N. Dhakal et al.: Study of Growth Rate and Lipid Content of Various Microalgae Species from Nepal
Study of Growth Rate and Lipid Content of Various Microalgae Species from Nepal Nirpesh Dhakal*, Sanjaya Lama, Angela Shrestha, Tika Bahadur Karki, and Parash Mani Timilsina Department of Biotechnology, Kathmandu University Abstract— More than 40% of foreign currency reserve of Nepal is required to import fossil fuels every year. HSD is largely consumed as compared to other petroleum product. Import of HSD is increasing every year with an average of 92424.5 kL/yr and is expected increase inevitably further along with development of the country. Microalgae as fast growing species with better oil content could address above issue. With very pleasant sunshine, climatic condition of this country could support algal culture. DOB/KU with support of RenewableNepal programme has been conducting R&D work on identifying potential species of microalgae for biodiesel production. Study on 18 different varieties of microalgae selects five potential species, namely Chlorella vulgaris, Chlamydomonas sp.,Euglena sp., Scenedesmus cf bijuga and Chlamydomonas cf orbicularis, on the basis of their growth rate and lipid content. Among these C. vulgaris showed the better result with maximum average specific growth rate of 0.529 gm cells/day and average lipid content of 28% of dry weight. Whereas other four species with maximum average growth rate of 0.351 gm cells/day (average of all four samples) and lipid content varied from 25 to 30 %. Along with growth rate and lipid content, other techniques on carbon source, flocculation, cell disruption and lipid extraction were also studied on C. vulgaris.1 Index Terms— Microalgae, growth rate, lipid, biodiesel
I. INTRODUCTION The evidence of climate change and environmental impacts due to excessive use of fossil fuels is accumulating. With the increasing demand in energy, fossil fuels as nonrenewable resources will be depleted soon. Oil and natural gas storage on earth has been estimated to be depleted in 40 and 64 years, respectively [1]. Nepal’s dependency upon petroleum product is increasing yearly. Out of total 12.66% commercial fuel consumed 9.02% is comprised with petroleum products [2]. More than 40% of foreign currency reserve is required to import fossil fuels every year [3]. Among different types of petroleum fuel, country imports High Speed Diesel (HSD) in comparatively large amount. Further, this import of HSD is increasing every year with an average of 92424.5 kL/yr [4]. There is a high possibility of inevitable increase in the demand of HSD and other petroleum products as country steps towards its development. With increased demand and dependency on fossil fuel corresponding to depleting natural storage may lead to devastating situation, unless other alternative measures are taken. Microalgae culture could address above problems related to fossil fuel and climate change. Biofuels have received wide attention in recent years, especially algae derived biofuels. Oil from microalgae could be transesterified to produce biodiesel. Algae reproduce quickly, produce oils more efficiently than crop plants, and require relatively few * Corresponding author:
[email protected] Rentech Symposium Compendium, Volume 3, September 2013
nutrients for growth. These nutrients can potentially be derived from inexpensive waste sources such as flue gas and wastewater, which provides a mutual benefit to mitigate carbon dioxide waste. Microalgae are capable of all year round production; therefore, oil productivity of microalgae cultures exceeds the yield of the best oilseed crops, e.g. oil yield upto 10,000 Gallons/acre [5]. It can fix CO2 comparatively faster and higher than any other terrestrial plant, i.e. 1 kg of dry algal biomass utilizes about 1.83 kg of CO2 [6]. Further, it could address other major problems like food security, land security and waste water treatment. Microalgae haven’t been much explored in context of Nepal, especially in the field of its mass cultivation, oil extraction or biodiesel production. Technically country could provide favorable climatic condition for algal bloom. Nepal receives very pleasant sunshine, i.e. 6.8 hours per day with the intensity of solar insolation ranging from 3.6 to 5.9 kW/sq.m/day [7]. Algae’ being a photoautotroph this light energy is major source of its growth. Along with this, country has abundant natural water resources (rich in minerals) which could be used for microalgae production in large scale. Department of Biotechnology, Kathmandu University (DOB/KU) with support of RenewableNepal Programme has been conducting research on microalgae. This paper discuss about the growth rate and lipid content of few microalgae species collected from different part of country (mid hill and terai region). Among the isolated algal species 5 species are thought to be potential one, among which Chlorella vulgaris is thought to be more potential species for biodiesel production in moderate environmental condition because of its faster growth rate and better lipid yield. Along with growth rate and lipid content, other techniques on carbon source, flocculation, cell disruption and lipid extraction were also studied on C. vulgaris.
II. MATERIALS AND METHOD Sampling and isolation Samples were collected from 8 different location of the country (Table I). Net tows of 10um were used to obtain live cells from the selected location and stored in 15 ml centrifuge tube containing 10 ml of 1 X Bold Basal Medium (BBM) [8] keeping it in dark condition. On arrival to laboratory, all samples were then filtered through 200 um plankton net to remove zooplankton and any foreign particles. Cells retained were washed with autoclaved 1X BBM media and brought in sterile condition suspending in 1X BBM Media up to approximately 30 ml. All the samples were examined microscopically for the presence of at least one species. For isolation of single species, streak plate technique was carried out in 1X BBM media containing 2% bacteriological 43
N. Dhakal et al.: Study of Growth Rate and Lipid Content of Various Microalgae Species from Nepal agar incubating in growth chamber at 26 ± 20 C and 2000 lux. Colonies from streak plates were individually inoculated in separate test tubes containing 10ml of sterilized 1X BBM and incubated at 2000 lux and 26 ± 20 C for 2-3 weeks. Inoculated cultures were observed under microscope for single species. Species were sent to Dr. S. K. Rai at Department of Botany, Post Graduate Campus, Biratnagar for identification Scale up cultures 18 single species cultures were washed three times with 1 X BBM followed by centrifugation at 2000 rpm for 5 min. Cell pellet was cultured into new sterile 100 ml culture flask containing 30 ml of media for 2-3 week in a growth chamber at temperature 26±20 C and light intensity of 4000 lux to obtain a sufficient amount of pure culture for further analysis of growth kinetics. Cell density was determined by haemocytometer count and 15ml of each culture was taken from conical flask and washed for 3 times with 1 X BBM followed by centrifugation at 2000 rpm for 5 min. Cell pellet was then cultured into sterile 250 ml conical flask containing 100 ml 1X BBM. Cultures were then incubated in Orbital Shaker BOD incubator under 12:12 h light:dark photoperiod at 26±20 C, 4000 lux and 50 rpm. Other two batch of the selected species from 18 species were cultured in a Growth Chamber with aeration in 250 ml conical flask containing 200 ml 1x BBM at 26±20 C, 4000 lux and 12:12 h light:dark ratio. Cultures growths were monitored spectrophotometrically at 438 nm and 750 nm, on daily basis. For C. vulgaris effect of different concentration of carbon in growth and lipid content was monitored by using NaHCO3 as carbon source. Concentration of 50 mg/l, 70 mg/l and 90 mg/l NaHCO3 was prepared in 1X BBM. C. vulgaris was cultured in four 250 ml conical flask containing 200 ml with 0 mg/l (1X BBM as blank), 50 mg/l, 70mg/l
and 90 mg/l of above prepared NaHCO3 solution. Culture were provided with aeration and incubated in growth chamber at culture condition of 26±20 C, 4000 lux and 12:12 h light:dark ratio. Optical density of culture was taken in daily basis at 438 nm and 750 nm. Harvesting and extraction Fully grown cultures were flocculated using FeSO4, FeCl3, CuSO4, ZnSO4 and maintaining high pH at 11. Flocculated samples were filtered through Whatman no.1 filters and dried at 60°C in hot air oven. Lipid content was extracted by modified of Bligh and Dyer method [9] with 1:1:0.8 ratio of chloroform:methanol:water mixture in duplicate[2]. Total lipids (TLs) were measured gravimetrically and reported as percentage of dry weight basis. Further, TLs of C. vulgaris was extracted using different procedures for cell disruption and lipid extraction for optimization. Autoclave at 1200C and 15 psi for 5 minutes, osmotic shock using 2 M NaCl for 24 hours, maceration using liquid nitrogen, direct heating at 1040C in hot air oven for 2 hours and ultrasonication for 5, 10, 15 & 30 mins at 20 kHz were used for cell disruption. For extraction, methanol, hexane, chloroform, diethyl ether, acetone and isopropanol at different solvent concentration and ratio were performed for cold extraction in room temperature and in soxhlet for warm extraction. All culture experiments and treatments were performed in duplicate and excellent reproducibility was demonstrated.
III. RESULT AND DISCUSSION List of the isolated species are given in Table I. We were able to isolate various species from same location and also, similar species from different locations. Cultures of similar species from the different location were carried out separately and monitored.
TABLE I ISOLATED SINGLE SPECIES ACCORDING TO THEIR LOCATION
1
Sample Code BNP i
Banepa, Kavre
Fish Pond
Chlorella vulgaris
2 3 4 5 6 7 8 9 10 11 12
KU ii GW iii HM iv DM v GW vi DM vii SMRK viii DM ix BNP x DM xi SMKU xii
Dhulikhel, Kavre Godavari, Lalitpur Buddhanagar, Ktm Gahana Pokharai, Ktm Godavari, Lalitpur Gahana Pokharai, Ktm SemlarVDC, Rupandehi Gahana Pokharai, Ktm Banepa, Kavre Gahana Pokharai, Ktm SemlarVDC, Rupandehi
Water drainage outside Algae lab, KU Pond Waste 5 liter water jar Pond Pond Pond Tinahu River Pond Fish Pond Pond Stream (Kulo)
13 14 15 16 17 18
BM xiii BM xiv DM xv GW xvii KU xvii DM vviii
Banglamukhi, Lalitpur Banglamukhi, Lalitpur Gahana Pokharai, Ktm Godavari, Lalitpur Dhulikhel, Kavre Gahana Pokharai, Ktm
Floor of Dhunge Dhara Floor of Dhunge Dhara Pond Pond KU’s water collection site (near Block 8) Pond
Chlorococcum sp. Chlorella sp. Chlorella sp. Chlamydomonas sp. Chlorella sp. Chlamydomonas sp. Chlamydomonas sp. Scenedesmus opoliensis Richter Euglena sp. Tetraedron caudatum (Corda) Hansg. Scenedesmus cf bijuga Coccomyxa cf. gloeobotrydiformis Scenedesmus opoliensis Richter Euglena sp. Chlamydomonas cf orbicularis Geminella sp. (not confirmed)
SN
Location
Description of Location
Rentech Symposium Compendium, Volume 3, September 2013
Isolated Species
Remarks
Species NI Unidentified Species NI Species NI Species NI Species NI Species NI Species NI Species NI
Species NI
44
N. Dhakal et al.: Study of Growth Rate and Lipid Content of Various Microalgae Species from Nepal
Five samples (BNP i, GW vi, DM xi, BM xiii, and KU xvii), selected from preliminary screening for further studies on growth kinetics and lipid content, were grown in growth chamber. Along with these sample cultures, standard sample cultures were also established in identical conditions (Nutrients, light:dark cycle, temperature) in order to compare and analyze the growth characteristics and lipid content of selected samples. During second round of culture, specific growth rate of BNP i significantly increased to 0.529 gm cells/day within 10 days achieving the late exponential phase (Fig. 2). Specific growth rate of 0.351 gm cells/day (average of all four samples) was obtained for GW vi, DM xi, BM xiii, and KU xvii which is higher than in preliminary screening. In overall, improvement in growth characteristics was observed for all selected samples during second round of culture. This achievement was primarily due to optimized culture condition in this round of cultivation as it was carried out in self designed growth chamber with controlled environment. The main source of energy for photosynthetic microalgal cells are light, carbon source, minerals, nutrients, and well mixing mechanism required for homogenous distribution of energy source to all cells in a culture. All these requirements were well maintained during second round culture in growth chamber compared to BOD incubator culture during preliminary screening. This analysis was done on the basis of replicating the experiment in triplicate for all selected samples in identical culture conditions. Rentech Symposium Compendium, Volume 3, September 2013
a3.5
BNP i
3
HM iv
Optical Density
Preliminary screening for potential species was done by growth kinetics study based on daily spectrophotometric reading of absorbance by cells and lipid content of cultured species analyzed by solvent extraction. Among 18 different samples cultured, chlorella vulgaris (BNP i) attained exponential phase of growth within 12 days. This was comparatively better as compared to other samples (Fig. 1a). Other seven samples (GW vi, DM vii, DM xi, SMKU xii, BM xiii, DM xv and KU xvii) had an average growth rate of 0.21 gm cells/day which was comparatively better that remaining 10 samples. Total of 9 samples were selected for second phase of monitoring on basis of their growth rate and lipid content. Although HM iv had comparatively very low specific growth rate i.e., 0.061 gm cells/day, it was selected for its high lipid content of about 35% (weight % of dry biomass), while rest of the samples had lipid content of average 27% (Table II). During second phase observation, again BNP i showed best specific growth rate among other species with specific growth rate of 0.326 gm cells/day. Similarly DM xi had specific growth rate of 0.309 gm cells/day within the same period (Fig. 1b). While slight lagging in growth was observed for GW vi, SMKU xii, and BM xiii with specific growth rate of 0.27 gm cells/day in average. Significant increase in growth kinetics was observed for HM iv during second round with specific growth rate of 0.238 gm cells/ day. From this preliminary screening, five samples (BNP i, GW vi, DM xi, BM xiii, and KU xvii) with the best to least growth kinetics and comparatively better lipid yield were selected for further round of kinetics study. Although HM iv was not selected for next phase, sample was preserved for further optimization (data not shown in this study).
GW vi
2.5
DM vii
2
DM xi
1.5
SMKU xii BM xiii
1
DM xv
0.5
KU xvii
0 0
10 Time (days)
20
b 3.5
BNP i
3
HM iv GW vi
Optical Density
Culture growth and lipid content
2.5
DM vii
2
DM xi SMKU xii
1.5
BM xiii
1
DM xv
0.5
KU xvii
0 0
10 20 Time (days)
30
Fig. 1: Optical density at 438 nm of (a) nine samples of microalgae among 18 single species incubated at BOD incubator & (b) selected 9 different microalgae varieties from 18 single species incubated at growth chamber. TABLE II AVERAGE LIPID CONTENT OF ALGAL SPECIES S.N.
Sample
1 2 3 4 5
BNP i HM vi GW vi DM vii DM xi SMKU xii BM xiii DM xv KU xvii
6 7 8 9
Avg. Dry Weight (15 ml Culture)
TLs Content
TLs weight %
24 11.7 14.3 21.5 23.1
6.8 4.1 4.3 5.8 6.2
28.3 35.0 30.1 27.0 26.8
23.6 26 27.5 12.6
4.9 6.9 7.1 3.7
20.8 26.5 25.8 29.4
45
Optical Density
N. Dhakal et al.: Study of Growth Rate and Lipid Content of Various Microalgae Species from Nepal 3.5 3 2.5 2 1.5 1 0.5 0
BNP i GW vi DM xi BM xiii KU xvii
0
10
20
Time (days) Fig. 2: Growth rate comparison of selected five algal varieties
Effect of carbon source on C. vulgaris
Optical Density
Optimization of culture media is essential to set up large scale culture because all media components used for lab scale culture will be less cost effective to be used in large scale. Thus optimization of culture condition is required for maximum yield and productivity. Manipulation of alternative source of media is required for commercial scale culture. However media and conditions optimized should not affect potentiality of culture system. 3.5 3 2.5 2 1.5 1 0.5 0
0 mg/l 50 mg/l 70 mg/l 90 mg/l
0
10 Time (day)
20
Fig. 3: Effect of carbon source on C. vulgaris
In our study of growth kinetics and lipid content determination among six different samples, Chlorella species stand out best with cultivation time period of 10-12 days and lipid content up to 30%. Thus further optimization was carried out for BNP i (Chlorella vulgaris). We studied the effect of sodium bicarbonate (NaHCO3) as an alternative source of carbon in addition to available CO2 in culture. Effect of NaHCO3 on culture growth and lipid content was compared with culture in standard 1X BBM. Specific growth rates and lipid contents of 0.322 gm cells/day (24.925 % lipid), 0.345 gm cells/day (22.584 % lipid), 0.333 gm cells/day (25.33 % lipid) and 0.339 gm cells/day (25.037 % lipid) were obtained for culture in 1X BBM (blank), 50mg/l NaHCO3, 70mg/l NaHCO3, and 90mg/l NaHCO3 respectively (Fig. 3). It can be concluded that overall growth rate was improved with supplementation of external carbon source in comparison to standard media while lipid content was almost constant with slight fluctuation. Effect of flocculants on biomass harvesting In our study 0.5 gm/l of dry biomass of BNP i was obtained with Whatman number 1 filter paper and the Rentech Symposium Compendium, Volume 3, September 2013
process was quite time consuming as it required at least 8-10 hours to filter 500 ml of culture. Cells penetrated into filter pores as the filtration proceeds, this result in clogging of filters and posed a significant problem. Centrifugation is more effective than filtration for biomass harvesting at lab scale with dry biomass yield of 0.61 gm/ l but it is more energy intensive for large scale processing. Gravity sedimentation was carried out by allowing cells (matured culture in 100 ml measuring cylinder with pH 9.6) to settle down. After 12 hours, 40 ml of culture supernatant was discarded thus reducing the working volume to 60 ml. Similarly, 100 ml matured culture of BNP i with pH adjusted to 11 was kept for 12 hours in dark. With this method significant settling of cells was observed with working volume reduced to 30 ml. To sum up, gravity sedimentation alone is not efficient enough to recover complete dry biomass. Additionally different flocculating agents (multivalent cations and cationic polymers) can be used if harvested biomass are not for food or feed purpose. The flocculants added should be non toxic, inexpensive and effective in low concentration without interfering downstream processing of the products. Microalgal cells bear negative charge on their surface creating repulsive force and preventing aggregation of cells. Different multivalent metal ion salts like ferric chloride (FeCl3), ferrous sulfate (FeSO4), copper sulfate (CuSO4) and zinc sulfate (ZnSO4) were used to neutralize the negative charge on surface and thus allowing flocculation of cells. Among different metal salts used, effective aggregation was observed (on culture at pH 10) with FeCl3 and FeSO4 at concentration of 1.05 mg/l and 20 mg/l. With this concentration, working volume was reduced to 350 ml from initial volume of 1000 ml after 12 hours. Similar result was obtained with 120 mg/l of CuSO4. Significant flocculation was not observed with ZnSO4 used at concentration of 100mg/l. After conducting all methods of harvesting individually, combination of different flocculants was tried. It was observed that flocculation prior to filtration yields well result than individual method. 1.32 gm dry biomass was obtained from two liters culture (at late exponential phase) of Chlorella species. Effect of different cell disruption and lipid extraction techniques Lipid extraction of dried biomass (sun drying or in oven at 60°C) using range of polar and non polar organic solvents like chloroform, methanol, ethanol, diethyl ether, hexane and acetone was carried out. Modified Bligh and Dyer extraction procedure is widely applied protocol to extract lipid from microalgae. Chloroform:Methanol:Water 1:1:0.8 was considered as best extraction solvent technique in our study. Following this method, lipid content of 24-30 % was obtained in all selected samples and also for HM iv which contain up to 35 % of its dry biomass. Further different pretreatment methods were applied to disrupt cells prior to solvent extraction. Pretreatment of cells resulted in cell lysis and easy access of solvent into cell thus yielding better result. After pretreatment cell was extracted with chloroform:methanol:water 1:1:0.8 ratio. Cells on osmotic shock shown best result with lipid content of 27% at 2M of 46
N. Dhakal et al.: Study of Growth Rate and Lipid Content of Various Microalgae Species from Nepal NaCl for 24 hours compared to other pretreatment techniques used, which are Autoclave at 1200C and 15 psi for 5 minutes, maceration using liquid nitrogen, direct heating at 1040C in hot air oven for 2 hours and ultrasonication for 5, 10, 15 & 30 mins at 20 kHz).
Prof Dr Panna Thapa, Project Administrator and Dean School of Science and Dr. S. K. Rai, Department of Botany, Post Graduate Campus, Tribhuwan University, Biratnagar.
Solvent extraction is widely followed method for lipid content determination from dry biomass in lab scale. Biomass harvesting and recovery of dry biomass is essential prior to lipid extraction. Biomass harvesting, separation of cells from liquid portion of media is one of the most crucial step. Harvesting at large scale covers up to 20-30% of overall cost of biodiesel production from microalgae. Biomass recovery is significantly problematic due to small size of microalgal cells (3-30 µm in diameter) and cost effective harvesting technology has not developed yet.
[1]
REFERENCES
[2] [3] [4] [5]
IV. CONCLUSION
[6]
Microalgae are considered as third generation biofuel. It is being counted for CO2 sequestration. With high growth rate and lipid content, there is better probability for it as renewable fuel source and atmospheric air improvement. Although it could be a potential source for biofuel due to its nature as microorganism there are various limitations. Slight change in culture parameters could significantly alter its growth cycle and yield. Along with this microalgae may not reproduce similar result in standard conditions. Harvesting technique is quite big challenge in current scenario. Although flocculation has been recommended, current flocculants used accounts about 40-60% of cells present in the culture to settle them completely. This increase overall cost of the process. Thus research on harvesting is necessary to use algae derived biodiesel in commercial level. Further R&D work is utmost necessity in current scenario to make algal fuel economically viable.
[7]
ACKNOWLEDGEMENT The authors gratefully acknowledge the contributions of RenewableNepal program office and its staffs Dr Bhupendra Bimal Chhetri, Rakesh Shrestha, Shujata Limbu, Sarina Shrestha and Sushma Maharjan. Also, the contribution of
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Xin L., Hu H.-ying, Ke G., and Sun Y.-xue. (2010). “Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalgae Scenedesmus sp.” Bioresource technology, Elsevier Ltd; 101(14): 5494-500. WECS (2006) Energy Synopsis Report, Nepal 2006, Water and Energy Commission Secretariat, Ministry of Water Resource, Nepal MOF (2007) Economic Survey, Fiscal Year 2006/07, Ministry of Finance, Nepal NOC (2012). Nepal Oil Corporation (Import and Sales) Retrieved from http://www.nepaloil.com.np/main/?opt1=supply&opt2=import Pienkos P T. (2007). The Potential for Biofuels from Algae. AlgaeBiomass Summit San Francisco, CA, November 15, 2007. NREL/PR-510-42414 Chisti Y. (2007). Biodiesel from microalgae. Biotechnology Advances; 25(3):294–306 Renewable Energy Technology for Rural Development-03/ Renewable Energy in Nepal- Progress at a Glance from 1998 to 2003 Stein J. (ED.) (1973 Handbook of Phycological methods: Culture methods and growth measurements. Cambridge University Press. 1:448 pp. Bligh,E.G. and Dyer,W.J. (1959). A rapid method for total lipid extraction and purification. Can.J.Biochem.Physiol. 37:911-917
BIOGRAPHIES Mr. Nirpesh Dhakal is a graduate in B.E. Biotechnology from VTU, Karnataka. He is currently enrolled in MS Biotechnology in Kathmandu University (KU). Mr. Sanjay Lama is a graduate in B.Tech. Biotechnology from Kathmandu University. He is currently enrolled in MS Biotechnology in Kathmandu University (KU). Mr. Parash Mani Timilsina has done his M.Sc. Biotechnology from Periyar University, Tamilnadu. He is currently working as Assistant Professor in Kathmandu University. Prof. Dr. Tika Bahadur Karki has done his PhD from Tokyo University. He is currently working as HOD at Department of Biotechnology, Kathmandu University. Ms. Angela Shrestha is a graduate in B. Tech. Biotechnology from Kathmandu University. She is currently enrolled in M.Tech. Biotechnology in Kathmandu University.
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