Bioresource Technology xxx (2014) xxx–xxx
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Short Communication
Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent Luis Guerrero-Cabrera ⇑, José A. Rueda, Hiram García-Lozano, A. Karin Navarro Universidad del Papaloapan, Av. Ferrocarril s/n, Loma Bonita, Oaxaca, Mexico
h i g h l i g h t s Chemical composition of three algae cultured in pisciculture effluent was studied. Nitrogen and phosphorus in effluent were efficiently absorbed by the algae. Effluent shows a lower productivity than a formulated medium in Scenedesmus sp. Protein percentage is higher when effluent is used but productivity is limited. Photobioreactors with lower volumes give a higher productivity and growth rate.
a r t i c l e
i n f o
Article history: Received 18 January 2014 Received in revised form 20 March 2014 Accepted 23 March 2014 Available online xxxx Keywords: Pisciculture effluents Wastewater as culture medium Secondary effluent cultures Fresh-water algae culture
a b s t r a c t Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae were cultured in three volumes of Tilapia Effluent Medium (TEM) in comparison with the Bold Basal Medium (BBM) (Nichols and Bold, 1965). Specific growth rate (l0 ), biomass dry productivity (Q), volumetric productivity (Qv) as well as lipid and protein content were measured. Then, volumetric productivities for both lipids and proteins were calculated (QVL and QVP). In Scenedesmus sp., BBM produced higher l0 and Qv than TEM in 1.5 L volume. Chlorella sp. showed a higher QVL for BBM than TEM. Any observed difference in protein or lipid productivities among volumes was in favor of a greater productivity for 1.5 L volume. Even when TEM had a larger protein content in Chlorella sp. than BBM, QVP was not different. Current results imply that TEM can be used as an alternative growth medium for algae when using Batch cultures, yet productivity is reduced. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction In aquatic ecosystems, phytoplankton growth depends upon light, temperature, salinity, pH and available nutrients. Nutrients such as nitrogen and phosphorus may be available in different chemical compounds. The major sources of nitrogen are nitrates and ammonium–nitrogen, but other inorganic compounds like nitric oxide, nitric acid and nitrites can be used, as well as organic compounds such as urea. The organic to inorganic ratio of nitrogen absorption may differ among phytoplankton species and it can be affected by environmental changes, by the nitrogen source, and by the concentration of phosphorus, carbon and oxygen (Barrera et al., 2008). Inside the cell, nitrate is turned into nitrite and then reduced to ammonium in a two-step reaction catalyzed by nitrate and nitrite reductases. Then, nitrogen incorporates into organic compounds ⇑ Corresponding author. Tel./fax: +52 281 8729230x220. E-mail address: [email protected] (L. Guerrero-Cabrera).
(Chen et al., 2009). Sodium nitrate and potassium nitrate are used in growing cultures in accordance with reports of high growth rate and high biomass productivity. In fact, most of the experiments in lipid accumulation induction are based on setting limits to nitrates as nitrogen source, thus reducing the nitrate level below the recommended concentrations for a culture medium (Griffiths and Harrison, 2009). Ammonium is not widely used as a nitrogen source in algae cultures, since growth rates and biomass productivity do not reach the levels achieved using nitrate instead of ammonium. Some of the media which use (NH4)2HPO4 and (NH4)2SO4 are Crammer Mayers for freshwater and PCR-S11 for marine water, respectively (Barsanti and Gualtieri, 2006). Both ammonium and urea are extensively used as fertilizers in field-cultivated crops, but they can also be found in high concentration in effluents from many productive activities and services. Nitrogen and phosphorus concentrations occur in a wide range in secondary effluents including livestock manure, human waste, and agricultural remnant waters. Municipal effluents and treated
http://dx.doi.org/10.1016/j.biortech.2014.03.127 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Guerrero-Cabrera, L., et al. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.127
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waters are some of the cases with higher concentrations. In particular wastewater may contain from 95 to 264 mg L1 of ammonium and 125 to 530 mg L1 of total phosphorus (Min et al., 2011). Domestic secondary effluent contains from 7 to 25 mg L1 of total nitrogen and from 0.04 to 1.93 mg L1 of total phosphorus (Li et al., 2010b; Wu et al., 2013). Municipal wastewater containing dairy sewage has 40 mg L1 of ammonium and 11 mg L1 of total phosphorus (Barrera et al., 2008). Livestock wastewaters represent a good alternative for microalgae cultures because of their low content of toxic elements. Furthermore, high phosphorus and nitrogen concentration in such effluents allow for their usage without dilution. Other examples are: pretreated piggery wastewater, which contains 268 mg L1 of ammonium and 56 mg L1 of orthophosphate (Gál et al., 2007); shrimp aquaculture wastewater, with 0.9 to 0.1 mg L1 ammonium (Da Silva et al., 2009) and marine fish effluent bio-filtration with 1 mg L1 of ammonium and 1.5 mg L1 of orthophosphate (Borges et al., 2005). Microalgae cultured in the above-mentioned effluents change their metabolic strategy from autotrophic to chemoheterotrophic or mixotrophic. Thus, photoautotrophic algae cultured in media containing organic compounds, use such substances as an alternative carbon source (Barsanti and Gualtieri, 2006). A variety of algae species have been studied using a secondary effluent as growth medium. Assessed variables range from growth rate, biomass productivity, chemical composition, through chlorophyll production, among others. Some of the more important purposes of such assays were biodiesel and lipid production with Monoraphidium sp. (Bogen et al., 2013), Scenedesmus sp. (Li et al., 2010a; Wu et al., 2013) and Chlorella sp. (Ji et al., 2013). Microalgae may provide a viable alternative to fossil biofuels, although this technology has yet to overcome a number of challenges including specie selection, growth media improvement and bioreactor design, in order to achieve a higher productivity. Several formulated media have been used for microalgae cultivation, in an attempt to increase the benefit–cost ratio. Recently, nonconventional media have been studied in hopes of finding an inexpensive alternative to commercial media. In particular, aquaculture effluents have been assessed in a continuous photo-bioreactor (PBR) or in outdoor conditions, but they have not been assessed in Batch cultures in controlled conditions, where it will be possible to measure accurately their potential in biomass production. Present work evaluates the viability of using the Nile tilapia effluent as an alternative medium for algae culture in order to reduce expenses in a scope of sustainability. 2. Methods 2.1. Algae The three algae species: Chlorella sp., Monoraphidium sp. and Scenedesmus sp. were isolated by sequential plate seeding method with Agar-Basal Bold Medium (BBM) from samples collected in natural freshwater ponds in Loma Bonita, Oax., Mexico. Algae morphological identification was completed through genera using specialized taxonomical guides (Shubert, 2003). The strains used were kept as Mon10UNPA-49, Sce10UNPA-44 and Chl10UNPA-45 from the Aquaculture Laboratory of the Universidad del Papaloapan, with re-seeding phase indicated after the dash.The three algae were grown in a 500 mL PBR with BBM. Algae were not pre-conditioned or adapted to the tilapia effluent for the Batch cultures in the assay. 2.2. Tilapia effluent medium (TEM) To obtain the TEM, a 20 kg m3 live weight stock of Oreochromis niloticus (line Gift) was kept in freshwater glass aquariums in
laboratory conditions for 78 h. The tilapia fishes were kept under intensive feeding regime (3% of live weight in food per day), temperature was 25 °C and air supply was steady. After this period, the effluent was collected, filtered through a 55 lm zooplankton mesh and sterilized by boiling it for 10 min. The effluent was cooled at room temperature and pH was adjusted to 7.5 (HannaÒ Instruments pH meter) with 1 M KOH and 1 M HCl. Finally, ammonium content (by the Nessler method modified) and orthophosphate content (by the Ascorbic Acid method) were measured so the initial conditions were known. These conditions in TEM are presented in Table 1. The nitrogen and phosphorus sources in BBM were 150 mg L1 of NaNO3, 175 mg L1 of KH2PO4 and 75 mg L1 of K2HPO4. 2.3. Algae culture Polyethylene terephthalate (PET) bottles with flat wall were used as PBR. Experimental dimensions were 1.5, 4 and 9 L. (diameter/height of 9/21, 14.5/22, and 19.5/29 cm, respectively). The experiments for each of the three algae were conducted simultaneously. PBRs were kept in laboratory under steady light and temperature conditions. Two parallel lines of 60 W fluorescent PhilipsÒ T12 lamps (6000 lm) 20 cm apart, were used and room temperature was 25 °C. Air supply, without CO2 enrichment, was adjusted from 1.5 to 4 L and from 4 to 9 L by a 1.7 factor according to preliminary assays, in order to optimize biomass productivity (data not shown). Air flow was established at 1.6, 2.9 and 4.9 L min1 for 1.5, 4 and 9 L, respectively. In order to estimate the nutrient removal, ammonium and orthophosphate contents were measured after the cultivation period. 2.4. Analytical methods Cumulated biomass was harvested at the end of the growth period. Biomass dry productivity (Q) was measured in an electronic moisture analyzer (scale MOC-120H, ShimadsuÒ). The volumetric productivity obtained in g L1 d1 (Qv) by means of Qv = l0 Q, where Q is biomass concentration in g L1 (Griffiths and Harrison, 2009; Li et al., 2010a). Dry matter samples were preserved to assess lipid concentration by the Bligh and Dyer method (1959) modified (Mandal and Mallick, 2009) through a 2:1 methanol/chloroform ratio by gravimetry. Protein content was obtained by the Bradford method. Both lipid productivity (QL) and protein productivity (QP) were then expressed as volumetric productivities in accordance with Griffiths and Harrison (2009):
Q VL ¼ ðQ V Þðpercentage of lipid contentÞ=100
ð1Þ
Q VP ¼ ðQ V Þðpercentage of protein contentÞ=100
ð2Þ
2.5. Kinetics parameters analysis The growth kinetics was studied by daily cell count in a Haematocytometer chamber during the 20 d period of Batch
Table 1 Characteristics of Tilapia Effluent Medium used for algae culture. Characteristic
Measure
N-NH4 NO3 NO2 pH PO4 Alkalinity
24 mg L1 ND ND 7.5 10 mg L1 80 mg L1
ND: Not detected.
Please cite this article in press as: Guerrero-Cabrera, L., et al. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.127
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culture, because at the end of this period the increase in cell density stops. Specific growth rate (l) was first calculated by the classical logistic model (Wu et al., 2013), thus relating starting to final cell count during exponential growth phase. However, data did not show such a phase and approach was changed to the numerical differentiation method to obtain l, the five point formula. This last method described kinetics as a smooth line which moves with data, allowing nonlinear fit by taking into account each point while reducing error and increasing accuracy (Burden and Douglas, 2011). Five point formula method is a numerical derivative, which implies there is always a near polynomial to a given continuous function close enough to all the data dots in that function for a specific interval (Burden and Douglas, 2011). The specified function was drawn from the data and fitted by a Lagrange polynomial, where error term is from order O(h4) where h equals time for each of the sample dots. This five point formula Eq. (3)includes x0 as the starting point for the derivative and s between x02h y x0 + 2h. To calculate numerical derivatives in each of the PBR, days 3, 6, 9, 12 and 15 were taken as the five reference points. The specific growth rate was computed as the quotient of the derivative of f(x0) by cell density (Cn) measured on the 9th day, as shown in Eq. (4).
f 0 ðx0 Þ ¼ ð1=12hÞ½f ðx0 2hÞ 8f ðx0 hÞ þ 8f ðx0 þ hÞ 4
f ðx0 þ 2hÞ þ ðh =30Þf ðv Þ
ð3Þ
l0 ¼ ð1=C n ÞðdC n =dtÞ
ð4Þ
Data was analyzed using a 2 3 factorial design, for two media (TEM and BBM) and three volumes (1.5, 4 and 9 L), with two replicates. Different analyses of variance were run for each alga-variable by the MIXED procedure and means comparison was done by a Tukey test in SAS software. 3. Results and discussion 3.1. Growth rate and productivity Specific growth rate (l), five-point-formula growth rate (l0 ), biomass dry productivity (Q) and volumetric productivity (Qv) are given in Table 2. Specific growth rate (l) obtained by classical methods was not related to Qv and means for l are presented just as a reference. Meanwhile, higher l0 values were closely associated with higher Qv in Scenedesmus sp. algae. For Chlorella sp. and Monoraphidium sp. algae, l0 was not related to Qv. Furthermore, data did
not show differences either for volumes or culture media for Qv or l0 . In Scenedesmus sp., there was no medium by volume interaction, either for l0 or for Qv. There was an evident advantage for the BBM with respect to TEM (l0 : 0.19 vs. 0.078, P < 0.05; Qv: 0.138 vs. 0.067, P < 0.05) for this alga (Fig. 1). In addition, l0 was different among PBR sizes, where the 1.5 L volume had higher l0 for Scenedesmus sp. with respect to 4 or 9 L (0.23 vs. 0.12 and 0.06, P < 0.05), corresponding with a higher Qv for the 1.5 L volume (0.25 vs. 0.04 and 0.017 g L1 d1, P < 0.05). No statistical differences in Qv were observed among media for Chlorella sp. (0.06 vs. 0.12 for TEM and BBM) or Monoraphidium sp. (0.4 vs. 0.049 for TEM and BBM). Furthermore Monoraphidium sp. showed a higher Qv in the 1.5 L volume (0.08 vs. 0.03 and 0.02) than in the other volumes as well. Biomass dry productivity (Q) was not different among media for any of the algae. Different approaches have been reported to approximate l. Nonetheless, culture conditions were so different in such assays that direct comparison is not supported. According to Wu et al. (2013), it is necessary to establish a relationship between l and dry productivity for each of the species. The nonlinear fit proposed in this work to calculate the growth rate is recommended for any condition where exponential phase is not evident, a common case when dealing with the use of pisciculture effluents as culture mediums. It has been found that Qv is affected, in both outdoor and in laboratory conditions PBRs by container height and diameter. This is mainly explained by medium optical depth and light requirements of algae cells (Lee, 2001). Wu et al. (2013) describe this fact as an exponential decay function. In addition, container or pond dimension affect air supply and air distribution (Grobbelaar, 2008). The data showed clear biomass productivity decay as volume increases, which according to the above mentioned can be explained in terms of differences in optical depth and air supply among volumes, expressed as a higher efficiency in smaller containers (1.5 vs. 4 and 9 in this particular case). Emphasis must be given to the fact that light remained steady in the run experiment, but dimensions of PBR have a clear effect on the mentioned variables. Higher Qv obtained in 1.5 L BBM than in 1.5 L TEM for Scenedesmus sp. and for Chlorella sp. is due to higher nitrogen and phosphorus contents in BBM than in TEM. The fact that Monoraphidium sp. was the exception to this difference in Qv among mediums may reflect interspecific differences in nutrients use. Borges et al. (2005) found that Tetraselmis sp. had three times as much productivity in snook
Table 2 Specific growth rate (l), dry biomass productivity (Q) and volumetric productivity (Qv) in three algae species cultured in Tilapia Effluent Medium and in Bold Medium. Specie/volume (L)
Q (g L1)
l0
l
Qv (g L1 d1)
TEM
BBM
TEM
BBM
TEM
BBM
TEM
BBM
Chlorella sp. 1.5 4 9 SE
0.006b 0.018a 0.015a
0.013 0.012 0.011 0.001
0.046c 0.367a 0.202b
0.143 0.166 0.177 0.027
1.036a 0.070b 0.437b
1.259a 0.788ab 0.308b 0.098
0.048 0.047 0.089
0.179a 0.127ab 0.054b 0.0175
Scenedesmus sp. 1.5 4 9 SE
0.006b 0.014a 0.010a
0.009 0.008 0.008 0.001
0.134a 0.075b 0.026b
0.322a 0.164b 0.092b 0.025
1.264a 0.344b 0.207b
1.032a 0.325b 0.325b 0.045
0.172a 0.026ab 0.005b
0.332a 0.054b 0.030b 0.027
Monoraphidium sp. 1.5 4 9 SE
0.011 0.013 0.007
0.012 0.012 0.012 0.001
0.146 0.118 0.119
0.158 0.165 0.156 0.030
0.600a 0.162b 0.111b
0.481a 0.274b 0.166c 0.0178
0.088a 0.019b 0.013b
0.076 0.045 0.027 0.01
a, b, c: Means in the same column with different letter are statistically different (P < 0.05) within each alga and variable; which shows differences among volumes. SE: Standard error of volume by medium interaction.
Please cite this article in press as: Guerrero-Cabrera, L., et al. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.127
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a
160
3.2. Lipid content
140
Lipid content expressed as a fraction of Q in percentage (%), as dry productivity per volume (QL) or as accumulated volumetric productivity per day (QVL) is shown in Table 3. Chlorella sp. showed a higher QVL for BBM than for TEM (0.0068 vs. 0.0031, P < 0.05). However, for Monoraphidium sp. or Scenedesmus sp., media did not show differences in lipid productivity either as percentage, as QL or as QVL. Regarding lipid content expressed as QL, there was no interaction volume by medium and 1.5 L PBR had a higher QL than 4 and 9 L volumes for Scenedesmus sp. (0.113, 0.031 and 0.014, P < 0.05) and Chlorella sp. (0.066, 0.024 and 0.013, P < 0.05). Furthermore, in Monoraphidium sp. interaction was present and it showed the same pattern in TEM (greater lipid productivity in the 1.5 L volume) only when expressed as volumetric productivity (QVL). According to the results of the present assay, higher QVL is reached by BBM than by TEM in Chlorella sp. Available nutrient content in each medium plays a central role in achieving the potential lipid productivity by algae. A lower biomass productivity is particularly expected when nitrogen is limited, which can be partially mended by the availability of a carbon source, such as glucose (Mandal and Mallick, 2009). Actual results agree with Ji et al. (2013), who report improved lipid production when using a formulated medium compared with piggery wastewater effluents for Chlorella vulgaris culture. Although ammonium–nitrogen in TEM is lower than nitrates–nitrogen in BBM, nitrogen does not seem to be limited in TEM, the concentration of this nutrient in the organic matter of TEM fills the cell requirements for development.
Cell count x 10-6
120 100 80 60 40
BBM
20
TEM
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 me (d)
b
0.35
Qv (g L -1d-1)
0.3 0.25 0.2 0.15
BBM
0.1
TEM
0.05 0
1.5
4
6.5
9
Volume (L)
μ'
c
0.35
3.3. Protein content
0.3
Protein content expressed as fraction of Q in percentage (%), as dry productivity per volume (QP), or as accumulated volumetric productivity per day (QVP) is shown in Table 4. QVP in 1.5 L volume was higher than in 4 and 9 L volumes for Monoraphidium sp. in BBM. In Chlorella sp. and Scenedesmus sp., independently of medium, the highest protein content in percentage was found for 4 L (43.45 vs. 22.3 and 32.1 for Chlorella sp. and 44.2 vs. 38.1 and
0.25
BBM
0.2
TEM
0.15 0.1 0.05 0
1.5
4
6.5
9
Volume (L) Fig. 1. (a) Daily cell count of Scenedesmus sp. algae using Tilapia Effluent as culture medium in comparison with Basal Bold Medium. (b) Volumetric productivity of Scenedesmus sp. algae cultured in Nile tilapia effluent and Basal Bold Medium. (c) Growth rate of Scenedesmus sp. algae cultured in Nile tilapia effluent and in Basal Bold Medium.
effluent than in the Guillard medium, while Phaeodactylum tricornutum had a major productivity in Guillard medium. Similarly, the data showed a higher Qv for Monoraphidium sp. in TEM than in BBM at 1.5 L volume, the opposite happening in 4 L volume, which could imply changes in nutrient usage when light is limited. Data for Scenedesmus sp. is completely coherent with respect to the relationship among the studied variables in the current assay, where the highest values for Qv and l0 concur in 1.5 L, as well as in the BBM. In contrast, lower values for Q and l0 occur in 4 and 9 L and for TEM. For this alga, there was no interaction volume by medium either for Qv or for l0 . Therefore, conclusions are given in general for media or volume. Differences in lipid or protein content (QL and QP) show increased productivity in 1.5 L, with respect to 4 or 9 L volumes in Scenedesmus sp.
Table 3 Lipid concentration in percentage, lipid dry productivity (QL) and lipid volumetric productivity (QVL) in three algae species cultured in Tilapia Effluent Medium and Bold Medium. Specie/volume (L)
Content (%)
QL (g L1)
QVL (g L1 d1)
TEM
BBM
TEM
BBM
TEM
BBM
Chlorella sp. 1.5 4 9 SE
5.2ab 9.2a 2.7b
6.0 4.8 6.0 0.86
0.054 0.012 0.012
0.007 0.036 0.014 0.01
0.002 0.004 0.002
0.011 0.006 0.003 0.002
Scenedesmus sp. 1.5 4 9 SE
8.4 10.3 8.5A
11.6a 8.2a 3.0Bb 0.67
0.107 0.035 0.018
0.120 0.027 0.001 0.006
0.015 0.003 0.0004
0.038a 0.00b 0.001b 0.003
Monoraphidium sp. 1.5 4 9 SE
17.8Aa 10.4b 8.9b
10.3B 11.4 13.1 0.96
0.106A 0.017 0.010
0.049Ba 0.032b 0.022b 0.005
0.015Aa 0.002 b 0.001 b
0.008B 0.005 0.004 0.001
a, b, c: Means in the same column with different letter are statistically different (P < 0.05) within each alga and variable; which shows differences among volumes. A,B: Means in the same row and variable with different letter are statistically diffident (P < 0.05), which shows differences among media. SE: Standard error of volume by medium interaction.
Please cite this article in press as: Guerrero-Cabrera, L., et al. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.127
L. Guerrero-Cabrera et al. / Bioresource Technology xxx (2014) xxx–xxx Table 4 Protein content in percentage, protein dry productivity (QP) and protein volumetric productivity (QVP) in three algae species cultured in Tilapia Effluent Medium and Bold Medium. Specie/volume (L)
Content (%)
QP (g L1)
QVP (g L1 d1)
TEM
BBM
TEM
BBM
TEM
BBM
Chlorella sp. 1.5 4 9 SE
23.4 61.9 35.7
21.1 25.0 28.5 5.88
0.243 0.079 0.158
0.249 0.205 0.088 0.047
0.011 0.029 0.033
0.036 0.030 0.015 0.008
Scenedesmus sp. 1.5 4 9 SE
39.9 50.4 20.9
36.3 38.0 31.6 5.3
0.499 0.175 0.044
0.381 0.124 0.103 0.054
0.040 0.008 0.004
0.019 0.020 0.001 0.002
Monoraphidium sp. 1.5 4 9 SE
46.8 45.4 33.7
22.8 45.2 40.5 5.24
0.281a 0.074b 0.037b
0.122B 0.123 0.064 0.017
0.064B 0.013 0.01
0.123Aa 0.019b 0.009b 0.016
a, b, c: Means in the same column with different letter are statistically different (P < 0.05) within each alga and variable; which shows differences only among volumes. A,B: Means in the same row and variable with different letter are statistically different (P < 0.05), which shows differences among media. SE: Standard error of volume by medium interaction.
26.3 for Scenedesmus sp.; 4, 1.5 and 9 L, in the same order, P < 0.05). In addition, TEM showed higher protein percentage than BBM (40.3 vs. 24.8, P < 0.05) for all the volumes as a whole. Despite higher percentage of proteins in Chlorella sp. this variable did not show differences among TEM and BBM for any of the algae when expressed as QVP. In the present experiment, protein content (%) was higher in TEM with respect to BBM in Chlorella sp. Nonetheless, lower Qv was recorded in TEM, being reflected in a low QVP. It is important to keep protein production expressed as QP and QVP, because a difference expressing higher protein concentration in percentage of dry biomass may disappear when expressed as QVP when Qv is low. For Scenedesmus sp. and Monoraphidium sp., higher protein accumulation in the 1.5 L PBR than in 4 or 9 L volumes was evident until concentration was expressed as QP. In addition, no differences in QP or QL were found among media for any of the three algae. Actually, recorded differences between such averages do not change in more than 30% of given values between media in any case. This last result may indicate an adaptation to a mixotrophic environment by the studied species. As no difference was found among media, further research could be conducted on the TEM utilization as an inexpensive way to produce protein for animal or human feeding purposes. On the other hand, in TEM produced from an outdoor pisciculture system, a natural high growth of phytoplankton is common, given the high nutrient availability. Therefore, growth rate of chosen species could be improved by Batch cultures where most important growth conditions, light and air supply, can be adjusted as needed.
3.4. Nutrient removal Ammonium and phosphate content were measured before and after algae culture. Measures of ammonium and phosphate absorption in TEM PBRs were 82.2 and 66.2; 92.3 and 80; 98 and 90% (same order), for Monoraphidium sp., Chlorella sp. and Scenedesmus sp., respectively. Regarding nutrient removal, the high nitrogen and phosphorus absorption rates, measured as a reduction in phosphates and
5
ammonium contents in TEM during algae development, imply a high ability to survive and produce under the TEM conditions. In Chlorella minutissima, Ördög et al. (2004) found 50% nitrogen removal from a 700 mg L1 nitrate content in a 20 L PBR, implying a high growth ability for this specie in mixotrophic conditions, such as municipal effluents, where high contents of carbon, nitrogen and phosphorus may be available (Min et al., 2011). Data collected in the present assay show a greater absorption rate than that reported by Barrera et al. (2008); who found a 61% and 65% of ammonium and phosphorus absorption (from a starting content 45 and 11 g L1, same order), studying 31 microalgae species in a 15 L PBR Batch culture under natural temperature and light conditions. In a different study, Gál et al. (2007) proposed a closed mixed system pisciculture-algae with a catfish extensive pond, intensive-production fish tanks and an algae pond, with nutrients and water recirculation. They found that nitrite and nitrate contents were reduced in the algae pond (and increased in the extensive fishpond of African catfish) but no phosphates or ammonium were widely used by algae in comparison to the high removal of such substances in the fishpond; they explain their results in terms of anaerobic conditions in the algae pond during night. Nevertheless, these results support the use of effluents for algae culture in commercial aquaculture. In the current experiment factors such as medium sterilization, steady air supply and continuous illumination might have played a central role in augmenting nutrient absorption by algae. None of those conditions were controlled in the assays mentioned before. Present study findings regarding phosphates and ammonium absorption agree with those found by Da Silva et al. (2009), who used Ulva clathrata (Roth) J. Agardh (Chlorophyceae) cultivated in 250 L ponds with shrimp waste water under outdoor conditions, where temperature and light were not controlled. These authors report that U. clathrata has a high efficiency to absorb ammonium and phosphate within a 15 h term, accounting for 70–80% of nitrogen removal and 50% of phosphates removal. Further studies shall be done in search for improving effluent media by adding sources of limiting nutrients or by diluting it with a formulated medium to achieve a synergic effect. Effluent media dilution may prevent high concentration of toxic elements and formulated mediums may help to achieve the nutritional requirements for algae (Mandal and Mallick, 2009). Assays could be run in low volume PBRs in order to avoid the exponential decay, because it might be difficult adjusting for air supply and optical depth in higher volumes.
4. Conclusions The three algae species were able to produce in the mixotrophic environment with Nile Tilapia effluent as aquaculture medium. In such conditions, both ammonium–nitrogen and faecal-nitrogen were absorbed. Tilapia effluent is suitable for algae culture when the objective is the production of biomass with high protein content. Batch cultures can be used to take advantage of the phytoplankton development that occurs naturally in pisciculture wastewaters (outdoor conditions). Furthermore, Batch conditions allow for better control of the variables affecting algae growth; such as temperature, light and air supply, which help to achieve a better absorption of nutrients from effluents.
Acknowledgements The authors would like to thank James Patrick Killough for revising the present manuscript regarding English language style.
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References Barrera, B.C., Vázquez, G., Barceló, Q.I., Bussy, A.L., 2008. Microalgal dynamics in batch reactors for municipal wastewater treatment containing dairy sewage water. Water Air Soil Pollut. 190, 259–270. Barsanti, L., Gualtieri, P., 2006. Algae Anatomy, Biochemistry and Biotechnology. Taylor & Francis Group, USA. Bogen, C., Klassen, V., Wichmann, J., La Russa, M., Doebbe, A., Grundmann, M., Uronen, P., Kruse, O., Mussgnug, J.H., 2013. Identification of Monoraphidium contortum as a promising species for liquid biofuel production. Bioresour. Technol. 133, 622–626. Borges, M.-T., Silva, P., Moreira, L., Soares, R., 2005. Integration of consumertargeted microalgal production with marine fish effluent biofiltration a strategy for mariculture sustainability. J. Appl. Phycol. 17, 187–197. Burden, L.R., Douglas, F.J., 2011. Numerical Differentiation and Integration. In: Julete, M. (Ed.), Numerical Analysis, nineth ed. CENGAGEL earning, Canada, pp. 174–191. Chen, W., Zhang, Q., Dai, S., 2009. Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa. J. Appl. Phycol. 21, 701–706. Da Silva, C.M., Tormena, T., Seeliger, U., 2009. Biofiltering efficiency, uptake and assimilation rates of Ulva clathrata (Roth) J. Agardh (Clorophyceae) cultivated in shrimp aquaculture waste water. J. Appl. Phycol. 21, 31–45. Gál, D., Pekár, F., Kerepeczki, É., Váradi, L., 2007. Experiments on the operation of a combined aquaculture-algae system. Aquac. Int. 15, 173–180. Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507. Grobbelaar, J.U., 2008. Upper limits of photosynthetic productivity and problems of scaling. J. Appl. Phycol. 21, 519–522. Ji, M.-K., Kim, H.-C., Sapìreddy, V.R., Yun, H.-S., Abou-Shanab, R.A.I., Choi, J., Lee, W., Timmes, T.C., Inamuddin, Jeon, B.-H., 2013. Simultaneous nutrient removal and
lipid production from pretreated piggery wastewater by Chlorella vulgaris YSW04. Appl. Microbiol. Biotechnol. 97, 2701–2710. Lee, Y.K., 2001. Microalgal mass culture systems and methods: Their limitation and potential. J. Appl. Phycol. 13, 307–315. Li, X., Hu, H.-Y., Yang, J., 2010a. Lipid accumulation and nutrient removal properties of a newly isolated freshwater microalga, Scenedesmus sp. LX1, growing in secondary effluent. N. Biotechnol. 27, 59–63. Li, X., Hu, H.-Y., Gan, K., Sun, Y.-X., 2010b. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Bioresour. Technol. 101, 5494–5500. Mandal, S., Mallick, N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84, 281–291. Min, M., Wang, L., Li, Y., Mohr, M.J., Hu, B., Zhou, W., Chen, P., Ruan, R., 2011. Cultivating Chlorella sp. in a pilot-scale photobioreactor using centrate wastewater for microalgae biomass production and wastewater nutrient removal. Appl. Biochem. Biotechnol. 165, 123–137. Nichols, H., Bold, H., 1965. Growth media. In: JR, S. (Ed.), Hand B. Physiol. Methods. Cambridge UniversityPress, Cambridge, pp. 7–24. Ördög, V., Stirk, W.A., Bálint, P., Staden, J.V., Lovász, C., 2004. Changes in lipid, protein and pigment concentrations in nitrogen-stressed Chlorella minutissima cultures. J. Appl. Phycol. 24, 907–914. Shubert, L.E., 2003. Non motile coccoid and colonial green algae. In: Wehr, J.D., Sheath, R.G. (Eds.), Freshwater Algae North Am. Ecol. Classif. Elsevier Science, San Diego, CA, pp. 253–309. Wu, Y.-H., Li, X., Yu, Y., Hu, H.-Y., Zhang, T.-Y., Li, F.-M., 2013. An integrated microalgal growth model and its application to optimize the biomass production of Scenedesmus sp. LX1 in open pond under the nutrient level of domestic secondary effluent. Bioresour. Technol. 144, 445–451.
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Short Communication
Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent Luis Guerrero-Cabrera ⇑, José A. Rueda, Hiram García-Lozano, A. Karin Navarro Universidad del Papaloapan, Av. Ferrocarril s/n, Loma Bonita, Oaxaca, Mexico
h i g h l i g h t s Chemical composition of three algae cultured in pisciculture effluent was studied. Nitrogen and phosphorus in effluent were efficiently absorbed by the algae. Effluent shows a lower productivity than a formulated medium in Scenedesmus sp. Protein percentage is higher when effluent is used but productivity is limited. Photobioreactors with lower volumes give a higher productivity and growth rate.
a r t i c l e
i n f o
Article history: Received 18 January 2014 Received in revised form 20 March 2014 Accepted 23 March 2014 Available online xxxx Keywords: Pisciculture effluents Wastewater as culture medium Secondary effluent cultures Fresh-water algae culture
a b s t r a c t Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae were cultured in three volumes of Tilapia Effluent Medium (TEM) in comparison with the Bold Basal Medium (BBM) (Nichols and Bold, 1965). Specific growth rate (l0 ), biomass dry productivity (Q), volumetric productivity (Qv) as well as lipid and protein content were measured. Then, volumetric productivities for both lipids and proteins were calculated (QVL and QVP). In Scenedesmus sp., BBM produced higher l0 and Qv than TEM in 1.5 L volume. Chlorella sp. showed a higher QVL for BBM than TEM. Any observed difference in protein or lipid productivities among volumes was in favor of a greater productivity for 1.5 L volume. Even when TEM had a larger protein content in Chlorella sp. than BBM, QVP was not different. Current results imply that TEM can be used as an alternative growth medium for algae when using Batch cultures, yet productivity is reduced. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction In aquatic ecosystems, phytoplankton growth depends upon light, temperature, salinity, pH and available nutrients. Nutrients such as nitrogen and phosphorus may be available in different chemical compounds. The major sources of nitrogen are nitrates and ammonium–nitrogen, but other inorganic compounds like nitric oxide, nitric acid and nitrites can be used, as well as organic compounds such as urea. The organic to inorganic ratio of nitrogen absorption may differ among phytoplankton species and it can be affected by environmental changes, by the nitrogen source, and by the concentration of phosphorus, carbon and oxygen (Barrera et al., 2008). Inside the cell, nitrate is turned into nitrite and then reduced to ammonium in a two-step reaction catalyzed by nitrate and nitrite reductases. Then, nitrogen incorporates into organic compounds ⇑ Corresponding author. Tel./fax: +52 281 8729230x220. E-mail address: [email protected] (L. Guerrero-Cabrera).
(Chen et al., 2009). Sodium nitrate and potassium nitrate are used in growing cultures in accordance with reports of high growth rate and high biomass productivity. In fact, most of the experiments in lipid accumulation induction are based on setting limits to nitrates as nitrogen source, thus reducing the nitrate level below the recommended concentrations for a culture medium (Griffiths and Harrison, 2009). Ammonium is not widely used as a nitrogen source in algae cultures, since growth rates and biomass productivity do not reach the levels achieved using nitrate instead of ammonium. Some of the media which use (NH4)2HPO4 and (NH4)2SO4 are Crammer Mayers for freshwater and PCR-S11 for marine water, respectively (Barsanti and Gualtieri, 2006). Both ammonium and urea are extensively used as fertilizers in field-cultivated crops, but they can also be found in high concentration in effluents from many productive activities and services. Nitrogen and phosphorus concentrations occur in a wide range in secondary effluents including livestock manure, human waste, and agricultural remnant waters. Municipal effluents and treated
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waters are some of the cases with higher concentrations. In particular wastewater may contain from 95 to 264 mg L1 of ammonium and 125 to 530 mg L1 of total phosphorus (Min et al., 2011). Domestic secondary effluent contains from 7 to 25 mg L1 of total nitrogen and from 0.04 to 1.93 mg L1 of total phosphorus (Li et al., 2010b; Wu et al., 2013). Municipal wastewater containing dairy sewage has 40 mg L1 of ammonium and 11 mg L1 of total phosphorus (Barrera et al., 2008). Livestock wastewaters represent a good alternative for microalgae cultures because of their low content of toxic elements. Furthermore, high phosphorus and nitrogen concentration in such effluents allow for their usage without dilution. Other examples are: pretreated piggery wastewater, which contains 268 mg L1 of ammonium and 56 mg L1 of orthophosphate (Gál et al., 2007); shrimp aquaculture wastewater, with 0.9 to 0.1 mg L1 ammonium (Da Silva et al., 2009) and marine fish effluent bio-filtration with 1 mg L1 of ammonium and 1.5 mg L1 of orthophosphate (Borges et al., 2005). Microalgae cultured in the above-mentioned effluents change their metabolic strategy from autotrophic to chemoheterotrophic or mixotrophic. Thus, photoautotrophic algae cultured in media containing organic compounds, use such substances as an alternative carbon source (Barsanti and Gualtieri, 2006). A variety of algae species have been studied using a secondary effluent as growth medium. Assessed variables range from growth rate, biomass productivity, chemical composition, through chlorophyll production, among others. Some of the more important purposes of such assays were biodiesel and lipid production with Monoraphidium sp. (Bogen et al., 2013), Scenedesmus sp. (Li et al., 2010a; Wu et al., 2013) and Chlorella sp. (Ji et al., 2013). Microalgae may provide a viable alternative to fossil biofuels, although this technology has yet to overcome a number of challenges including specie selection, growth media improvement and bioreactor design, in order to achieve a higher productivity. Several formulated media have been used for microalgae cultivation, in an attempt to increase the benefit–cost ratio. Recently, nonconventional media have been studied in hopes of finding an inexpensive alternative to commercial media. In particular, aquaculture effluents have been assessed in a continuous photo-bioreactor (PBR) or in outdoor conditions, but they have not been assessed in Batch cultures in controlled conditions, where it will be possible to measure accurately their potential in biomass production. Present work evaluates the viability of using the Nile tilapia effluent as an alternative medium for algae culture in order to reduce expenses in a scope of sustainability. 2. Methods 2.1. Algae The three algae species: Chlorella sp., Monoraphidium sp. and Scenedesmus sp. were isolated by sequential plate seeding method with Agar-Basal Bold Medium (BBM) from samples collected in natural freshwater ponds in Loma Bonita, Oax., Mexico. Algae morphological identification was completed through genera using specialized taxonomical guides (Shubert, 2003). The strains used were kept as Mon10UNPA-49, Sce10UNPA-44 and Chl10UNPA-45 from the Aquaculture Laboratory of the Universidad del Papaloapan, with re-seeding phase indicated after the dash.The three algae were grown in a 500 mL PBR with BBM. Algae were not pre-conditioned or adapted to the tilapia effluent for the Batch cultures in the assay. 2.2. Tilapia effluent medium (TEM) To obtain the TEM, a 20 kg m3 live weight stock of Oreochromis niloticus (line Gift) was kept in freshwater glass aquariums in
laboratory conditions for 78 h. The tilapia fishes were kept under intensive feeding regime (3% of live weight in food per day), temperature was 25 °C and air supply was steady. After this period, the effluent was collected, filtered through a 55 lm zooplankton mesh and sterilized by boiling it for 10 min. The effluent was cooled at room temperature and pH was adjusted to 7.5 (HannaÒ Instruments pH meter) with 1 M KOH and 1 M HCl. Finally, ammonium content (by the Nessler method modified) and orthophosphate content (by the Ascorbic Acid method) were measured so the initial conditions were known. These conditions in TEM are presented in Table 1. The nitrogen and phosphorus sources in BBM were 150 mg L1 of NaNO3, 175 mg L1 of KH2PO4 and 75 mg L1 of K2HPO4. 2.3. Algae culture Polyethylene terephthalate (PET) bottles with flat wall were used as PBR. Experimental dimensions were 1.5, 4 and 9 L. (diameter/height of 9/21, 14.5/22, and 19.5/29 cm, respectively). The experiments for each of the three algae were conducted simultaneously. PBRs were kept in laboratory under steady light and temperature conditions. Two parallel lines of 60 W fluorescent PhilipsÒ T12 lamps (6000 lm) 20 cm apart, were used and room temperature was 25 °C. Air supply, without CO2 enrichment, was adjusted from 1.5 to 4 L and from 4 to 9 L by a 1.7 factor according to preliminary assays, in order to optimize biomass productivity (data not shown). Air flow was established at 1.6, 2.9 and 4.9 L min1 for 1.5, 4 and 9 L, respectively. In order to estimate the nutrient removal, ammonium and orthophosphate contents were measured after the cultivation period. 2.4. Analytical methods Cumulated biomass was harvested at the end of the growth period. Biomass dry productivity (Q) was measured in an electronic moisture analyzer (scale MOC-120H, ShimadsuÒ). The volumetric productivity obtained in g L1 d1 (Qv) by means of Qv = l0 Q, where Q is biomass concentration in g L1 (Griffiths and Harrison, 2009; Li et al., 2010a). Dry matter samples were preserved to assess lipid concentration by the Bligh and Dyer method (1959) modified (Mandal and Mallick, 2009) through a 2:1 methanol/chloroform ratio by gravimetry. Protein content was obtained by the Bradford method. Both lipid productivity (QL) and protein productivity (QP) were then expressed as volumetric productivities in accordance with Griffiths and Harrison (2009):
Q VL ¼ ðQ V Þðpercentage of lipid contentÞ=100
ð1Þ
Q VP ¼ ðQ V Þðpercentage of protein contentÞ=100
ð2Þ
2.5. Kinetics parameters analysis The growth kinetics was studied by daily cell count in a Haematocytometer chamber during the 20 d period of Batch
Table 1 Characteristics of Tilapia Effluent Medium used for algae culture. Characteristic
Measure
N-NH4 NO3 NO2 pH PO4 Alkalinity
24 mg L1 ND ND 7.5 10 mg L1 80 mg L1
ND: Not detected.
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culture, because at the end of this period the increase in cell density stops. Specific growth rate (l) was first calculated by the classical logistic model (Wu et al., 2013), thus relating starting to final cell count during exponential growth phase. However, data did not show such a phase and approach was changed to the numerical differentiation method to obtain l, the five point formula. This last method described kinetics as a smooth line which moves with data, allowing nonlinear fit by taking into account each point while reducing error and increasing accuracy (Burden and Douglas, 2011). Five point formula method is a numerical derivative, which implies there is always a near polynomial to a given continuous function close enough to all the data dots in that function for a specific interval (Burden and Douglas, 2011). The specified function was drawn from the data and fitted by a Lagrange polynomial, where error term is from order O(h4) where h equals time for each of the sample dots. This five point formula Eq. (3)includes x0 as the starting point for the derivative and s between x02h y x0 + 2h. To calculate numerical derivatives in each of the PBR, days 3, 6, 9, 12 and 15 were taken as the five reference points. The specific growth rate was computed as the quotient of the derivative of f(x0) by cell density (Cn) measured on the 9th day, as shown in Eq. (4).
f 0 ðx0 Þ ¼ ð1=12hÞ½f ðx0 2hÞ 8f ðx0 hÞ þ 8f ðx0 þ hÞ 4
f ðx0 þ 2hÞ þ ðh =30Þf ðv Þ
ð3Þ
l0 ¼ ð1=C n ÞðdC n =dtÞ
ð4Þ
Data was analyzed using a 2 3 factorial design, for two media (TEM and BBM) and three volumes (1.5, 4 and 9 L), with two replicates. Different analyses of variance were run for each alga-variable by the MIXED procedure and means comparison was done by a Tukey test in SAS software. 3. Results and discussion 3.1. Growth rate and productivity Specific growth rate (l), five-point-formula growth rate (l0 ), biomass dry productivity (Q) and volumetric productivity (Qv) are given in Table 2. Specific growth rate (l) obtained by classical methods was not related to Qv and means for l are presented just as a reference. Meanwhile, higher l0 values were closely associated with higher Qv in Scenedesmus sp. algae. For Chlorella sp. and Monoraphidium sp. algae, l0 was not related to Qv. Furthermore, data did
not show differences either for volumes or culture media for Qv or l0 . In Scenedesmus sp., there was no medium by volume interaction, either for l0 or for Qv. There was an evident advantage for the BBM with respect to TEM (l0 : 0.19 vs. 0.078, P < 0.05; Qv: 0.138 vs. 0.067, P < 0.05) for this alga (Fig. 1). In addition, l0 was different among PBR sizes, where the 1.5 L volume had higher l0 for Scenedesmus sp. with respect to 4 or 9 L (0.23 vs. 0.12 and 0.06, P < 0.05), corresponding with a higher Qv for the 1.5 L volume (0.25 vs. 0.04 and 0.017 g L1 d1, P < 0.05). No statistical differences in Qv were observed among media for Chlorella sp. (0.06 vs. 0.12 for TEM and BBM) or Monoraphidium sp. (0.4 vs. 0.049 for TEM and BBM). Furthermore Monoraphidium sp. showed a higher Qv in the 1.5 L volume (0.08 vs. 0.03 and 0.02) than in the other volumes as well. Biomass dry productivity (Q) was not different among media for any of the algae. Different approaches have been reported to approximate l. Nonetheless, culture conditions were so different in such assays that direct comparison is not supported. According to Wu et al. (2013), it is necessary to establish a relationship between l and dry productivity for each of the species. The nonlinear fit proposed in this work to calculate the growth rate is recommended for any condition where exponential phase is not evident, a common case when dealing with the use of pisciculture effluents as culture mediums. It has been found that Qv is affected, in both outdoor and in laboratory conditions PBRs by container height and diameter. This is mainly explained by medium optical depth and light requirements of algae cells (Lee, 2001). Wu et al. (2013) describe this fact as an exponential decay function. In addition, container or pond dimension affect air supply and air distribution (Grobbelaar, 2008). The data showed clear biomass productivity decay as volume increases, which according to the above mentioned can be explained in terms of differences in optical depth and air supply among volumes, expressed as a higher efficiency in smaller containers (1.5 vs. 4 and 9 in this particular case). Emphasis must be given to the fact that light remained steady in the run experiment, but dimensions of PBR have a clear effect on the mentioned variables. Higher Qv obtained in 1.5 L BBM than in 1.5 L TEM for Scenedesmus sp. and for Chlorella sp. is due to higher nitrogen and phosphorus contents in BBM than in TEM. The fact that Monoraphidium sp. was the exception to this difference in Qv among mediums may reflect interspecific differences in nutrients use. Borges et al. (2005) found that Tetraselmis sp. had three times as much productivity in snook
Table 2 Specific growth rate (l), dry biomass productivity (Q) and volumetric productivity (Qv) in three algae species cultured in Tilapia Effluent Medium and in Bold Medium. Specie/volume (L)
Q (g L1)
l0
l
Qv (g L1 d1)
TEM
BBM
TEM
BBM
TEM
BBM
TEM
BBM
Chlorella sp. 1.5 4 9 SE
0.006b 0.018a 0.015a
0.013 0.012 0.011 0.001
0.046c 0.367a 0.202b
0.143 0.166 0.177 0.027
1.036a 0.070b 0.437b
1.259a 0.788ab 0.308b 0.098
0.048 0.047 0.089
0.179a 0.127ab 0.054b 0.0175
Scenedesmus sp. 1.5 4 9 SE
0.006b 0.014a 0.010a
0.009 0.008 0.008 0.001
0.134a 0.075b 0.026b
0.322a 0.164b 0.092b 0.025
1.264a 0.344b 0.207b
1.032a 0.325b 0.325b 0.045
0.172a 0.026ab 0.005b
0.332a 0.054b 0.030b 0.027
Monoraphidium sp. 1.5 4 9 SE
0.011 0.013 0.007
0.012 0.012 0.012 0.001
0.146 0.118 0.119
0.158 0.165 0.156 0.030
0.600a 0.162b 0.111b
0.481a 0.274b 0.166c 0.0178
0.088a 0.019b 0.013b
0.076 0.045 0.027 0.01
a, b, c: Means in the same column with different letter are statistically different (P < 0.05) within each alga and variable; which shows differences among volumes. SE: Standard error of volume by medium interaction.
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a
160
3.2. Lipid content
140
Lipid content expressed as a fraction of Q in percentage (%), as dry productivity per volume (QL) or as accumulated volumetric productivity per day (QVL) is shown in Table 3. Chlorella sp. showed a higher QVL for BBM than for TEM (0.0068 vs. 0.0031, P < 0.05). However, for Monoraphidium sp. or Scenedesmus sp., media did not show differences in lipid productivity either as percentage, as QL or as QVL. Regarding lipid content expressed as QL, there was no interaction volume by medium and 1.5 L PBR had a higher QL than 4 and 9 L volumes for Scenedesmus sp. (0.113, 0.031 and 0.014, P < 0.05) and Chlorella sp. (0.066, 0.024 and 0.013, P < 0.05). Furthermore, in Monoraphidium sp. interaction was present and it showed the same pattern in TEM (greater lipid productivity in the 1.5 L volume) only when expressed as volumetric productivity (QVL). According to the results of the present assay, higher QVL is reached by BBM than by TEM in Chlorella sp. Available nutrient content in each medium plays a central role in achieving the potential lipid productivity by algae. A lower biomass productivity is particularly expected when nitrogen is limited, which can be partially mended by the availability of a carbon source, such as glucose (Mandal and Mallick, 2009). Actual results agree with Ji et al. (2013), who report improved lipid production when using a formulated medium compared with piggery wastewater effluents for Chlorella vulgaris culture. Although ammonium–nitrogen in TEM is lower than nitrates–nitrogen in BBM, nitrogen does not seem to be limited in TEM, the concentration of this nutrient in the organic matter of TEM fills the cell requirements for development.
Cell count x 10-6
120 100 80 60 40
BBM
20
TEM
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 me (d)
b
0.35
Qv (g L -1d-1)
0.3 0.25 0.2 0.15
BBM
0.1
TEM
0.05 0
1.5
4
6.5
9
Volume (L)
μ'
c
0.35
3.3. Protein content
0.3
Protein content expressed as fraction of Q in percentage (%), as dry productivity per volume (QP), or as accumulated volumetric productivity per day (QVP) is shown in Table 4. QVP in 1.5 L volume was higher than in 4 and 9 L volumes for Monoraphidium sp. in BBM. In Chlorella sp. and Scenedesmus sp., independently of medium, the highest protein content in percentage was found for 4 L (43.45 vs. 22.3 and 32.1 for Chlorella sp. and 44.2 vs. 38.1 and
0.25
BBM
0.2
TEM
0.15 0.1 0.05 0
1.5
4
6.5
9
Volume (L) Fig. 1. (a) Daily cell count of Scenedesmus sp. algae using Tilapia Effluent as culture medium in comparison with Basal Bold Medium. (b) Volumetric productivity of Scenedesmus sp. algae cultured in Nile tilapia effluent and Basal Bold Medium. (c) Growth rate of Scenedesmus sp. algae cultured in Nile tilapia effluent and in Basal Bold Medium.
effluent than in the Guillard medium, while Phaeodactylum tricornutum had a major productivity in Guillard medium. Similarly, the data showed a higher Qv for Monoraphidium sp. in TEM than in BBM at 1.5 L volume, the opposite happening in 4 L volume, which could imply changes in nutrient usage when light is limited. Data for Scenedesmus sp. is completely coherent with respect to the relationship among the studied variables in the current assay, where the highest values for Qv and l0 concur in 1.5 L, as well as in the BBM. In contrast, lower values for Q and l0 occur in 4 and 9 L and for TEM. For this alga, there was no interaction volume by medium either for Qv or for l0 . Therefore, conclusions are given in general for media or volume. Differences in lipid or protein content (QL and QP) show increased productivity in 1.5 L, with respect to 4 or 9 L volumes in Scenedesmus sp.
Table 3 Lipid concentration in percentage, lipid dry productivity (QL) and lipid volumetric productivity (QVL) in three algae species cultured in Tilapia Effluent Medium and Bold Medium. Specie/volume (L)
Content (%)
QL (g L1)
QVL (g L1 d1)
TEM
BBM
TEM
BBM
TEM
BBM
Chlorella sp. 1.5 4 9 SE
5.2ab 9.2a 2.7b
6.0 4.8 6.0 0.86
0.054 0.012 0.012
0.007 0.036 0.014 0.01
0.002 0.004 0.002
0.011 0.006 0.003 0.002
Scenedesmus sp. 1.5 4 9 SE
8.4 10.3 8.5A
11.6a 8.2a 3.0Bb 0.67
0.107 0.035 0.018
0.120 0.027 0.001 0.006
0.015 0.003 0.0004
0.038a 0.00b 0.001b 0.003
Monoraphidium sp. 1.5 4 9 SE
17.8Aa 10.4b 8.9b
10.3B 11.4 13.1 0.96
0.106A 0.017 0.010
0.049Ba 0.032b 0.022b 0.005
0.015Aa 0.002 b 0.001 b
0.008B 0.005 0.004 0.001
a, b, c: Means in the same column with different letter are statistically different (P < 0.05) within each alga and variable; which shows differences among volumes. A,B: Means in the same row and variable with different letter are statistically diffident (P < 0.05), which shows differences among media. SE: Standard error of volume by medium interaction.
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L. Guerrero-Cabrera et al. / Bioresource Technology xxx (2014) xxx–xxx Table 4 Protein content in percentage, protein dry productivity (QP) and protein volumetric productivity (QVP) in three algae species cultured in Tilapia Effluent Medium and Bold Medium. Specie/volume (L)
Content (%)
QP (g L1)
QVP (g L1 d1)
TEM
BBM
TEM
BBM
TEM
BBM
Chlorella sp. 1.5 4 9 SE
23.4 61.9 35.7
21.1 25.0 28.5 5.88
0.243 0.079 0.158
0.249 0.205 0.088 0.047
0.011 0.029 0.033
0.036 0.030 0.015 0.008
Scenedesmus sp. 1.5 4 9 SE
39.9 50.4 20.9
36.3 38.0 31.6 5.3
0.499 0.175 0.044
0.381 0.124 0.103 0.054
0.040 0.008 0.004
0.019 0.020 0.001 0.002
Monoraphidium sp. 1.5 4 9 SE
46.8 45.4 33.7
22.8 45.2 40.5 5.24
0.281a 0.074b 0.037b
0.122B 0.123 0.064 0.017
0.064B 0.013 0.01
0.123Aa 0.019b 0.009b 0.016
a, b, c: Means in the same column with different letter are statistically different (P < 0.05) within each alga and variable; which shows differences only among volumes. A,B: Means in the same row and variable with different letter are statistically different (P < 0.05), which shows differences among media. SE: Standard error of volume by medium interaction.
26.3 for Scenedesmus sp.; 4, 1.5 and 9 L, in the same order, P < 0.05). In addition, TEM showed higher protein percentage than BBM (40.3 vs. 24.8, P < 0.05) for all the volumes as a whole. Despite higher percentage of proteins in Chlorella sp. this variable did not show differences among TEM and BBM for any of the algae when expressed as QVP. In the present experiment, protein content (%) was higher in TEM with respect to BBM in Chlorella sp. Nonetheless, lower Qv was recorded in TEM, being reflected in a low QVP. It is important to keep protein production expressed as QP and QVP, because a difference expressing higher protein concentration in percentage of dry biomass may disappear when expressed as QVP when Qv is low. For Scenedesmus sp. and Monoraphidium sp., higher protein accumulation in the 1.5 L PBR than in 4 or 9 L volumes was evident until concentration was expressed as QP. In addition, no differences in QP or QL were found among media for any of the three algae. Actually, recorded differences between such averages do not change in more than 30% of given values between media in any case. This last result may indicate an adaptation to a mixotrophic environment by the studied species. As no difference was found among media, further research could be conducted on the TEM utilization as an inexpensive way to produce protein for animal or human feeding purposes. On the other hand, in TEM produced from an outdoor pisciculture system, a natural high growth of phytoplankton is common, given the high nutrient availability. Therefore, growth rate of chosen species could be improved by Batch cultures where most important growth conditions, light and air supply, can be adjusted as needed.
3.4. Nutrient removal Ammonium and phosphate content were measured before and after algae culture. Measures of ammonium and phosphate absorption in TEM PBRs were 82.2 and 66.2; 92.3 and 80; 98 and 90% (same order), for Monoraphidium sp., Chlorella sp. and Scenedesmus sp., respectively. Regarding nutrient removal, the high nitrogen and phosphorus absorption rates, measured as a reduction in phosphates and
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ammonium contents in TEM during algae development, imply a high ability to survive and produce under the TEM conditions. In Chlorella minutissima, Ördög et al. (2004) found 50% nitrogen removal from a 700 mg L1 nitrate content in a 20 L PBR, implying a high growth ability for this specie in mixotrophic conditions, such as municipal effluents, where high contents of carbon, nitrogen and phosphorus may be available (Min et al., 2011). Data collected in the present assay show a greater absorption rate than that reported by Barrera et al. (2008); who found a 61% and 65% of ammonium and phosphorus absorption (from a starting content 45 and 11 g L1, same order), studying 31 microalgae species in a 15 L PBR Batch culture under natural temperature and light conditions. In a different study, Gál et al. (2007) proposed a closed mixed system pisciculture-algae with a catfish extensive pond, intensive-production fish tanks and an algae pond, with nutrients and water recirculation. They found that nitrite and nitrate contents were reduced in the algae pond (and increased in the extensive fishpond of African catfish) but no phosphates or ammonium were widely used by algae in comparison to the high removal of such substances in the fishpond; they explain their results in terms of anaerobic conditions in the algae pond during night. Nevertheless, these results support the use of effluents for algae culture in commercial aquaculture. In the current experiment factors such as medium sterilization, steady air supply and continuous illumination might have played a central role in augmenting nutrient absorption by algae. None of those conditions were controlled in the assays mentioned before. Present study findings regarding phosphates and ammonium absorption agree with those found by Da Silva et al. (2009), who used Ulva clathrata (Roth) J. Agardh (Chlorophyceae) cultivated in 250 L ponds with shrimp waste water under outdoor conditions, where temperature and light were not controlled. These authors report that U. clathrata has a high efficiency to absorb ammonium and phosphate within a 15 h term, accounting for 70–80% of nitrogen removal and 50% of phosphates removal. Further studies shall be done in search for improving effluent media by adding sources of limiting nutrients or by diluting it with a formulated medium to achieve a synergic effect. Effluent media dilution may prevent high concentration of toxic elements and formulated mediums may help to achieve the nutritional requirements for algae (Mandal and Mallick, 2009). Assays could be run in low volume PBRs in order to avoid the exponential decay, because it might be difficult adjusting for air supply and optical depth in higher volumes.
4. Conclusions The three algae species were able to produce in the mixotrophic environment with Nile Tilapia effluent as aquaculture medium. In such conditions, both ammonium–nitrogen and faecal-nitrogen were absorbed. Tilapia effluent is suitable for algae culture when the objective is the production of biomass with high protein content. Batch cultures can be used to take advantage of the phytoplankton development that occurs naturally in pisciculture wastewaters (outdoor conditions). Furthermore, Batch conditions allow for better control of the variables affecting algae growth; such as temperature, light and air supply, which help to achieve a better absorption of nutrients from effluents.
Acknowledgements The authors would like to thank James Patrick Killough for revising the present manuscript regarding English language style.
Please cite this article in press as: Guerrero-Cabrera, L., et al. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.127
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L. Guerrero-Cabrera et al. / Bioresource Technology xxx (2014) xxx–xxx
References Barrera, B.C., Vázquez, G., Barceló, Q.I., Bussy, A.L., 2008. Microalgal dynamics in batch reactors for municipal wastewater treatment containing dairy sewage water. Water Air Soil Pollut. 190, 259–270. Barsanti, L., Gualtieri, P., 2006. Algae Anatomy, Biochemistry and Biotechnology. Taylor & Francis Group, USA. Bogen, C., Klassen, V., Wichmann, J., La Russa, M., Doebbe, A., Grundmann, M., Uronen, P., Kruse, O., Mussgnug, J.H., 2013. Identification of Monoraphidium contortum as a promising species for liquid biofuel production. Bioresour. Technol. 133, 622–626. Borges, M.-T., Silva, P., Moreira, L., Soares, R., 2005. Integration of consumertargeted microalgal production with marine fish effluent biofiltration a strategy for mariculture sustainability. J. Appl. Phycol. 17, 187–197. Burden, L.R., Douglas, F.J., 2011. Numerical Differentiation and Integration. In: Julete, M. (Ed.), Numerical Analysis, nineth ed. CENGAGEL earning, Canada, pp. 174–191. Chen, W., Zhang, Q., Dai, S., 2009. Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa. J. Appl. Phycol. 21, 701–706. Da Silva, C.M., Tormena, T., Seeliger, U., 2009. Biofiltering efficiency, uptake and assimilation rates of Ulva clathrata (Roth) J. Agardh (Clorophyceae) cultivated in shrimp aquaculture waste water. J. Appl. Phycol. 21, 31–45. Gál, D., Pekár, F., Kerepeczki, É., Váradi, L., 2007. Experiments on the operation of a combined aquaculture-algae system. Aquac. Int. 15, 173–180. Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507. Grobbelaar, J.U., 2008. Upper limits of photosynthetic productivity and problems of scaling. J. Appl. Phycol. 21, 519–522. Ji, M.-K., Kim, H.-C., Sapìreddy, V.R., Yun, H.-S., Abou-Shanab, R.A.I., Choi, J., Lee, W., Timmes, T.C., Inamuddin, Jeon, B.-H., 2013. Simultaneous nutrient removal and
lipid production from pretreated piggery wastewater by Chlorella vulgaris YSW04. Appl. Microbiol. Biotechnol. 97, 2701–2710. Lee, Y.K., 2001. Microalgal mass culture systems and methods: Their limitation and potential. J. Appl. Phycol. 13, 307–315. Li, X., Hu, H.-Y., Yang, J., 2010a. Lipid accumulation and nutrient removal properties of a newly isolated freshwater microalga, Scenedesmus sp. LX1, growing in secondary effluent. N. Biotechnol. 27, 59–63. Li, X., Hu, H.-Y., Gan, K., Sun, Y.-X., 2010b. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Bioresour. Technol. 101, 5494–5500. Mandal, S., Mallick, N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84, 281–291. Min, M., Wang, L., Li, Y., Mohr, M.J., Hu, B., Zhou, W., Chen, P., Ruan, R., 2011. Cultivating Chlorella sp. in a pilot-scale photobioreactor using centrate wastewater for microalgae biomass production and wastewater nutrient removal. Appl. Biochem. Biotechnol. 165, 123–137. Nichols, H., Bold, H., 1965. Growth media. In: JR, S. (Ed.), Hand B. Physiol. Methods. Cambridge UniversityPress, Cambridge, pp. 7–24. Ördög, V., Stirk, W.A., Bálint, P., Staden, J.V., Lovász, C., 2004. Changes in lipid, protein and pigment concentrations in nitrogen-stressed Chlorella minutissima cultures. J. Appl. Phycol. 24, 907–914. Shubert, L.E., 2003. Non motile coccoid and colonial green algae. In: Wehr, J.D., Sheath, R.G. (Eds.), Freshwater Algae North Am. Ecol. Classif. Elsevier Science, San Diego, CA, pp. 253–309. Wu, Y.-H., Li, X., Yu, Y., Hu, H.-Y., Zhang, T.-Y., Li, F.-M., 2013. An integrated microalgal growth model and its application to optimize the biomass production of Scenedesmus sp. LX1 in open pond under the nutrient level of domestic secondary effluent. Bioresour. Technol. 144, 445–451.
Please cite this article in press as: Guerrero-Cabrera, L., et al. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.127
