as the surface of treetrunks, water pots and damp walls. Chlorella parasitca is foundsymbiotically in the cells of Paramecium and Hydra. Chlorella vul-garis, Chlorella conductrix, Chlorella gonglomerata and C. parasitcaare the common Indian species. Chlorella is represented by onlyeight species [19].

Its cells are solitary, very small (2.0–10.0 lm) and spherical,globular or ellipsoidal in shape. The cells are surrounded by a thincellulose wall, which encloses a parietal and cup shaped chloro-plast with a pyrenoid. In certain species the pyrenoids are absent.The cells are devoid of flagella, stigma and contractile vacuoles, butcontain a centrally located nucleus. When dried it is about 20% fat,45% protein, 20% carbohydrate, 10% various minerals and vitamins[3].

Chlorella is of immense economic importance ranging from hu-man food to applications in space travel. Bio-energy generationfrom Chlorella is a new facet in renewable energy research. Illmanet al. [21] had studied calorific values of Chlorella strains grown inlow nitrogen medium including four fresh water strains (Chlorellaprotothecoides, C. vulgaris, Chlorella emersonii and Chlorella sorokini-ana) and one marine strain (Chlorella minutissima) and suggestedChlorella strains may be suitable for diesel replacements. Scragget al. [22] successfully used an emulsion consisting of transesteri-fied rape seed oil, a surfactant and slurry of C. vulgaris in anunmodified single cylinder diesel engine. Xu et al. [23] obtainedhigh quality biodiesel production from heterotrophic microalgaeC. protothecoides.

3. Materials and methods

3.1. Micro-organism and growth medium

Chlorella sp. MP-1 isolated from water samples collected fromLake Joysagar, one of the largest manmade lake, Sibsagar, Assam,India (26�5701200N and 94�3703400E) was used in this study. Stockcultures of Chlorella sp. MP-1 were maintained routinely on bothliquid and agar slants of BG-11 media [24] by regular sub-culturingat 15 days interval. The species under investigation was also cul-tured in BBM [25], modified Chu-13 and [26] and Basal [24] mediato estimate the biomass yield in the respective media. The compo-sition of the various culture media is presented in Table 2.

3.2. Isolation and purification

The microalgae were subjected to purification by serial dilutionfollowed by streaking. The individual colonies were isolated andinoculated into liquid medium (BG-11). The purity of the culturewas established by repeated streaking and routine microscopicexamination.

3.3. Culture conditions

The microalgal cultures were carried out in 500 ml Erlenmeyerflasks with shaking at 90 rpm; at 26 ± 2 �C, light intensity 1200 lux;(16:8) light and dark cycle; and inoculation at 10% (v/v).

3.4. Growth evaluation

The growth of Chlorella sp. MP-1 was monitored spectrophoto-metrically (Beckman DU530) by reading the culture absorbance at682 nm.

3.5. Biomass estimation

The cultures were harvested by centrifugation at 7000 rpm for15 min. The cells were washed twice with distilled water after cen-trifugation. The pellet was dried at 80 �C for 24 h. The dry weight ofthe algal biomass was determined gravimetrically and growth wasexpressed in terms of dry weight.

M.M. Phukan et al. / Applied Energy 88 (2011) 3307–3312 3309

3.6. Determination of Gross calorific value (GCV)

Calorific value (CV) was determined using an automaticadiabatic bomb calorimeter (Changsha Kaiyuan Instruments Co.,5E-1AC/ML). The sample (dried algal pellet) was oxidized bycombustion in an adiabatic bomb containing 3.4 Mpsi oxygenunder pressure. The assays were carried out in triplicates and themean values are reported.

3.7. Determination of Net calorific value (NCV)

The NCV was calculated from the following equation [27]:

NCV ¼ GCV� 1� w100

� �� 2:444� w

100

� �� 2:444

H100

� �� 8:936

� 1� w100

� �; MJ=kg;w:b:ð Þ

where 2.444 = Enthalpy difference between gaseous and liquidwater at 25 �C.

8:936 ¼ MH2O

MH2

; i:e: the molecular mass relation between

H2O and H2:

where NCV is the Net calorific value, GCV is the Gross calorific va-lue, h is the concentration of hydrogen in weight%, w is the Moisturecontent of the fuel in weight%.

3.8. CHN analysis

C, H, N analysis was carried out in a CHN analyzer and the oxy-gen content was calculated by difference.

3.9. Proximate analysis

The moisture, volatile matter and ash content of the dry algalbiomass were determined according to ASTM D 3173, ASTM D3175 and ASTM D 3174 protocols. Finally the fixed carbon contentwas calculated by difference.

3.10. Carbohydrate estimation

The anthrone method was used for the determination of totalcarbohydrates [28]. A calibration curve was prepared using D+ glu-cose dissolved in distilled water. The glucose concentration (Cglc,

mg/ml) and optical density had the following relationship:

Cglc ¼1

6:165ðOD630nm þ 0:015Þ

3.11. Protein estimation

Ten milliliter algal culture was centrifuged at 6000 rpm for10 min. The cell pellet was re-suspended in 5 ml of 1 M NaOHand boiled for 10 min. The protein content was determined usingthe method of Lowry [29]. A calibration curve was prepared usingBSA dissolved in distilled water. The BSA concentration (Cbsa,

mg/ml) and optical density had the following relationship:

Cbsa ¼1

0:005ðOD660nm þ 0:001Þ

3.12. Determination of total lipids

Total lipid was determined using the method of Bligh and Dyer[30].

3.13. FTIR analysis

The IR spectrum of dried algal biomass was recorded on NicoletIR spectrometer at room temperature. The dried algal powder wasblended with potassium bromide (KBr) powder, and pressed intotablets before measurement. A region of 4000–400 cm�1 was usedfor scanning.

3.14. Thermal analysis

For thermal analysis microalgae from late exponential phasewere harvested by centrifugation at 7000 rpm for 15 min. The pel-let was washed twice with distilled water and then dried at 80 �Cfor 24 h. The samples were pulverized in a mortar to fine particlesin order to eliminate heat transfer effects during pyrolysis and thenfinally stored in a desiccator. Thermogravimetric analysis (TGA)was done in order to study the combustion behavior of algal bio-mass. Algal biomass and de-oiled cake was subjected to thermo-gravimetric analysis in nitrogen atmosphere at heating rates of10 �C/min and 30 �C/min. Sample weighing approximately 10 mgwas heated at the preselected heating rate from ambient tempera-ture to 750 �C in a Pyris diamond TG/DT analyzer (PERKIN ELMER).A high purity nitrogen gas (99.99%) was fed at a constant flow rateof 100 ml/min as an inert purge gas to displace air in the pyrolyticzone, thereby avoiding unwanted oxidation of the sample. Thecontinuous on-line records of weight loss and temperature wereobtained to plot the TGA curve and the derivative thermogravimet-ric analysis (DTG) curves.

4. Results and discussions

The successful implementation of algal biomass as a potentialbio-energy feedstock is largely governed by the quantum of pro-ducible biomass. Therefore enhancement of the growth rate of al-gae in terms of biomass productivity is one of the most importantparameters. The growth of Chlorella sp. MP-1 was tested in four dif-ferent culture media namely, BG-11, Basal, BBM and modified Chu-13 media. Among all the tested media the highest biomass yield of824 mg/l was obtained in BG-11 media. The study only takes intoaccount BG-11 media with the highest gross biomass yield for cul-turing Chlorella sp. MP-1 and subsequent investigations. The bio-mass yield of Chlorella sp. MP-1 in different culture media isshown in Fig. 1.

The characterization of algal biomass as an energy source is ta-ken into consideration in the present investigation. Table 3 showsthe biomass properties of Chlorella sp. MP-1 with their averagecharacteristic composition. The moisture content of biomass differsignificantly, depending on the type of biomass and biomass stor-age. Moisture content is of great importance with regard to selec-tion of biomass conversion technology. Biomass fuels with lowmoisture content are more suited for thermal conversion technol-ogy while those with high moisture content are more suited forbiochemical process such as fermentation conversion [31]. On thisbasis Chlorella sp. MP-1 with moisture content of 6.8% seems to bea potential candidate for direct thermo-chemical conversion.

Ash content of biomass affects both the handling and processingcosts of overall biomass energy conversion. The ash content in mic-roalgae may vary considerably in different species and also withgeographical location and season. In this investigation ash valuewas determined using ASTM D 3174 protocol. The characterizationof ash was not done and is a part of planned future research activ-ity. The ash content was found to be 5.93% and volatile matter con-tent was 72.19%. The high amount of volatile matter in Chlorella sp.MP-1 biomass strongly influences its combustion behavior andthermal decomposition. The fixed carbon content was 15.08%.

Fig. 1. Biomass yield of cake. Chlorella sp. MP-1 in various culture media.

Table 3Properties of Chlorella sp. MP-1.

Properties Chlorella spp.

Gross calorific value (MJ/Kg) 18.59 ± 0.42Net calorific value (MJ/Kg) 15.88 ± 0.39Empirical formulae (on ash free basis) C8.25H14.79NO5.02

H/C molar ratio (on ash free basis) 1.79O/C molar ratio (on ash free basis) 0.6

Elemental analysis (wt.%) Carbon 47.54Hydrogen 7.1Nitrogen 6.73Oxygen (by difference) 38.63

Proximate analysis (wt.%) Moisture 6.8 ± 1.11Volatile matter 72.19 ± 1.73Fixed carbon 15.08 ± 1.21Ash 5.93 ± 0.81

Biochemical analysis Total carbohydrate 9.46 ± 0.25Protein content 43.22 ± 0.33Lipid content 28.82 ± 0.72

Fig. 2. FTIR spectra of Chlorella sp. MP-1 biomass and deoiled cake.

Fig. 3. TG-DTG of Chlorella sp. MP-1 biomass at heating rate 10 �C and 30 �C.

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The biomass had high percentage of volatile matter and low ashcontent which is important with respect to their application in gas-ification and pyrolysis process.

The elemental content of carbon, hydrogen, oxygen and nitro-gen in the algal biomass was 47.54%, 7.1%, 38.63% and 6.73%respectively. The empirical formula of the algal biomass isC8.25H14.79NO5.02. The H/C and O/C molar ratios (on an ash freedry basis) were calculated from elemental composition as 1.79and 0.6 respectively. The Gross calorific value (GCV) and Net calo-rific value (NCV) for Chlorella sp. MP-1 biomass was 18.59 MJ/kgand 15.88 MJ/kg respectively. The Gross calorific value in theexamined sample was higher than 18 MJ/kg, as reported by Illmanet al. [21]. An increase in calorific value in algae is linked to in-crease in lipid content rather than any change in other cell compo-nents such as carbohydrates and proteins [21].

The FTIR spectrum of Chlorella sp. MP-1 biomass and de-oiledcake is shown in Fig. 2. The region 3300–3000 cm�1 is characteris-tic for CAH stretching vibrations of C„C, C@C and ArAH, whereasthe region from 3000 to 2700 cm�1 is dominated by CAH stretch-ing vibrations of ACH3, >CH2, CH and CHO functional groups,respectively [32,33]. The olephinic CAH stretching vibration be-tween 3600 and 3300 cm�1 indicates unsaturate. The absorptionat 1652 cm�1 implies the presence of C@O of carboxylic acid andderivatives. The region between 1800 and 1500 cm�1 demonstratecharacteristic bands for proteins, whereas 1700–1600 cm�1 is

specific for amide-I bands [33], which is mainly due to C@Ostretching vibrations of peptide bond [34]. The bands in the amideI region provide insight into the protein secondary structure [35].On the other hand the region from 1600 to 1500 cm�1 is specificfor amide-II bands, which is due to NAH bending vibrations [36].The region from 1200 to 900 cm�1 signifies a sequence of bandsdue to CAO, CAC, CAOAC and CAOAP stretching vibrations ofpolysaccharides [37,38] as well as CH3, CH2 rocking modes [39].CH2 stretching vibrations in the range of 3100–2800 cm�1 impliesthe presence of lipid [40]. The absorption at 2928 and 2860 cm�1

implies CH2 asymmetric and symmetric stretching in lipid.All the above mentioned bonds which were prominent in the al-

gal biomass showed progressive degradation in their intensity inthe spectra of the de-oiled cake. There was a general decrease inprotein and carbohydrate content indicated by a decrease in theintensity of absorption bands in the 1800–800 cm�1 region. Thisregion is specific for proteins and carbohydrates [33]. Declinationin the intensity of absorption in the range of 3100–2800 cm�1 isindicative of decrease in the lipid content.

As shown in Fig. 3 the TG-DTG profile of Chlorella sp. MP-1 bio-mass reveals an initial weight loss between ambient temperatureand about 130 �C and 160 �C for 10 and 30 �C/min. This couldpossibly be due to elimination of physically absorbed water inthe biomass and due to external or superficial water bounded bysurface tension. This was followed by continuous decrease in

Fig. 4. TG-DTG of Chlorella sp. MP-1 de-oiled cake at heating rate 10 �C and 30 �C.

M.M. Phukan et al. / Applied Energy 88 (2011) 3307–3312 3311

sample weight (where main degradation occurred) which ended byapproximately 380–390 �C for the lower heating rate and 410–435 �C for the higher heating rate. These zones (130–390 �C) and(160–435 �C) has been referred to as the zone of active pyrolysis.For the two heating rates significant changes in the slope of thethermogram were observed at around 400 �C and 440 �C, whichindicated the initiation of the passive pyrolysis zone that termi-nated at around 525 �C for the former and 650 �C for the later. Avery slow loss of weight occurred until 750 �C which indicates thatthere was further reaction involving char. This implies that themain pyrolysis reactions occurred between 160–525 �C and 160–650 �C for the stated heating rates.

The temperature range for the active pyrolysis zone as depictedin Fig. 3 corresponds to the findings of Shuping et al. [41]. The anal-ysis of the thermogram shows that during the main pyrolysis pro-cess, only one strong peak and henceforth only one decompositionprocess corresponding to the degradation of crude protein was ob-served [41]. Microalgae contains very high amount of proteins,43.22% in the case of Chlorella sp. MP-1 and therefore the majordegradation corresponds to protein.

As shown in Fig. 4 the TG-DTG profile of Chlorella sp. MP-1 de-oiled cake reveals an initial slight weight loss between ambienttemperature and about 110 �C for 10 and 30 �C/min. This couldpossibly be due to moisture evolution. This was followed by con-tinuous decrease in sample weight (where main degradation oc-curred) which ended by approximately 330–340 �C for the lowerheating rate and 350–365 �C for the higher heating rate. No majorobservable difference was noticed in the thermogram of the de-oiled cake at the two heating rates. The degradation of de-oiledcake terminated earlier than algal biomass due to loss of crude cellcomponents in the lipid extraction procedure. The shorter thermaldegradation profile of the algal de-oiled cake suggests that it can bean ideal feedstock for thermo-chemical conversion.

5. Conclusion

Microalgae Chlorella sp. MP-1 exhibits several important attri-butes for futuristic research on renewable energy. With simpleand inexpensive nutrient regime to culture, faster growth rate ascompared to terrestrial energy crops, high biomass productivity,attractive biochemical profile and good energy content (18.59 MJ/kg) Chlorella sp. MP-1 offers strong candidature as a bioenergysource. The robust nature of pyrolysis technology can be efficientlyapplied to algal de-oiled cakes for the co-production of biochar

with bio-oil. The characterization of Chlorella biomass in light ofbioenergy production ensures that it can be, used as renewablefeedstock for biochemical and thermo-chemical conversion andmay serve the demands of second generation biofuels. Moreoverthe production of these biofuels from microalgae can be coupledwith flue gas CO2 mitigation, waste water treatment and produc-tion of high value chemicals. The potential of energy productionfrom Chlorella is vast and necessitates further research.

Acknowledgements

The first author would like to offer his sincere thanks to theONGC, Jorhat, India for providing fund in the form of a fellowshipproject. The authors are also thankful to Dr. J.R. Chetia and S.Banerjee for TGA analysis.

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