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Microalgae Chlorella as a potential bio-energy feedstock
Applied Energy 88 (2011) 3307–3312
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Applied Energy journal homepage: www.elsevier.com/locate/apenergy
Microalgae Chlorella as a potential bio-energy feedstock Mayur M. Phukan a, Rahul S. Chutia b, B.K. Konwar a,⇑, R. Kataki b a b
Department of Molecular Biology & Biotechnology, School of Science & Technology, Tezpur University, Assam, Napaam 784 028, India 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
Article history: Received 15 September 2010 Received in revised form 1 November 2010 Accepted 18 November 2010 Available online 6 January 2011 Keywords: Microalgae Biomass Thermogravimetry Chlorella Biofuel
a b s t r a c t Microalgae are promising biomass species owing to their fast growth rate and high CO2 fixation ability as compared to terrestrial plants. Microalgae have long been recognized as potentially good source for biofuel 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 spectroscopy and thermogravimetric study at 10 °C/min and 30 °C/min to investigate its feasibility for thermo-chemical conversion. The present investigation suggests that within the realm of biomass energy technologies the algal biomass can be used as feedstock for bio and thermo-chemical whereas the de-oiled cake for thermo-chemical conversion thereby serving the demand of second generation biofuels. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction As conventional energy sources across the globe are fast depleting, unless the renewable and non-conventional energy sources are tapped, coupled with prudent use and management of energy, humanity is bound to engender a horrific specter of a global energy vacuum. The quest for renewable energy has geared up and one of the facets with great potential for satisfying mankind’s primary energy demand is energy derived from biomass. The present century has witnessed major emphasis on the use of biomass as an alternative to fossil fuels due to its renewable nature and reduced CO2 emissions. Biomass energy sources are amongst the most promising, most hyped and most heavily subsidized renewable energy sources. Biomass can be sustainable, environmentally benign and economically sound. It can provide heat, power and transportation fuels in an environmentally friendly manner, by reducing green house gas emissions and thus can aid in achieving renewable energy targets. A major insight into the search operation for new sources of biomass energy can be offered by microalgae. The generic term microalgae refer to a large group of very diverse photosynthetic micro-organisms of microscopic dimensions. They are sunlight driven oil factories which convert carbon dioxide into potential biofuels, feeds, foods and high value bioactives [1–9]. Algae are the most efficient biological producer of oil on the planet and a versatile
⇑ Corresponding author. Tel.: +91 9954449458; fax: +91 371 2267005/6. E-mail address: [email protected] (B.K. Konwar). 0306-2619/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apenergy.2010.11.026
biomass source and may soon be one of the Earths most important renewable fuel crops [10]. Microalgae are a major natural source for an enormous array of valuable compounds, including a diversity of pigments, for which these photosynthetic micro-organisms represent an almost exclusive biological resource [11]. The potential of microalgae as the most efficient primary producer of biomass still requires comprehensive understanding, but there is little doubt that they will eventually become one of the most important renewable energy sources. The use of microalgae as bio-energy feedstock seems to be promising because: (1) Biomass doubling times in microalgae during exponential growth are commonly as short as 3.5 h [13]. (2) Due to their simple cellular structure, algae have higher rates of biomass and oil production than conventional crops [12]. Oil content in microalgae can exceed 80% by weight of dry biomass [13]. (3) Their lipid content could be adjusted through altering growth media composition [14]. (4) Salty or waste water can be used for the culture of microalgae [15]. (5) Can be harvested batch-wise nearly all-year-around providing a reliable and continuous supply of oil [15]. (6) Atmospheric carbon dioxide is the source of carbon for the growth of microalgae [15]. (7) Depending on species microalgae produce many kinds of lipids, hydrocarbons and other complex oils [5,16,17]. (8) Algae can produce 30–100 times more energy per hectare as compared to terrestrial crops [18].
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(9) The production of biofuels from algae can be coupled with flue gas CO2 mitigation, waste water treatment and production of high value chemicals [18]. The potential value of any biomass depends on the chemical and physical properties of molecules from which it is made. This work is an endeavor to study the biomass properties of Chlorella spp. as several characteristics affect the performance of biomass fuel including the calorific value, moisture content and physicochemical properties and to evaluate the potential of the above as feedstock for the production of biofuel basing on their chemical and physical characteristics. The study also investigates the feasibility of algal de-oiled cake for thermo-chemical conversion.
2. Description of the species Chlorella Beijerinck (Gr. Chloros, green; ella, diminutive). The taxonomic position of Chlorella is depicted in Table 1. Chlorella is a single celled, spherical non-motile green alga 2.0–10.0 lm in diameter. Chlorella occurs in both fresh and marine water. Some call Chlorella ubiquitous since it occurs in various different habitats. They are generally found in fresh water of ponds and ditches, in moist soil or other damp situations such as the surface of tree trunks, water pots and damp walls. Chlorella parasitca is found symbiotically in the cells of Paramecium and Hydra. Chlorella vulgaris, Chlorella conductrix, Chlorella gonglomerata and C. parasitca are the common Indian species. Chlorella is represented by only eight 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 thin cellulose wall, which encloses a parietal and cup shaped chloroplast with a pyrenoid. In certain species the pyrenoids are absent. The cells are devoid of flagella, stigma and contractile vacuoles, but contain 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 human food to applications in space travel. Bio-energy generation from Chlorella is a new facet in renewable energy research. Illman et al. [21] had studied calorific values of Chlorella strains grown in low nitrogen medium including four fresh water strains (Chlorella protothecoides, C. vulgaris, Chlorella emersonii and Chlorella sorokiniana) and one marine strain (Chlorella minutissima) and suggested Chlorella strains may be suitable for diesel replacements. Scragg et al. [22] successfully used an emulsion consisting of transesterified rape seed oil, a surfactant and slurry of C. vulgaris in an unmodified single cylinder diesel engine. Xu et al. [23] obtained high quality biodiesel production from heterotrophic microalgae C. protothecoides.
Table 1 Taxonomic position of Chlorella [20]. Systematic position
Fritsch (1935)
Bold and Wyne (1978)
Division Class Order Family Genus
Chlophyceae Chlorococcales Chlorellaceae Chlorella
Chlorophycophyta Chlorophyceae Chlorellales Chlorellaceae Chlorella
Table 2 Media composition used for culturing Chlorella sp. MP-1 (g L1). Composition
Modified Chu 13
BBM
BG11
Basal
KNO3 NaNO3 K2HPO4 KH2PO4 CaCl22H2O MgSO47H2O Na2CO3 NaCl FeSO4 EDTA Citric acid Ammonium ferric citrate Ferric citrate Ca(NO3)24H2O b-Na2glyserophosphate EDTA-Na2 Vitamin B12 Biotin Thiamine-HCL H3BO3 MnCl24H2O ZnSO47H2O Na2MoO42H2O CuSO45H2O Co(NO3)26H2O FeCl36H2O CoCl26H2O Trisaminomethane
200 – 40 – 80 100 – – – – 100 – 10 – – – – – – – – – – – – – – –
– 250 74 17.5 24 73 – 25 5 45 –
– 1500 40 – 36 75 20 – –
100 – – – – 40 – – – – –
– – – – – – – – – – – – – – – –
6 6 – – – 1 – – – 2.86 1.81 0.22 0.39 0.08 0.05 – – –
– 150 50 2.71 0.0001 0.0001 0.01 – 0.108 0.066 0.0075 – – 5.888 0.012 500
3.2. Isolation and purification The microalgae were subjected to purification by serial dilution followed by streaking. The individual colonies were isolated and inoculated into liquid medium (BG-11). The purity of the culture was established by repeated streaking and routine microscopic examination. 3.3. Culture conditions The microalgal cultures were carried out in 500 ml Erlenmeyer flasks 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. Materials and methods
3.4. Growth evaluation
3.1. Micro-organism and growth medium
The growth of Chlorella sp. MP-1 was monitored spectrophotometrically (Beckman DU530) by reading the culture absorbance at 682 nm.
Chlorella sp. MP-1 isolated from water samples collected from Lake Joysagar, one of the largest manmade lake, Sibsagar, Assam, India (26°570 1200 N and 94°370 3400 E) was used in this study. Stock cultures of Chlorella sp. MP-1 were maintained routinely on both liquid and agar slants of BG-11 media [24] by regular sub-culturing at 15 days interval. The species under investigation was also cultured in BBM [25], modified Chu-13 and [26] and Basal [24] media to estimate the biomass yield in the respective media. The composition of the various culture media is presented in Table 2.
3.5. Biomass estimation The cultures were harvested by centrifugation at 7000 rpm for 15 min. The cells were washed twice with distilled water after centrifugation. The pellet was dried at 80 °C for 24 h. The dry weight of the algal biomass was determined gravimetrically and growth was expressed in terms of dry weight.
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3.6. Determination of Gross calorific value (GCV)
3.13. FTIR analysis
Calorific value (CV) was determined using an automatic adiabatic bomb calorimeter (Changsha Kaiyuan Instruments Co., 5E-1AC/ML). The sample (dried algal pellet) was oxidized by combustion in an adiabatic bomb containing 3.4 Mpsi oxygen under pressure. The assays were carried out in triplicates and the mean values are reported.
The IR spectrum of dried algal biomass was recorded on Nicolet IR spectrometer at room temperature. The dried algal powder was blended with potassium bromide (KBr) powder, and pressed into tablets before measurement. A region of 4000–400 cm1 was used for scanning.
3.7. Determination of Net calorific value (NCV) The NCV was calculated from the following equation [27]:
w w H 8:936 NCV ¼ GCV 1 2:444 2:444 100 100 100 w 1 ; ðMJ=kg; w:b:Þ 100 where 2.444 = Enthalpy difference between gaseous and liquid water at 25 °C.
8:936 ¼
M H2 O ; i:e: the molecular mass relation between M H2
H2 O and H2 : where NCV is the Net calorific value, GCV is the Gross calorific value, h is the concentration of hydrogen in weight%, w is the Moisture content of the fuel in weight%. 3.8. CHN analysis C, H, N analysis was carried out in a CHN analyzer and the oxygen content was calculated by difference. 3.9. Proximate analysis The moisture, volatile matter and ash content of the dry algal biomass were determined according to ASTM D 3173, ASTM D 3175 and ASTM D 3174 protocols. Finally the fixed carbon content was calculated by difference. 3.10. Carbohydrate estimation The anthrone method was used for the determination of total carbohydrates [28]. A calibration curve was prepared using D+ glucose dissolved in distilled water. The glucose concentration (Cglc, mg/ml) and optical density had the following relationship:
C glc ¼
1 ðOD630nm þ 0:015Þ 6:165
3.11. Protein estimation Ten milliliter algal culture was centrifuged at 6000 rpm for 10 min. The cell pellet was re-suspended in 5 ml of 1 M NaOH and boiled for 10 min. The protein content was determined using the method of Lowry [29]. A calibration curve was prepared using BSA dissolved in distilled water. The BSA concentration (Cbsa, mg/ml) and optical density had the following relationship:
C bsa ¼
1 ðOD660nm þ 0:001Þ 0:005
3.12. Determination of total lipids Total lipid was determined using the method of Bligh and Dyer [30].
3.14. Thermal analysis For thermal analysis microalgae from late exponential phase were harvested by centrifugation at 7000 rpm for 15 min. The pellet was washed twice with distilled water and then dried at 80 °C for 24 h. The samples were pulverized in a mortar to fine particles in order to eliminate heat transfer effects during pyrolysis and then finally stored in a desiccator. Thermogravimetric analysis (TGA) was done in order to study the combustion behavior of algal biomass. Algal biomass and de-oiled cake was subjected to thermogravimetric analysis in nitrogen atmosphere at heating rates of 10 °C/min and 30 °C/min. Sample weighing approximately 10 mg was heated at the preselected heating rate from ambient temperature 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 rate of 100 ml/min as an inert purge gas to displace air in the pyrolytic zone, thereby avoiding unwanted oxidation of the sample. The continuous on-line records of weight loss and temperature were obtained to plot the TGA curve and the derivative thermogravimetric analysis (DTG) curves. 4. Results and discussions The successful implementation of algal biomass as a potential bio-energy feedstock is largely governed by the quantum of producible biomass. Therefore enhancement of the growth rate of algae in terms of biomass productivity is one of the most important parameters. The growth of Chlorella sp. MP-1 was tested in four different culture media namely, BG-11, Basal, BBM and modified Chu13 media. Among all the tested media the highest biomass yield of 824 mg/l was obtained in BG-11 media. The study only takes into account BG-11 media with the highest gross biomass yield for culturing Chlorella sp. MP-1 and subsequent investigations. The biomass yield of Chlorella sp. MP-1 in different culture media is shown in Fig. 1. The characterization of algal biomass as an energy source is taken into consideration in the present investigation. Table 3 shows the biomass properties of Chlorella sp. MP-1 with their average characteristic composition. The moisture content of biomass differ significantly, depending on the type of biomass and biomass storage. Moisture content is of great importance with regard to selection of biomass conversion technology. Biomass fuels with low moisture content are more suited for thermal conversion technology while those with high moisture content are more suited for biochemical process such as fermentation conversion [31]. On this basis Chlorella sp. MP-1 with moisture content of 6.8% seems to be a potential candidate for direct thermo-chemical conversion. Ash content of biomass affects both the handling and processing costs of overall biomass energy conversion. The ash content in microalgae may vary considerably in different species and also with geographical location and season. In this investigation ash value was determined using ASTM D 3174 protocol. The characterization of ash was not done and is a part of planned future research activity. The ash content was found to be 5.93% and volatile matter content was 72.19%. The high amount of volatile matter in Chlorella sp. MP-1 biomass strongly influences its combustion behavior and thermal decomposition. The fixed carbon content was 15.08%.
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Fig. 1. Biomass yield of cake. Chlorella sp. MP-1 in various culture media.
Table 3 Properties of Chlorella sp. MP-1. Properties
Chlorella spp.
Gross calorific value (MJ/Kg) Net calorific value (MJ/Kg) Empirical formulae (on ash free basis) H/C molar ratio (on ash free basis) O/C molar ratio (on ash free basis)
18.59 ± 0.42 15.88 ± 0.39 C8.25H14.79NO5.02 1.79 0.6
Elemental analysis (wt.%)
Carbon Hydrogen Nitrogen Oxygen (by difference)
47.54 7.1 6.73 38.63
Proximate analysis (wt.%)
Moisture Volatile matter Fixed carbon Ash
6.8 ± 1.11 72.19 ± 1.73 15.08 ± 1.21 5.93 ± 0.81
Biochemical analysis
Total carbohydrate Protein content Lipid content
9.46 ± 0.25 43.22 ± 0.33 28.82 ± 0.72
The biomass had high percentage of volatile matter and low ash content which is important with respect to their application in gasification and pyrolysis process. The elemental content of carbon, hydrogen, oxygen and nitrogen in the algal biomass was 47.54%, 7.1%, 38.63% and 6.73% respectively. The empirical formula of the algal biomass is C8.25H14.79NO5.02. The H/C and O/C molar ratios (on an ash free dry basis) were calculated from elemental composition as 1.79 and 0.6 respectively. The Gross calorific value (GCV) and Net calorific value (NCV) for Chlorella sp. MP-1 biomass was 18.59 MJ/kg and 15.88 MJ/kg respectively. The Gross calorific value in the examined sample was higher than 18 MJ/kg, as reported by Illman et al. [21]. An increase in calorific value in algae is linked to increase in lipid content rather than any change in other cell components such as carbohydrates and proteins [21]. The FTIR spectrum of Chlorella sp. MP-1 biomass and de-oiled cake is shown in Fig. 2. The region 3300–3000 cm1 is characteristic for CAH stretching vibrations of C„C, C@C and ArAH, whereas the region from 3000 to 2700 cm1 is dominated by CAH stretching vibrations of ACH3, >CH2, CH and CHO functional groups, respectively [32,33]. The olephinic CAH stretching vibration between 3600 and 3300 cm1 indicates unsaturate. The absorption at 1652 cm1 implies the presence of C@O of carboxylic acid and derivatives. The region between 1800 and 1500 cm1 demonstrate characteristic bands for proteins, whereas 1700–1600 cm1 is
Fig. 2. FTIR spectra of Chlorella sp. MP-1 biomass and deoiled cake.
specific for amide-I bands [33], which is mainly due to C@O stretching vibrations of peptide bond [34]. The bands in the amide I region provide insight into the protein secondary structure [35]. On the other hand the region from 1600 to 1500 cm1 is specific for amide-II bands, which is due to NAH bending vibrations [36]. The region from 1200 to 900 cm1 signifies a sequence of bands due to CAO, CAC, CAOAC and CAOAP stretching vibrations of polysaccharides [37,38] as well as CH3, CH2 rocking modes [39]. CH2 stretching vibrations in the range of 3100–2800 cm1 implies the presence of lipid [40]. The absorption at 2928 and 2860 cm1 implies CH2 asymmetric and symmetric stretching in lipid. All the above mentioned bonds which were prominent in the algal biomass showed progressive degradation in their intensity in the spectra of the de-oiled cake. There was a general decrease in protein and carbohydrate content indicated by a decrease in the intensity of absorption bands in the 1800–800 cm1 region. This region is specific for proteins and carbohydrates [33]. Declination in the intensity of absorption in the range of 3100–2800 cm1 is indicative of decrease in the lipid content. As shown in Fig. 3 the TG-DTG profile of Chlorella sp. MP-1 biomass reveals an initial weight loss between ambient temperature and about 130 °C and 160 °C for 10 and 30 °C/min. This could possibly be due to elimination of physically absorbed water in the biomass and due to external or superficial water bounded by surface tension. This was followed by continuous decrease in
Fig. 3. TG-DTG of Chlorella sp. MP-1 biomass at heating rate 10 °C and 30 °C.
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with bio-oil. The characterization of Chlorella biomass in light of bioenergy production ensures that it can be, used as renewable feedstock for biochemical and thermo-chemical conversion and may serve the demands of second generation biofuels. Moreover the production of these biofuels from microalgae can be coupled with flue gas CO2 mitigation, waste water treatment and production of high value chemicals. The potential of energy production from Chlorella is vast and necessitates further research. Acknowledgements The first author would like to offer his sincere thanks to the ONGC, Jorhat, India for providing fund in the form of a fellowship project. The authors are also thankful to Dr. J.R. Chetia and S. Banerjee for TGA analysis. References Fig. 4. TG-DTG of Chlorella sp. MP-1 de-oiled cake at heating rate 10 °C and 30 °C.
sample weight (where main degradation occurred) which ended by approximately 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 the thermogram were observed at around 400 °C and 440 °C, which indicated the initiation of the passive pyrolysis zone that terminated at around 525 °C for the former and 650 °C for the later. A very slow loss of weight occurred until 750 °C which indicates that there was further reaction involving char. This implies that the main 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 depicted in Fig. 3 corresponds to the findings of Shuping et al. [41]. The analysis of the thermogram shows that during the main pyrolysis process, only one strong peak and henceforth only one decomposition process corresponding to the degradation of crude protein was observed [41]. Microalgae contains very high amount of proteins, 43.22% in the case of Chlorella sp. MP-1 and therefore the major degradation corresponds to protein. As shown in Fig. 4 the TG-DTG profile of Chlorella sp. MP-1 deoiled cake reveals an initial slight weight loss between ambient temperature and about 110 °C for 10 and 30 °C/min. This could possibly be due to moisture evolution. This was followed by continuous decrease in sample weight (where main degradation occurred) which ended by approximately 330–340 °C for the lower heating rate and 350–365 °C for the higher heating rate. No major observable difference was noticed in the thermogram of the deoiled cake at the two heating rates. The degradation of de-oiled cake terminated earlier than algal biomass due to loss of crude cell components in the lipid extraction procedure. The shorter thermal degradation profile of the algal de-oiled cake suggests that it can be an ideal feedstock for thermo-chemical conversion.
5. Conclusion Microalgae Chlorella sp. MP-1 exhibits several important attributes for futuristic research on renewable energy. With simple and inexpensive nutrient regime to culture, faster growth rate as compared 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 bioenergy source. The robust nature of pyrolysis technology can be efficiently applied to algal de-oiled cakes for the co-production of biochar
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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 b
Department of Molecular Biology & Biotechnology, School of Science & Technology, Tezpur University, Assam, Napaam 784 028, India 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
Article history: Received 15 September 2010 Received in revised form 1 November 2010 Accepted 18 November 2010 Available online 6 January 2011 Keywords: Microalgae Biomass Thermogravimetry Chlorella Biofuel
a b s t r a c t Microalgae are promising biomass species owing to their fast growth rate and high CO2 fixation ability as compared to terrestrial plants. Microalgae have long been recognized as potentially good source for biofuel 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 spectroscopy and thermogravimetric study at 10 °C/min and 30 °C/min to investigate its feasibility for thermo-chemical conversion. The present investigation suggests that within the realm of biomass energy technologies the algal biomass can be used as feedstock for bio and thermo-chemical whereas the de-oiled cake for thermo-chemical conversion thereby serving the demand of second generation biofuels. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction As conventional energy sources across the globe are fast depleting, unless the renewable and non-conventional energy sources are tapped, coupled with prudent use and management of energy, humanity is bound to engender a horrific specter of a global energy vacuum. The quest for renewable energy has geared up and one of the facets with great potential for satisfying mankind’s primary energy demand is energy derived from biomass. The present century has witnessed major emphasis on the use of biomass as an alternative to fossil fuels due to its renewable nature and reduced CO2 emissions. Biomass energy sources are amongst the most promising, most hyped and most heavily subsidized renewable energy sources. Biomass can be sustainable, environmentally benign and economically sound. It can provide heat, power and transportation fuels in an environmentally friendly manner, by reducing green house gas emissions and thus can aid in achieving renewable energy targets. A major insight into the search operation for new sources of biomass energy can be offered by microalgae. The generic term microalgae refer to a large group of very diverse photosynthetic micro-organisms of microscopic dimensions. They are sunlight driven oil factories which convert carbon dioxide into potential biofuels, feeds, foods and high value bioactives [1–9]. Algae are the most efficient biological producer of oil on the planet and a versatile
⇑ Corresponding author. Tel.: +91 9954449458; fax: +91 371 2267005/6. E-mail address: [email protected] (B.K. Konwar). 0306-2619/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apenergy.2010.11.026
biomass source and may soon be one of the Earths most important renewable fuel crops [10]. Microalgae are a major natural source for an enormous array of valuable compounds, including a diversity of pigments, for which these photosynthetic micro-organisms represent an almost exclusive biological resource [11]. The potential of microalgae as the most efficient primary producer of biomass still requires comprehensive understanding, but there is little doubt that they will eventually become one of the most important renewable energy sources. The use of microalgae as bio-energy feedstock seems to be promising because: (1) Biomass doubling times in microalgae during exponential growth are commonly as short as 3.5 h [13]. (2) Due to their simple cellular structure, algae have higher rates of biomass and oil production than conventional crops [12]. Oil content in microalgae can exceed 80% by weight of dry biomass [13]. (3) Their lipid content could be adjusted through altering growth media composition [14]. (4) Salty or waste water can be used for the culture of microalgae [15]. (5) Can be harvested batch-wise nearly all-year-around providing a reliable and continuous supply of oil [15]. (6) Atmospheric carbon dioxide is the source of carbon for the growth of microalgae [15]. (7) Depending on species microalgae produce many kinds of lipids, hydrocarbons and other complex oils [5,16,17]. (8) Algae can produce 30–100 times more energy per hectare as compared to terrestrial crops [18].
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(9) The production of biofuels from algae can be coupled with flue gas CO2 mitigation, waste water treatment and production of high value chemicals [18]. The potential value of any biomass depends on the chemical and physical properties of molecules from which it is made. This work is an endeavor to study the biomass properties of Chlorella spp. as several characteristics affect the performance of biomass fuel including the calorific value, moisture content and physicochemical properties and to evaluate the potential of the above as feedstock for the production of biofuel basing on their chemical and physical characteristics. The study also investigates the feasibility of algal de-oiled cake for thermo-chemical conversion.
2. Description of the species Chlorella Beijerinck (Gr. Chloros, green; ella, diminutive). The taxonomic position of Chlorella is depicted in Table 1. Chlorella is a single celled, spherical non-motile green alga 2.0–10.0 lm in diameter. Chlorella occurs in both fresh and marine water. Some call Chlorella ubiquitous since it occurs in various different habitats. They are generally found in fresh water of ponds and ditches, in moist soil or other damp situations such as the surface of tree trunks, water pots and damp walls. Chlorella parasitca is found symbiotically in the cells of Paramecium and Hydra. Chlorella vulgaris, Chlorella conductrix, Chlorella gonglomerata and C. parasitca are the common Indian species. Chlorella is represented by only eight 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 thin cellulose wall, which encloses a parietal and cup shaped chloroplast with a pyrenoid. In certain species the pyrenoids are absent. The cells are devoid of flagella, stigma and contractile vacuoles, but contain 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 human food to applications in space travel. Bio-energy generation from Chlorella is a new facet in renewable energy research. Illman et al. [21] had studied calorific values of Chlorella strains grown in low nitrogen medium including four fresh water strains (Chlorella protothecoides, C. vulgaris, Chlorella emersonii and Chlorella sorokiniana) and one marine strain (Chlorella minutissima) and suggested Chlorella strains may be suitable for diesel replacements. Scragg et al. [22] successfully used an emulsion consisting of transesterified rape seed oil, a surfactant and slurry of C. vulgaris in an unmodified single cylinder diesel engine. Xu et al. [23] obtained high quality biodiesel production from heterotrophic microalgae C. protothecoides.
Table 1 Taxonomic position of Chlorella [20]. Systematic position
Fritsch (1935)
Bold and Wyne (1978)
Division Class Order Family Genus
Chlophyceae Chlorococcales Chlorellaceae Chlorella
Chlorophycophyta Chlorophyceae Chlorellales Chlorellaceae Chlorella
Table 2 Media composition used for culturing Chlorella sp. MP-1 (g L1). Composition
Modified Chu 13
BBM
BG11
Basal
KNO3 NaNO3 K2HPO4 KH2PO4 CaCl22H2O MgSO47H2O Na2CO3 NaCl FeSO4 EDTA Citric acid Ammonium ferric citrate Ferric citrate Ca(NO3)24H2O b-Na2glyserophosphate EDTA-Na2 Vitamin B12 Biotin Thiamine-HCL H3BO3 MnCl24H2O ZnSO47H2O Na2MoO42H2O CuSO45H2O Co(NO3)26H2O FeCl36H2O CoCl26H2O Trisaminomethane
200 – 40 – 80 100 – – – – 100 – 10 – – – – – – – – – – – – – – –
– 250 74 17.5 24 73 – 25 5 45 –
– 1500 40 – 36 75 20 – –
100 – – – – 40 – – – – –
– – – – – – – – – – – – – – – –
6 6 – – – 1 – – – 2.86 1.81 0.22 0.39 0.08 0.05 – – –
– 150 50 2.71 0.0001 0.0001 0.01 – 0.108 0.066 0.0075 – – 5.888 0.012 500
3.2. Isolation and purification The microalgae were subjected to purification by serial dilution followed by streaking. The individual colonies were isolated and inoculated into liquid medium (BG-11). The purity of the culture was established by repeated streaking and routine microscopic examination. 3.3. Culture conditions The microalgal cultures were carried out in 500 ml Erlenmeyer flasks 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. Materials and methods
3.4. Growth evaluation
3.1. Micro-organism and growth medium
The growth of Chlorella sp. MP-1 was monitored spectrophotometrically (Beckman DU530) by reading the culture absorbance at 682 nm.
Chlorella sp. MP-1 isolated from water samples collected from Lake Joysagar, one of the largest manmade lake, Sibsagar, Assam, India (26°570 1200 N and 94°370 3400 E) was used in this study. Stock cultures of Chlorella sp. MP-1 were maintained routinely on both liquid and agar slants of BG-11 media [24] by regular sub-culturing at 15 days interval. The species under investigation was also cultured in BBM [25], modified Chu-13 and [26] and Basal [24] media to estimate the biomass yield in the respective media. The composition of the various culture media is presented in Table 2.
3.5. Biomass estimation The cultures were harvested by centrifugation at 7000 rpm for 15 min. The cells were washed twice with distilled water after centrifugation. The pellet was dried at 80 °C for 24 h. The dry weight of the algal biomass was determined gravimetrically and growth was expressed in terms of dry weight.
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3.6. Determination of Gross calorific value (GCV)
3.13. FTIR analysis
Calorific value (CV) was determined using an automatic adiabatic bomb calorimeter (Changsha Kaiyuan Instruments Co., 5E-1AC/ML). The sample (dried algal pellet) was oxidized by combustion in an adiabatic bomb containing 3.4 Mpsi oxygen under pressure. The assays were carried out in triplicates and the mean values are reported.
The IR spectrum of dried algal biomass was recorded on Nicolet IR spectrometer at room temperature. The dried algal powder was blended with potassium bromide (KBr) powder, and pressed into tablets before measurement. A region of 4000–400 cm1 was used for scanning.
3.7. Determination of Net calorific value (NCV) The NCV was calculated from the following equation [27]:
w w H 8:936 NCV ¼ GCV 1 2:444 2:444 100 100 100 w 1 ; ðMJ=kg; w:b:Þ 100 where 2.444 = Enthalpy difference between gaseous and liquid water at 25 °C.
8:936 ¼
M H2 O ; i:e: the molecular mass relation between M H2
H2 O and H2 : where NCV is the Net calorific value, GCV is the Gross calorific value, h is the concentration of hydrogen in weight%, w is the Moisture content of the fuel in weight%. 3.8. CHN analysis C, H, N analysis was carried out in a CHN analyzer and the oxygen content was calculated by difference. 3.9. Proximate analysis The moisture, volatile matter and ash content of the dry algal biomass were determined according to ASTM D 3173, ASTM D 3175 and ASTM D 3174 protocols. Finally the fixed carbon content was calculated by difference. 3.10. Carbohydrate estimation The anthrone method was used for the determination of total carbohydrates [28]. A calibration curve was prepared using D+ glucose dissolved in distilled water. The glucose concentration (Cglc, mg/ml) and optical density had the following relationship:
C glc ¼
1 ðOD630nm þ 0:015Þ 6:165
3.11. Protein estimation Ten milliliter algal culture was centrifuged at 6000 rpm for 10 min. The cell pellet was re-suspended in 5 ml of 1 M NaOH and boiled for 10 min. The protein content was determined using the method of Lowry [29]. A calibration curve was prepared using BSA dissolved in distilled water. The BSA concentration (Cbsa, mg/ml) and optical density had the following relationship:
C bsa ¼
1 ðOD660nm þ 0:001Þ 0:005
3.12. Determination of total lipids Total lipid was determined using the method of Bligh and Dyer [30].
3.14. Thermal analysis For thermal analysis microalgae from late exponential phase were harvested by centrifugation at 7000 rpm for 15 min. The pellet was washed twice with distilled water and then dried at 80 °C for 24 h. The samples were pulverized in a mortar to fine particles in order to eliminate heat transfer effects during pyrolysis and then finally stored in a desiccator. Thermogravimetric analysis (TGA) was done in order to study the combustion behavior of algal biomass. Algal biomass and de-oiled cake was subjected to thermogravimetric analysis in nitrogen atmosphere at heating rates of 10 °C/min and 30 °C/min. Sample weighing approximately 10 mg was heated at the preselected heating rate from ambient temperature 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 rate of 100 ml/min as an inert purge gas to displace air in the pyrolytic zone, thereby avoiding unwanted oxidation of the sample. The continuous on-line records of weight loss and temperature were obtained to plot the TGA curve and the derivative thermogravimetric analysis (DTG) curves. 4. Results and discussions The successful implementation of algal biomass as a potential bio-energy feedstock is largely governed by the quantum of producible biomass. Therefore enhancement of the growth rate of algae in terms of biomass productivity is one of the most important parameters. The growth of Chlorella sp. MP-1 was tested in four different culture media namely, BG-11, Basal, BBM and modified Chu13 media. Among all the tested media the highest biomass yield of 824 mg/l was obtained in BG-11 media. The study only takes into account BG-11 media with the highest gross biomass yield for culturing Chlorella sp. MP-1 and subsequent investigations. The biomass yield of Chlorella sp. MP-1 in different culture media is shown in Fig. 1. The characterization of algal biomass as an energy source is taken into consideration in the present investigation. Table 3 shows the biomass properties of Chlorella sp. MP-1 with their average characteristic composition. The moisture content of biomass differ significantly, depending on the type of biomass and biomass storage. Moisture content is of great importance with regard to selection of biomass conversion technology. Biomass fuels with low moisture content are more suited for thermal conversion technology while those with high moisture content are more suited for biochemical process such as fermentation conversion [31]. On this basis Chlorella sp. MP-1 with moisture content of 6.8% seems to be a potential candidate for direct thermo-chemical conversion. Ash content of biomass affects both the handling and processing costs of overall biomass energy conversion. The ash content in microalgae may vary considerably in different species and also with geographical location and season. In this investigation ash value was determined using ASTM D 3174 protocol. The characterization of ash was not done and is a part of planned future research activity. The ash content was found to be 5.93% and volatile matter content was 72.19%. The high amount of volatile matter in Chlorella sp. MP-1 biomass strongly influences its combustion behavior and thermal decomposition. The fixed carbon content was 15.08%.
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Fig. 1. Biomass yield of cake. Chlorella sp. MP-1 in various culture media.
Table 3 Properties of Chlorella sp. MP-1. Properties
Chlorella spp.
Gross calorific value (MJ/Kg) Net calorific value (MJ/Kg) Empirical formulae (on ash free basis) H/C molar ratio (on ash free basis) O/C molar ratio (on ash free basis)
18.59 ± 0.42 15.88 ± 0.39 C8.25H14.79NO5.02 1.79 0.6
Elemental analysis (wt.%)
Carbon Hydrogen Nitrogen Oxygen (by difference)
47.54 7.1 6.73 38.63
Proximate analysis (wt.%)
Moisture Volatile matter Fixed carbon Ash
6.8 ± 1.11 72.19 ± 1.73 15.08 ± 1.21 5.93 ± 0.81
Biochemical analysis
Total carbohydrate Protein content Lipid content
9.46 ± 0.25 43.22 ± 0.33 28.82 ± 0.72
The biomass had high percentage of volatile matter and low ash content which is important with respect to their application in gasification and pyrolysis process. The elemental content of carbon, hydrogen, oxygen and nitrogen in the algal biomass was 47.54%, 7.1%, 38.63% and 6.73% respectively. The empirical formula of the algal biomass is C8.25H14.79NO5.02. The H/C and O/C molar ratios (on an ash free dry basis) were calculated from elemental composition as 1.79 and 0.6 respectively. The Gross calorific value (GCV) and Net calorific value (NCV) for Chlorella sp. MP-1 biomass was 18.59 MJ/kg and 15.88 MJ/kg respectively. The Gross calorific value in the examined sample was higher than 18 MJ/kg, as reported by Illman et al. [21]. An increase in calorific value in algae is linked to increase in lipid content rather than any change in other cell components such as carbohydrates and proteins [21]. The FTIR spectrum of Chlorella sp. MP-1 biomass and de-oiled cake is shown in Fig. 2. The region 3300–3000 cm1 is characteristic for CAH stretching vibrations of C„C, C@C and ArAH, whereas the region from 3000 to 2700 cm1 is dominated by CAH stretching vibrations of ACH3, >CH2, CH and CHO functional groups, respectively [32,33]. The olephinic CAH stretching vibration between 3600 and 3300 cm1 indicates unsaturate. The absorption at 1652 cm1 implies the presence of C@O of carboxylic acid and derivatives. The region between 1800 and 1500 cm1 demonstrate characteristic bands for proteins, whereas 1700–1600 cm1 is
Fig. 2. FTIR spectra of Chlorella sp. MP-1 biomass and deoiled cake.
specific for amide-I bands [33], which is mainly due to C@O stretching vibrations of peptide bond [34]. The bands in the amide I region provide insight into the protein secondary structure [35]. On the other hand the region from 1600 to 1500 cm1 is specific for amide-II bands, which is due to NAH bending vibrations [36]. The region from 1200 to 900 cm1 signifies a sequence of bands due to CAO, CAC, CAOAC and CAOAP stretching vibrations of polysaccharides [37,38] as well as CH3, CH2 rocking modes [39]. CH2 stretching vibrations in the range of 3100–2800 cm1 implies the presence of lipid [40]. The absorption at 2928 and 2860 cm1 implies CH2 asymmetric and symmetric stretching in lipid. All the above mentioned bonds which were prominent in the algal biomass showed progressive degradation in their intensity in the spectra of the de-oiled cake. There was a general decrease in protein and carbohydrate content indicated by a decrease in the intensity of absorption bands in the 1800–800 cm1 region. This region is specific for proteins and carbohydrates [33]. Declination in the intensity of absorption in the range of 3100–2800 cm1 is indicative of decrease in the lipid content. As shown in Fig. 3 the TG-DTG profile of Chlorella sp. MP-1 biomass reveals an initial weight loss between ambient temperature and about 130 °C and 160 °C for 10 and 30 °C/min. This could possibly be due to elimination of physically absorbed water in the biomass and due to external or superficial water bounded by surface tension. This was followed by continuous decrease in
Fig. 3. TG-DTG of Chlorella sp. MP-1 biomass at heating rate 10 °C and 30 °C.
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with bio-oil. The characterization of Chlorella biomass in light of bioenergy production ensures that it can be, used as renewable feedstock for biochemical and thermo-chemical conversion and may serve the demands of second generation biofuels. Moreover the production of these biofuels from microalgae can be coupled with flue gas CO2 mitigation, waste water treatment and production of high value chemicals. The potential of energy production from Chlorella is vast and necessitates further research. Acknowledgements The first author would like to offer his sincere thanks to the ONGC, Jorhat, India for providing fund in the form of a fellowship project. The authors are also thankful to Dr. J.R. Chetia and S. Banerjee for TGA analysis. References Fig. 4. TG-DTG of Chlorella sp. MP-1 de-oiled cake at heating rate 10 °C and 30 °C.
sample weight (where main degradation occurred) which ended by approximately 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 the thermogram were observed at around 400 °C and 440 °C, which indicated the initiation of the passive pyrolysis zone that terminated at around 525 °C for the former and 650 °C for the later. A very slow loss of weight occurred until 750 °C which indicates that there was further reaction involving char. This implies that the main 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 depicted in Fig. 3 corresponds to the findings of Shuping et al. [41]. The analysis of the thermogram shows that during the main pyrolysis process, only one strong peak and henceforth only one decomposition process corresponding to the degradation of crude protein was observed [41]. Microalgae contains very high amount of proteins, 43.22% in the case of Chlorella sp. MP-1 and therefore the major degradation corresponds to protein. As shown in Fig. 4 the TG-DTG profile of Chlorella sp. MP-1 deoiled cake reveals an initial slight weight loss between ambient temperature and about 110 °C for 10 and 30 °C/min. This could possibly be due to moisture evolution. This was followed by continuous decrease in sample weight (where main degradation occurred) which ended by approximately 330–340 °C for the lower heating rate and 350–365 °C for the higher heating rate. No major observable difference was noticed in the thermogram of the deoiled cake at the two heating rates. The degradation of de-oiled cake terminated earlier than algal biomass due to loss of crude cell components in the lipid extraction procedure. The shorter thermal degradation profile of the algal de-oiled cake suggests that it can be an ideal feedstock for thermo-chemical conversion.
5. Conclusion Microalgae Chlorella sp. MP-1 exhibits several important attributes for futuristic research on renewable energy. With simple and inexpensive nutrient regime to culture, faster growth rate as compared 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 bioenergy source. The robust nature of pyrolysis technology can be efficiently applied to algal de-oiled cakes for the co-production of biochar
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