VIETNAM JOURNAL OF CHEMISTRY VOL. 51(3) 384-389 JUNE 2013
CULTURING CHLORELLA VULGARIS IN ANAEROBICALLY DIGESTED PIGGERY WASTEWATER FOR CONCOMITANT NUTRIENT REMOVAL
AND BIODIESEL FEEDSTOCK PRODUCTION
Doan Thi Thai Yen*, Nguyen Minh Giang
Institute for Environmental Science and Technology, Hanoi University of Science and Technology Received 14 September 2012
Abstract
Anaerobic digestion of piggery manure generates effluents which containing high concentration of inorganic nitrogen and phosphorus and causes eutrophication when discharged into natural water bodies. Microalgae can be cultured in an anaerobic eflluent as a required tertiary treatment as well as for biodiesel feedstock production. In this study, anaerobic digested piggery effluent was used as nutrients source for culturing green microalgae Chlorella vulgaris. The effect of dilution of the anaerobically digested piggery effluent nutrients removal efficiency of Chlorella vulgaris was determined During 8 days in batch culture, Chlorella vulgaris grew faster m the more diluted wastewater, which related to initial COD concentration of about 400 mg/L, The maximum specific growth rate and biomass concentration were 0.306 day"' and 0.191 g/L, respectively, Microalgae removed ammonia, total phosphorus and COD by 60-95.8%, 22-68% and 34-73.8%, respectively. The lipid contents of algal biomass after 8 day were 28-30% of dry weight, which could be utilized as a feedstock for biodiesel production.
Keywords' Piggery wastewater, Chlorella vulgaris, biodiesel feedstock, nutrient removal, lipid content.
1, INTRODUCTION
The potential of microalgae as a base biofuel resource has been recognised with exploratory research projects developed in the 1980's and early 1990's [1]. Currently, the rationale for extensive development of renewable fuel resources is seen as key to management of world energy demands in a sustainable manner [2], and microalgae have the capability to produce bigh-lipid content biomass, as precursor for liquid biofuels that does not conflict with food production [3].
To date, great efforts have been concenfrating on reducing the costs of producing biodiesel from microalgae. One of them is reuse of digested piggery wastewater for microalgae cultivation for biofuel feedstock [4, 5], Digested piggery effluent could be an alternative nutrient source for mass microalgal production since anaerobically treated waste contains nutinents such as N and P (NH,*, NO3", PO,,^") which are suitable for growing algae.
Additional, pathogens in the digested effluent were eliminated particular after two-stage (thermophilic and mesopbilic) digestion. Therefore, digested effluent as medium of algal culhire could alternative costly mineral medium, which cause to less utilize
fresh water and remove effectively piggery wastewater.
The sfrain of Chlorella vulgaris used in the present study is popular for wastewater freatment and biodiesel feedstock [6, 7]. C. vulgaris was found to mixofropbically grow in diluted piggery wastewater from a local pig farm. The relationships between the initial COD values and properties such as the growth rate, the elimination of nutiients (NH4'-N, TKN, P04'"-P, total P and COD) and the algal lipid content were individually investigated.
2. MATERL\LS AND METHODS 2.1. Microalgal strain and culture conditions
Chlorella vulgaris was locally isolated from a pond in Co Dong pig farm. Son Tay, Hanoi. The sti^in was cultured in Bold' Basal Medium with vitamins (BBM+V) as described in [8]. Culture flasks were maintained at room temperature (25- 32°C) under static condition. Light irradiation was 1500 lux using 6 cool-white fluorescence tubes, with a 12:12 h of light:dark cycle. Light intensity was measured at the surface of the culture broth using a Testo 545 lux meter (Testo GmbH & Co, Germany).
VJCVoL 51(3), 2013
2.2. Sampling site of piggery wastewater The samples of piggery wastewater were collected at an effluent from an upflow anaerobic sludge blanket (UASB) freatment system in a pig farm at Sontay, Hanoi, as displayed in Fig. 1. The samples were ti-ansported to laboratory, and then filtered over a glass cotton layer to removal suspended solids. Before culturing microalgae, the samples were diluted witb distilled water to get several levels of COD concenfrations.
-^-xir*'fffti^f&-
Fig. I: Sampling site in the pig farm at Son Tay, Hanoi
2.3. Diluted wastewater to various COD concentrations
Digested wastewater was diluted to different concentrations of COD, i.e. 250, 300, 400, 530, 600 and 740 mg/L, C vulgaris was inoculated in 250 mL conical flasks containing 100 mL of various diluted piggery wastewater (triplicate cultures for each COD concenfration). The expenment flasks were manually shaken three limes a day to ensure microalgal cells in suspension. All expenmenls were terminated at day 8' .
2.4. Determination of cell growth rate The growth rate of each sti^in was characterized based on measurement of cell density every days, using a specfrophotometiy UV/VIS Lambda EZ210 (PerkinElmer, USA), at X = 680 nm. The specific growth rate of each microalgae sfrain was calculated from the slope of linear regression of time (days) and cell density (cells/mL):
\nOD,-\nOD.
Doan Thi Thai Yen, et al.
2.5. Biomass dry weight (DW)
Biomass dry weight was determined as follows:
On the terminated day of experiments, 50 mL of microalgae suspension was sampled and filtered on a pre-weigbed Whatman GF/F glass fiber filter (0.7 pm pore size and 47 mm diameter; Whatman, USA) using a Kontec Ulfra-ware microfilfration apparatus.
The algal pellets were dried overnight at 80°C and weighed,
2.6. Total lipid content
Total lipid content of microalgae was exfracted from the dry biomass using a modified solvent based on die method descnbed by Folch et al. [9] and then quantified gravimefrically. The dry biomass was fransferred into a cenfrifuge tube containing a mixture of chloroform :methanol (2:1, v/v), vigorously shaked and placed into an ulfrasonic water bath (TELSONIC Ulfrasonic Inc., USA) for 2 hours. Then, the supernatant was collected by pipette and transferred into another cenfrifiige tube. The extraction procedure was repeated twice to ensure adequate lipid extiaction. NaCI 0.9% solution was added to supernatant to make the ratio of lipid solution: solvent to be 1:5, v/v. The centnfuge tube was then vortex mixed for 5 minutes and allowed to phase separation for 15 minutes. Collecting the bottom layer of the centiifuge tiabe and deposited into a pre-weigbed aluminum fray. The aluminum tray was placed into an fume hood to evaporate solvent, then was dned overnight at 80°C and re- weighed,
2.7. Nutrients removal efficiency
C. vulgaris was cultivated in diluted wastewater for 8 days and then efficiencies of nufrient removal, i.e. chemical oxygen demand (COD), total Kjeldahl nifrogen (TKN) and total phosphorus (Total P), were examined. The analysis of COD, NH/-N and total P was conducted according to Standard Methods for the Examination of Water and Wastewater [10]. The nutiient removal efficiency was calculated as follows:
<100 t-t^
u /J ,^ .1- r, ^«,.rtS r^t<> o n i<; where n (%) is the nutrient removal efficiency, Co is where u (day-1) is the specific growth rate, UUt is Elicit •; ^ / ^f ^nitivaiinn
« .• i j V, f 1 1 11 n i ^ ^ rti ^nH o n i<; nufrient concenfration at the first day ot cultivation S r - r / f a g l o t i r r i h e i r o n h f j c g (^g/U and C ,s nu.,en. co„cen.a..o„ of the . s . phase. day (mg/L).
VJC, Vol. 51(3), 2013 3. RESULTS AND DISCUSSION 3.1. Characteristics of piggery wastewater
Piggery wastewater used for microalgae culturing was taken from effluent sfream after a UASB treatment system (Fig. 1). Analytical results of UASB effluent were displayed in table 1.
Concenfrations of pollutants such as BOD, COD, nitrogen and phosphorus after UASB treatment were still higher than Viemamese Environmental Standard for wastewater discharged, QCVN 40: 2011/
BTNMT (column B). The values of COD and BOD were 10-13 times and 5-8 times greater than the Standard, respectively. The digested wastewater of pig farm still contained very high concenfration of nifrogen and phosphorus, i.e. 22-24 times greater than Standard for TKN and 49.5-105 times for total P. The ratio of N:P fluctuated from 3-6.5:1 during sampling periods. It was mainly due to the differences of feed composition for young and mature pigs. The ratio was around 6.5:1 for young pig as feed in this penod was rich of protein. The ratios were decreased in the samples of growing pigs (i.e. 4.9:1) and mature pigs (i.e. about 3:1). It can be seen from table 1 that most of samples contained the N:P < 5. It meaned that nifrogen was the limited factor in these samples of piggery of wastewater.
Culturing Chlorella vulgaris in anaeruoically...
3.2. Culturing C vulgaris in piggery wastewater with different COD concentrations
The piggery wastewater samples were diluted in six levels of COD from 250 mg/L to 740 mg/L. in preliminary experiments, origin wastewater with high concenfrations of NHj^-N, COD (see table 1) and dark color inhibited the growth of C.vulgaris.
Thus, wastewater samples were diluted to get initial COD < 800 mg/L. The dilution ratios also ensured ammonium concenfration approximately 150 mg/L, which did not inhibited microalgae growth. The initial density of C. vulgaris inoculum in the culture medium was equivalent to 13 mg/mL for all the various COD solutions. The growth curves of the microalga were presented in Fig. 2.
Table I: Characteristics of piggery Co Dong pig farm. Son Tay,
wastewater at . Hanoi
Parameters
pH TS (mg/L)
TSS (mg/L)
COD (mg/L)
BOD, (mg/L) P O / - P (mg/L) Total P (mg/L) N H / -N
(mg/L) TKN (mg/L)
N:P'
Sampling days 21/3/
2012 7,68 3470 134 1995 390 188 635 863 966 3.4,1
06/04 /2012 7,89 3243 256 1879 240
297
872 6.5:1
17/04/
2012 7,82 3012 273 1632 310
374
834 4.9:1
3/5/
2012 8,45 3323 242 1768
246 541 756 762 3.1:1
QCVN 4 0 : 2 0 1 1 / BTNMT (column B)
5,5-9
-
100 150 50
-
6 10 40
'N = TKN/14,P = TotaIP/31
Culturing days Fig. 2: Growth curves of Chlorella vulgaris in BBM's solution and diluted piggery wastewater with
various initial COD values As can be seen in Fig. 2, C. vulgaris grew well in the high diluted wastewater with initial COD concentrations in the range of 250-400 mg/L. The optimized concentration range of initial COD for C.
vulgaris robustly growing were 300-400 mg/L. In the high COD levels (i.e. 500-740 mgCOD/L) the high TKN concenfration was the limiting factor and biomass production were comparatively low since inhibit algal growth. C. vulgaris increased more rapidly in high diluted wastewater than in BBM solution, which implied that C. vulgaris could grow mixofropbically. The culture media containing initial COD of 400 mg/L (corresponding to TKN of 226 mg/L) resulted in better C. vulgaris production. This indicated that the available organic carbon and TKN
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VJC, VoL 51(3), 2013
were being used at a maximum rate and an accelerated productivity was achieved at level of 400 mg COD/L. The COD concenfrations for the best growth of C. vulgaris in this study (COD:300-400 mg/L) were lower than the ones in Wang's report [11], i.e. COD:700-1000 mg/L. It is possible that Chlorella pyrenoidosa in Wang's study might have higher tolerance and growing capability in piggery wastewater.
Specific growth rates of diluted wastewaters with initial COD concenfration of 300 mg/L and 400 mg/L were highest (i.e. 0.26d"'), equivalent to highest biomass concenfration after 8 day of culturing (i.e. -0.2 g/L). Lag phase of of algal growth was 2 days for COD concentration of 600 and 740 mg/l, but only 1 days for the rest of the culture mediums (Fig. 2).
3 J . Nutrient removal efficiencies
Equivalent to microalga growth in various diluted wastewater, the efficiencies of nutnent removals were also vaned. The removal rates of COD were all about 34-73.8% in different samples of diluted wastewater (Fig. 3a). The best reduction of COD of 66-74% took place in an initial COD concentration of 250-400 mg/L. The good growth and considerable COD reduction in diluted piggery wastewater indicate that C. vulgaris could grow irnxofrophically since it used organic carbon as a source of energy and a subsfrate for cell growth. The most effective removal (73.7%) at day 8 for an initial COD concenfration of 400 mg/L. After 8 day inoculated, C. vulgaris in solution of initial COD
< 400 mg/L, removed COD level to lower than requirement of the National Standard (Fig. 3a).
Nifrogen is limited factor therefore removal efficiency of NH/-N achieved 95.8% for initial
Doan Thi Thai Yen, et al.
COD level of 400 mg/L (see Fig. 3b) while it was 60% for solution of initial COD of 740 mg/L. That means the higher the initial COD concenfrations , were, the lesser efficiencies of NH4*-N removal obtained, hi this stiidy, the efficiency of NH4*-N removal were greater than Park's stiidy (i.e. 95.8%
vs. 47%, respectively), but lesser for POj^-P removal (i.e. 56% vs. 68%, respectively) [12].
Remarkable reductions in total nifrogen TKN and total phosphorus were also found for all four diluted wastewater samples (Fig, 3c and d). It is obvious that the removal rate of both total nitrogen and total phosphorus increased along witb the COD value lesser than 530 mg/L.
3.4. Total lipid content
Microalgae have been known as a promising feedstock for biofuels, especial for biodiesel due to high intracellular lipid production. In this study, lipid content of biomass harvested from wastewater cultures was determined. The lipid contents produced in BBM's solution and diluted piggery wastewater of 400 mg COD/L were roughly at equal levels, i.e. 28.3 and 28.8% DW, respectively (table 2). However, lipid contents for the solution of 250 and 300 mg COD/L were slightiy higher, i.e 30% of DW. It might be due to a limitation of nitrogen in these solution. The results showed that C. vulgaris accumulated mfracellular lipid during cultivation period m wastewater. The rest solutions of wastewater-algae cultunng contained rather low lipid content, i,e.
< 14%, due to C vulgaris grew poorly in these solutions. With the lipid content about 28-30%, biomass of C -vulgaris can be used as a source for biodiesel production. For high quality of biodiesel feedstock, more study to optimize both biomass and lipid productivities have to be carried out.
Table 2: Total lipid content of biomass cultivated in BBM solution and diluted piggery wastewater.
Data expressed in mean values ±SD (n=3) Lipid
content (%DW)
BBM 28.3±0.5
Diluted wastewater with various initial COD concentration COD-250
30.0±0.4
COD-300 30.1+0.5
COD-400 28.8±0.8
COD-530 14.0+0.7
COD-600 13.8±0.5
COD-740 13.2±0.7 4. CONCLUSIONS
C -vulgaris can removed efficiently nutnents in digested piggery wastewater. The highest growth rate of C vulgaris was occurred in diluted wastewater with initial COD concenfration of 300- 400 mg/L. These diluted solutions were also
displayed the best efficiencies of nutnent removals, I.e. 66.4-73.7% for COD, 95-95,8% for NH^-N, 78.9-86.7% for TKN, 54.4.56,1% for P04^'-P and 58.8-59.2 for total P. During cultivation in diluted wastewater the lipid content of C. vulgaris was about 28-30% which can be used for biodiesel feedstock.
VJC, Vol. 51(3), 2013 Cullwing Chlorella vulgaris in anaerobically.
800 ^ C O C ( d a y O ) COD (ctav 8) -^700 National Slanda f 600
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Fig. 3: The removal rates of (a) COD, (b) ammonium, (c) total nitrogen, (d) phosphate and (e) total phosphorus calculated from their contents at the initial and final stages of the cultivation of Chlorella
vulgaris in various diluted piggery wastewaters
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VJC, Vol. 51(3), 2 0 1 3
Acknowledgement: Financial supports from Hanoi Department of Science and Technology. Vietnam (Grant code OlC-09/02-2012-2) is gratefully
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Corresponding author: D o a n T h i T h a i Y e n
H a n o i University of Science and Technology 1 Dai C o Viet, Hai Ba Trung, Hanoi Vietnam
Email; y e n h a n b n g u y e t @ y a h o o , c o m .
