BIOFUEL PROSPECTS OF MICROALGAL COMMUNITY IN URBAN WETLANDS

1、biofuel prospects of microalgal community in urban wetlandsramachandra t.v 1,2,3,*, alakananda b 1, supriya g.1 1energy & wetlands research groupcentre for ecological sciences2centre for sustainable technologies (astra)3centre for infrastructure, sustainable transportation and urban planning cistupi

2、ndian institute of science,bangalore 560 012, india 1cestvrces.iisc.ernet.in (*corresponding author),2alkaces.iisc.ernet.in, 3supriyaces.iisc.ernet.in,abstract: microalgae are emerging as one of the most promising sources of biofuel because of their high photosynthetic efficiency and faster replicat

3、ion as compared to any other energy crops. although, the concept of using microalgal lipid as a source of fuel is very mature, its approach in benefiting both environmental and energy-related is a frontier research area today. algal community for the production of lipid depends on the physical, chem

4、ical as well as biological variables of aquatic ecosystems. this communication focuses on achieving the lipid characterization of the microalgal community collected from four wetlands and one agricultural field of bangalore, karnataka with a wide range of environmental characteristics. results revea

5、l significant change in lipid component with change in algal community and chlorophyll content which was explained by community structure analysis and chlorophyll estimation. the presence of triacyl glycerol (tag) was examined through thin layer chromatography (tlc). the profile of tag was further c

6、onfirmed through gas chromatography mass spectroscopy (gc-ms). this study confirms the potential of algal community towards meeting growing demand for alternate sustainable fuel. keywords: microalgae, community structure, lipid, gas chromatography mass spectroscopy.i. introductionwater occupies most

7、 part of the earths surface amounting to a volume of 1.38x109 km3 of which freshwater contributes to approximately 0.0013% of the global water1. freshwater ecosystems encompass an extensive range of habitats viz., rivers, lakes, and wetlands, with constant interaction of biotic with abiotic componen

8、ts. studies have revealed that the use of freshwater in agricultural purposes is 4000 km3 of water by 20502, for domestic purposes (during 1987 2003) is estimated to be 325 billion cubic meter3 while industrial consumption was 665 billion m3 during the same period4. however, in the 21st century, fre

9、shwater ecosystems are vulnerable to and by climate change5,6,7, increase in burgeoning human population coupled with growing food requirements, industrialization and urban sprawl8. this turns fresh water into wastewater polluting the environment and creating health hazards to the aquatic life in th

10、e freshwater bodies making it unfit for human consumption. these polluted aquatic ecosystems are neglected owing to decline in water quality and quantity, nutrient and hence impeding species diversity, photosynthesis, chlorophyll and the biochemical composition which includes lipids, carbohydrates a

11、nd proteins. this has directed towards the threshold of water crisis and the urgent need for developing appropriate water management plans. along with water management the utilization of biotic components like macrophytes9,10, micro11,12 and macrolage13 as sources of energy has gained prominence in

12、recent years in an era of global warming in addressing production and utilization of renewable energy while dealing with the social and ecological problems.biodiesel is a proven fuel and the technology for more than a decade now14. water is the primary factor in the development of biofuel feedstock

13、production15. numerous researches have been carried out on the production of biodiesel through vegetable oils16 and other plant oils17,18. but due to the high cost of these oleaginous materials, the commercial production of biodiesel is hindered. therefore, finding cheaper way of producing biodiesel

14、 is the need of the hour.lipids, the important secondary metabolite owing to specific cell functions and cell signaling pathways play a role in biodiesel production19. major feedstock of biodiesel include soybeans, canola oil, animal fat, palm oil, corn oil, waste cooking oil and jatropha oil20. the

15、se crop based biofuels have limitations like low biomass productivity (table 1), requirement of large land area and its non renewability21. the other limitation includes the inadequacy of these crops and animal fats oil to meet the existing demand for fuels21. micro algae are efficient biological fa

16、ctories capable of taking zero-energy form of carbon and synthesize it into a high density liquid form of energy (natural oil) and are capable of storing carbon in the form of natural oils or as a polymer of carbohydrates22. microalgae as primary producers form the basis of the food web and play a s

17、ignificant role in the biotic and abitoic interactions of any aquatic ecosystem. the variation in water chemistry and biotic components of an aquatic ecosystem consequent to anthropogenic stress attributes to changes in the structure of microalgae at community level. the concept of using microalgal

18、lipid as a source of fuel is very mature, but its approach in benefiting multiple needsboth environmental and energy-related is an upcoming area of research. hence characterizing the microalgal community is critical for better understanding of the ecological as well as biogeochemical processes23. ov

19、er the past few decades, several thousand algae and cyanobacterial species have been screened for high lipid content of which several hundred oleaginous species have been isolated and characterized under laboratory and or outdoor conditions12,21,24,25. the current investigation focuses on lipid char

20、acterization of the micro algal community in bangalore collected from 4 wetlands with a wide range of environmental characteristics and an agricultural field.table 1biofuel source comparison21, 37feedstockcropoil yield (l/ha)corn172soybean446canola1190jatropa1892coconut2689oil palm5950microalgae a13

21、6900microalgae b58700a= 70% oil (by wt) in biomass and b= 30 % oil (by wt) in biomassii. study areaselected four lakes and an agricultural field for the microalgal community and lipid characterization investigation fall within bangalore (fig. 1), the capital city of karnataka and is the fifth larges

22、t city in india. bangalore city is located at 12.9406990n and 77.7465960e geographic position, at an elevation of 900 meters and a surface area of 741 sq. km (as per 2007). mean annual temperature being 24 c with extremes ranging from 15 c (in winter) to 37 c (in summer). the average annual rainfall

23、 is 859 millimeters26. the selection of lakes for the study was based on the levels of anthropogenic stress upon the lake, covering different environmental condition. samples from each lake were sampled at inlet channel so as to record the pollution level on microalgal lipid content. the doddabidare

24、kallu lake (nagasandra lake) with an area of 13.07 ha is situated in the industrial area (peenya industrial area), receives industrial effluents representing the industrial waste sample. hesaraghatta (371.24 ha) and hoskere lake (also known as soolekere) with a surface area of 15.54 ha is situated o

25、n the city boundary (as per bbmp) and is relatively clean without any sewage and industrial waste into the lake channel while, varthur with an area of 180.4 ha receives about 40% of the citys sewage. agriculture field was selected near the varthur lake to see the effects of inorganic fertilizers wit

26、h high phosphates and nitrates. hoskere lakevarthur agriculturevarthur lakehesaraghatta lakedoddabidarekallu lake1-doddabidarekallu2-hoskere3-hesaraghatta4- varthur lake5-varthur agriculturenote: sampling sites are 1- doddabidarekallu, 2- hoskere, 3- hesaraghatta, 4- varthur lake, 5- varthur agricul

27、ture)fig 1. sampling locations in bangalore, karnataka, indiaiii. materials and methodsa. water sampling and analysis four lakes (fig. 1) were selected based on the exploratory survey of 15 lakes during eight months (september 2009- april 2010) which includes water quality analysis of both inlet and

28、 outlet channels. water samples were collected from four lakes and one agricultural field in bangalore during may 2010. they were selected based on the anthropogenic stress (industrial runoff, sewage runoff, unpolluted, high nutrient load) influencing on it. triplicates were collected at each sampli

29、ng point in 1l polythene bottle. on site physical parameters like ph, water temperature (wt), electric conductivity (ec), salinity and total dissolved solids (tds) were analyzed using ph/ec probe. dissolved oxygen (do) was estimated following wrinklers method. samples were brought to aquatic ecology

30、 laboratory for further analysis of chemical variables such as nitrates, phosphates, alkalinity, total hardness, calcium hardness, magnesium hardness, chlorides, sodium, potassium, biological oxygen demand (bod) and chemical oxygen demand (cod). these variables were estimated as per standard procedu

31、re27.b. microalgae sampling microalgae were sampled from aquatic plant at all sampling points by shaking vigorously and then squeezed in the plastic bag. the resulting brown suspension is transferred into a polythene sample bottle and preserved. community structure analysis: 0.5 ml of the preserved

32、microalgal sample was observed under light microscope (100x magnification). the entire coverslip was covered to record the presence/absence data of the taxa and photographed for identification. c. chlorophyll estimation 25 ml of the microalgal sample was centrifuged t 300 rpm and was filtered. the f

33、iltered sample was then processed for chlorophyll estimation following apha method (apha 10200 h).d. lipid characterization25 ml of the microalgae sample was sonicated28 in water bath for 2 hours at room temperature in order to disrupt the cell membranes, chloroform: methanol (2:1) was added as the

34、extraction solvent. the chloroform layer was evaporated using rotary evaporator (eppendorf vacuum concentrator 5301) to obtain lipids. thin layer chromatography: all samples were reconstituted in chloroform to make stock solutions. the stock solutions were spotted in bands onto silica gel tlc plates

35、 (merck kgaa). the mobile phase consisted of a solvent system of hexane/diethyl ether/acetic acid (70:30:1 by volume)29. the plates were developed by exposing the vapors of iodine crystals to stain the plates for visualizing neutral lipids. the samples were extracted and stored in -20 c until furthe

36、r analysis30.e. gas chromatographymass spectrometry analysisafter the initial thin layer chromatography (tlc) lipid screening, the extracts were converted into fatty acid methyl esters (fame) using boron trifluoride-methanol and was heated in water bath at a temperature of 60 c for 1 hour. the methy

37、lated sample was then purified further for gc-ms. the main focus of using gc-ms was purely for lipid identification rather than quantification. the injector and detector temperatures were set at 250 c while the initial column temperature was set at 40 c for 1 min. a 1l sample volume was injected int

38、o the column and ran using a 50:1 split ratio. after 1 min, the oven temperature was raised to 150 c at a ramp rate of 10 c min-1. the oven temperature was then raised to 230 c at a ramp rate of 3 c min-1, and finally the oven temperature was increased to 300 c at a ramp rate of 10 c min-1 and maint

39、ained at this temperature for 2 min. the total run time was programmed for 47.667 min. the mass spectra were acquired and processed using agilent chem station (5975 c; agilent, usa).acbdfig. 2 variation in water physical and chemical variables a): ph, b)ec,sal and tds, c) n and p, d) chlorophyll com

40、position across sites (mg/l)iv. results and discussiona. water qualityphysical and chemical variables analyzed across the sampling sites (lakes) are listed in table 2. the ph ranged from neutral to alkaline (7.13 9.42 as in fig. 2a), highest being in the hesaraghatta lake (9.42) due to the increased

41、 acid neutralizing capacity. ionic concentration was low at hesarghatta lake (150.7 s), hoskere lake (337 s) and fairly high at doddabidderakallu lake (3320 s) owing the industrial pollution. difference between hesarghatta and doddabidderakallu lakes was significant by ec, sal and tds (fig. 2b). amo

42、ng water chemistry variables, phosphates, chlorides, hardness and alkalinity showed a high value in doddabidderakallu followed by varthur akin toconditions in agriculture site while hesarghatta andhoskere showed low range reflecting clean water compared to the former sites. nitrate levels of agricul

43、tural field (1.203 mgl-1) encompassed the low range as observed in doddabidderakallu (0.84 mgl-1), varthur (0.594 mgl-1), hoskere (0.246 mgl-1) and hesarghatta (0.215 mgl-1) lakes. high amount of phosphate was sensible in doddabidderakallu (1.93 mgl-1) compared to other lakes (fig. 2c). this high am

44、ount of nutrients and ionic concentrations, mainly alkalinity and hardness in doddabidderakallu can be attributed to the untreated industrial effluents and sewage into the inlet channel. even though varthur showed moderate water quality, high amounts of contamination has been reported in the past31.

45、 hoskere and hesarghatta showed a negligible amount of anthropogenic activities except for few local disturbances. the elevated nitrate and phosphate concentrations in agriculture site were evident from the intrusion of fertilizers. table 2:water quality variables of 5 sampling sites (1-5 as describ

46、ed in study area)12345ph8.218.559.427.237.13wt (oc)27.0030.333.132.5029.80ec ( s)3320337.1501122127tds (mgl-1)2370.0230.0102.7781.00886.00sal (mgl-1)1640.0159.075.70560.00623.00turbidity (ntu)139.0013.2015.0071.7044.30do (ppm)0.009.3512.2013.339.35cod (mgl-1)240.00213.3117.3128.00250.67bod (mgl-1)1.

47、56.25.52.533.52n (mgl-1)0.840.2460.2150.5941.203p (mgl-1)1.930.080.170.761.40chlorides (mgl-1)610.6062.4822.72187.44249.92total ha (mgl-1)680.0096.0080.00232.00332.00ca. ha (mgl-1)439.8167.9839.9759.86147.85mg (mgl-1)107.3116.599.7514.6136.08alkalinity (mgl-1)1080.0160.0380.0440.00540.00b. community

48、 structure analysis the community structure of microalgae through microscopic analysis resulted with 27 genus belonging to 4 classes with 2 unidentified filamentous algae (table 3). the class bacillariophyta (diatoms) and chlorophyta dominated at hoskere and varthur lake as well as agricultural samp

49、le with achnanthidium ktzing, gomphonema ehrenberg, nitzschia hassall, navicula bory de saint-vincent, chlamydomonas ehrenberg, scenedesmus meyen and anabaena bory de saint-vincent ex bornet & flahault accounting more in number (occurrence number in microscopic field). dodabidarekallu was represente

50、d by nitzschia sp. alone, whose presence justifies high ionic and organic nutrients load. hoskere was well occupied by diatoms viz., fragiallria lyngbye, sellaphora mereschowsky, surirella turpin along with the former species. significant relation of ecology of microalgae such as nitzschia sp., sell

51、aphora sp., chlorella m.beijerinck and phacus dujardin (varthur and agricultural field samples) with the extent of pollution load was observed. table 3community structure of 5 sampling sites (1-5 as described in study area. + indicates presence and indicates absence of species)sampling sites12345bac

52、illariophytaachnanthidium ktzing-+-cyclotella (ktzing) brbisson -+-+-cymbella c.agardh-+-diploneis ehrenberg ex cleve-+-fragillaria lyngbye-+-gomphonema ehrenberg-+-+navicula bory de saint-vincent-+-+nitzschia hassall+-+rhopalodia otto mller-+-sellaphora mereschowsky-+-+surirella turpin-+-chlorophyt

53、achlamydomonas ehrenberg-+-chlorella m.beijerinck-+chlorogonium ehrenberg-+-closterium nitzsch ex ralfs-+-cosmarium corda ex ralfs-+-monoraphidium komrkov-legnerov-+-+-pandorina bory de saint-vincent-+-scenedesmus meyen-+-euglenophytaeuglena ehrenberg-+-phacus dujardin-+trachelomonas ehrenberg-+-fil

54、amentous algaefilamentous algae 1-+-filamentous algae 2-+-cyanophytaanabaena bory de saint-vincent ex bornet & flahault-+-cylindrospermopsis seenayya & subba raju+-merismopedia meyen-+-+-c. water quality and community structure nitzschia sp. was prevalent in doddabidderakallu with the high quantum o

55、f nutrients and ionic concentrations. compared to this varthur showed moderate water quality, while, hoskere and hesarghatta showed a negligible amount of anthropogenic activities except for few local disturbances. the elevated nitrate and phosphate concentrations is observed in agriculture sites. t

56、he class bacillariophyta (diatoms) and chlorophyta dominated at hoskere and varthur lake as well as agricultural sample. occurrence of microalgae such as nitzschia sp., sellaphora sp., chlorella m.beijerinck and phacus dujardin with the extent of pollution load show significant correlation (p0.05). d. lipid analysisthe neutral lipid profile of the microalgal community revealed characteristic profile of the given community. the neutral lipid profile of each lake which is characteristic feature of the thriving microalgal community is given