Post on 31-Aug-2018
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
78
Cultivation of microalgae using municipal wastewater as a nutritional source
Eman, A. Mahmoud.1; Laila, A. Farahat.1 ; Zeinab, K. Abdel Aziz.2 ; Nesreen, A. Fatthallah1; Samy B. Elhenawy1 and Rawheya, A. Salah El Din2. 1 Egyptian Petroleum Research Institute, Process Development Department, Petroleum Biotechnology Lab 2 Al-Azhar University (Girls Branch), Faculty of Science, Botany and Microbiology Department
Eman, A. Mahmoud, Laila, A. Farahat, Zeinab, K.
Abdel Aziz., Nesreen, A. Fatthallah, Samy B.
Elhenawy and Rawheya, A. Salah El Din. 2016.
Cultivation of microalgae using municipal wastewater
as a nutritional source. J. Algal Biomass Utln. 7 (1):
78-85
Key words: Wastewater, Treatments, Microalgae
Abstract
Using wastewater to grow algae is probably the most promising way to reduce biodiesel production cost associated with nutrients and water. In the present study, three municipal waste water (MWW) from different effluents were collected and subjected to physical and chemical analysis. From about 21 isolates, only two promising microalgal isolates, Chlorella vulgaris and Scenedesmus quadricauda were tested for their ability to grow on the collected waste waters. Different treatments were performed for the collected (MWW) to determine the most suitable one for cultivation of microalgae. The results showed that supplementation with NaNo3 is the best treatment at least for Chlorella vulgaris.
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Corresponding Author: Eman, A. Mahmoud.;
Em_micro81@yahoo.com
1. Introduction
Biodiesel production from microalgae has been expected to displace the petroleum-based energy sources because of
its high aerial productivity, lipid contents and the higher energy yield: which is about 7–31 times higher than palm oil
[1] and hundred times than other oily plants such as corn, soybean, canola, jatropha, coconut and palm oil [2]. However,
the cultivation of algae on large-scale for bioenergy production should be significantly effective to compete with the cost
of energy production from other resources, especially petroleum based fuel. The main spending would have to cover
the costs of large amount of water with nutrients supplement, biomass harvest, and lipid extraction from biomass. In
order to reduce the cost for nutrients and freshwater, it has been suggested to mix algal cultivation with wastewater
treatment [3], [4]. Many species of microalgae are capable of effectively growing in wastewater conditions through their
ability to utilize abundant organic carbon and inorganic N and P in the wastewater. Microalgae are efficient in removing
N, P and toxic metals from wastewater [5], [6] and therefore have potential to play an important remediation role
particularly during the final (tertiary) treatment stage of wastewater. The significant advantage of algal processes in
wastewater treatment over the conventional chemical-based treatment methods is the potential cost saving and the
lower level technology that is utilized, therefore making this approach more attractive to developing countries. The
efficient growth of microalgae in wastewater depends on a variety of variables. As with any growth medium, critical
variables are the pH and temperature of the growth medium, the concentration of essential nutrients, including N, P
and organic carbon, and the availability of light, O2 and CO2. For example, increased temperature decreased algal
biomass [7]. Some biotic factors like pathogenic bacteria or predatory zooplankton may have negatively effect on algal
growth. Moreover, other microorganisms in the waste- water might out-compete the microalgae for essential nutrients.
The starting density of microalgae in the wastewater is also expected to be a critical factor for the growth of the whole
population [8]. These variables will clearly differ depending on the wastewater type and from one wastewater treatment
site to another. Additionally, there will be variation in the ability of different algal species to tolerate a particular
wastewater condition. Unicellular chlorophytic microalgae have been shown to be particularly tolerant to many
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
79
wastewater conditions and very efficient at accumulating nutrients from wastewater [9], [10]. A broad range of studies
have analyzed the growth of microalgae under a variety of wastewater conditions, mainly growth in municipal sewage
wastewater and agricultural manure wastewater. These studies have mainly been focused on evaluating the potential
of algae for removing N and P, and in some cases metals from wastewater [11]. The ability of microalgae to grow well
under definite wastewater conditions, as described above, has showed the potential of these resources as suitable
sustainable growth medium for biofuel feedstock. In this study we will briefly evaluate how effective wastewater
resources can provide significant algal biomass and which wastewater treatment will improve generation of high
amounts of algal biomass.
2. Materials and Methods
2.1. Microalgal isolates collection and identification
Two algal stains Chlorella vulgaris and Scenedesmus sp were isolated from brackish water in Cairo and Qalubia,
Governorates, Egypt, respectively. Algal identification has been done according to the keys of identification by Komárek
and Fott [12]. A further confirmation on the identification were done using18s rRNA. All identification steps were carried
out at Sigma Scientific Services Co., Cairo, Egypt. Genomic DNA was isolated from all algal species via a phenol
chloroform method on a pellet obtained by centrifugation of 10 mL of algal culture at the late-log phase. DNA
amplification from genomic DNA containing a partial 18S ribosomal RNA region was performed by PCR using the
following primers:
Forward: 5ʹ-GCGGTAATTCCAGCTCCAATAGC–3ʹ
and Reverse: 5ʹ -GACCATACTCCCCCCGGAACC-3ʹ.
Briefly, DNA was denatured at 94°C for 5 min and amplified by 30 cycles of denaturation at 95°C for 30 s, annealing at
58 °C for 30 s, and extension at 72 °C for 1 min. There was a final extension period at 72 °C for 10 min prior to a 4 °C
hold. The PCR product was isolated using a Gel PCR Clean-Up Kit (Qiagen). For sequencing reactions, 25 ng of PCR
product was used as template with 10 pmol of the above primers in separate reactions in a final volume of 12 μL. The
samples were then sent to the Biosearch tec. Company in USA for sequencing. Ribosomal RNA nucleotide sequences
from the isolates were obtained from the NCBI database based on the BLAST results of each microalga sequenced in
this study. When sequences from multiple isolates of a species were available, two nucleotide sequences were chosen:
(i) highest maximum score sequence, (ii) highest maximum score sequence with identified genus and species.
2.2. Wastewater
The wastewater used in this study were collected from three different effluent lines of a final settling tank. The first
sample was collected from oxidation station in Saadat city, Meonufia (OS), where, the second was collected from yellow
Mountain station in El salaam city, Cairo Governorate (Y) and the last sample was collected from zenin station, Giza
Governorate (Z). All are Sewage treatment plants. The samples were pre-filtrated using Whatman No.1 filters before
analysis. They were subjected to complete physical and chemical analysis at Central Lab, Egyptian Petroleum
Research Institute. Anions and cations were determined according to ASTM D-4327 and 6919, respectively using ion
chromatography. The instrument used was Dionex IC model ICS 1100 equipped with high capacity columns (AS9 and
CS12) for anion and cations, respectively.
Heavy metals were determined using Flame Atomic Absorption Spectrophotometer model Zenit 700p
according to ASTM D4691.
Physical properties including density, specific gravity, pH, etc.., were measured according to the following
standards procedures:
Total dissolved solids (TDS) were determined according to ASTM D-1888.
Density and specific gravity were determined according to ASTM D-1429
pH was determined according to ASTM D-1293 using digital pH meter model metler Toledo-Seven Go.
Alkalines (CO3, OH, and HCO3) were measured according to ASTM D-3875. Calculations were done using
Alkalinity calculator Ver. 2.10 (USGS).
2.3. Microalgal cultivation in different wastewaters with different treatments.
The two selected microalgal isolates were cultivated on the three tested waste waters S, Y and Z with ten different
treatments. (1) 100% waste water. (2) 100% waste water + complete BG11 elements (A+B) without fresh water.
(3)100% waste water + B solution only. (4) 100% waste water + 1 g/l NaNo3. (5) 100% waste water + soil extract. (6)
100% waste water + 5g/l glucose. (7) 50% waste water + 50% BG11. (8)50% waste water + 50% tap water + 100%
BG11 components. (9) 70% waste water + 30% BG11. (10) 70% waste water + 30% tap water + 100%BG11
components.
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
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All Flask cultures were completed to 250 mL in Erlenmeyer flasks containing the wastewater of 100 mL as a working
volume at a shaking incubator of 25 ± 1˚C, rotation speed of 150 rpm and continuous illumination with 60 µ mol cool-
white fluorescent light for about 20 day. The initial pH was controlled to pH 6.5 ± 0.1.The growth of the two isolates
were compared with their growth on BG11 medium. The microalgae growth was determined by measuring optical
density at a wavelength of 685 nm [11] (denoted as OD 685) using a spectrophotometer (model jenway 6300, Eu). The
dry cell weight (DCW) of microalgae biomass was also obtained by filtering 50 ml aliquots of culture through a cellulose
acetate membrane filter (0.45 μm pore size, 47 mm in diameter). Each loaded filter was dried at 105 ˚C until the stability
of weight. The dry weight of the blank filter was subtracted from that of the loaded filter to obtain the microalgae dry cell
weight.
2.4. Cultivation profile on wastewater
In order to evaluate the growth performance of microalgae in waste water, the selected isolates were cultivated on it
for maximum growth time (about 22 day). Cell counts were conducted on 0, 2, 4, 6 day and every two days until 22
day.
3. Results and discussion
3.1. Phylogenetic characterization of candidate microalgal strains.
Two water samples were collected from diverse aquatic habitats from Cairo and Qalubia, Egypt. Visual microscopy
confirmed the isolation of axenic cultures. Morphological comparisons to other described microalgae suggested that
these strains belonged to the genera Chlorella and Scenedesmus. To specify the identity of the microalgae strains
used in our experiments, a partial 18S region of the ribosomal RNA gene was amplified by PCR and sequenced. The
obtained sequences were then compared to existing sequences in the NCBI database. Sequence identity searches
confirmed a close relationship of the isolated candidate strains to Chlorella vulgaris strain KCTC AG10191and
Scenedesmus sp CCAP 217 7 as shown in (Figs.1 and 2).
Fig. 1: phylogenetic tree of 18S rRNA gene sequences from microalgae Chlorella vulgaris strain KCTC AG10191
Fig. 2: phylogenetic tree of 18S rRNA gene sequences from microalgae Scenedesmus sp CCAP 217 7
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
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2.2. Wastewater samples collection and analysis
The samples were collected from three different water stations in three different areas. All of them were subjected
to complete physical and chemical analysis to determine the elements content ratio in each one (Table1). In aquatic
ecosystems, nitrogen and phosphorus are the most important, as they are most often in short supply relative to
the needs of plants, algae and microorganisms. Other elements, like iron, manganese and copper, are needed in
small amounts [13]. Aforesaid output data showed the variation of nitrate and phosphate percentages from sample
to another which were affected the Microalgal isolates biomass. One of the most important factors for algal growth
is the nitrogen source and its concentration, so, it has been investigated through the previous analysis. The results
show the absence of nitrogen in (OS) sample and presence in low levels in (Y) and (Z) samples. For this reason,
the supplementation of wastewater with external nitrogen source often will be very useful. This result agree with
the studies showing that the combination of several waste water would be a choice for optimization of wastewater
for microalgae cultivation [14] and [15]. Phosphorus is an essential nutrient in biological wastewater treatment, it
was found only in OS station and absent in the two other stations. The results also show nearly the absence of
most heavy metals which enhance algal growth. Furthermore, some physical properties such as pH and TDS, were
suitable for algal growth.
Table (1): Physical and chemical properties of the collected water samples
Water sample
Analysis OS Y Z
physical properties
Total dissolved solids
(TDS) mg/l
2080 1220
746
pH
at 25˚ C
7.32 7.58
7.87
Salinity
mg/l
1133.3 449.9 433.1
Hardness
mg/l
612.1 410.0 274.9
Chemical properties(mg/l)
Nitrate Nil 18.12 19.6
Phosphate 60.99 Nil Nil
Chloride 686.84 272.65 262.5
Sodium 414.21 211.68 116.38
Magnesium 20.51 26.28 15.95
Heavy metals (mg/l)
Iron Nil Nil 10.68
Cupper 0.05 Nil 0.01
Barium Nil Nil Nil
Cadmium Nil Nil Nil
Lead Nil Nil Nil
Nickle Nil Nil Nil
3.3 Application of the previously mentioned treatments for three different waste waters on Chlorella vulgaris
strain KCTC AG10191 and Scenedesmus sp. CCAP 217 7
Ten treatments were applied on all collected wastewater. The results indicated that (OS) water is the best one for
microalgal cultivation and the supplementation with nitrogen source (NaNO3) is the best treatment. The growth of the
two promising algal strains were enhanced by this addition but the yield was different due to the wastewater used. The
growth limitation in the wastewaters of Chlorella vulgaris strain KCTC AG10191 (C.V) and Scenedesmus sp. CCAP
217 7 (S.S) could be partially explained by nutrients such as nitrogen and phosphorus depletion, but it seemed that
there were other factors besides the N and P limitation in the culture because the nutrients used were enough during
the total cultivation. However, the highest biomass production was achieved in the culture of (OS) wastewater
supplemented with nitrogen source. Therefore the results indicate that the best waste water is (OS) as a nutrient source
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
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and the most promising treatment is treatment (4) (fig.3). This result was similar to that studies which reported the
importance of nitrogen source for microalgal growth [16], [17], [18] and [19] .As shown in fig. 4 and 5, the biomass
yield is nearly similar in all treatments and the supplementation with external nitrogen source have no promising
increasing effect which mean that the algal growth on (Y) and (z) wastewaters with all treatments is not the best in
compare with BG11 media. Also we noted that the treatment 6 (supplementation with glucose) have a bad effect on
both microalgal isolates. This result was not agree with Liang et al., 2009, [20] who reported the ability of many algae
to assimilate a variety of organic carbon sources.
Fig.3: Application of the ten treatments on (OS) water and the two promising microalgal strains Chlorella vulgaris strain
KCTC AG10191and Scenedesmus sp CCAP 217 7
Fig.4: Application of the ten treatments on (Y) water and the two promising microalgal strains Chlorella vulgaris strain
KCTC AG10191and Scenedesmus sp CCAP 217 7
Fig.5: Application of the ten treatments on (Z) water and the two promising microalgal strains Chlorella vulgaris strain
KCTC AG10191and Scenedesmus sp CCAP 217 7
0
2
1 2 3 4 5 6 7 8 9 10
Dry
wei
gt (
g/l)
Treatments
(OS) Water
C.V S.S
0
0.5
1
1 2 3 4 5 6 7 8 9 10
Dry
wei
ght
(g/l
)
Treatments
(Z) Water
C.V S.S
0
0.5
1
1 2 3 4 5 6 7 8 9 10
Dry
wei
ght
(g/l
) Treatments(Y) Water
C.V S.S
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
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3.4. Growth profile of microalgal strains on waste water
Algal growth in terms of optical density O.D680 on the (OS) wastewater under axenic condition were plotted in (Figs.
6and 7). Short lag phase was observed specially for the organism (C.V.) indicating that this algal isolate could adapt
well in OS. waste water. Moreover, the algal growth was significantly enhanced in the OS + (O.S waste water
supplemented with NaNo3 as a nitrogen source) because of its higher levels of nitrogen. The final algal biomass reach
to 6.2 O.D in Chlorella vulgaris strain KCTC AG10191 and 2.6 O.D in Scenedesmus sp CCAP 217 7 under the condition
of OS+. In the treatment OS-( O.S waste water without NaNo3) the final algal biomass reach to 3.2 O.D in Chlorella
vulgaris strain KCTC AG10191 and 1.82 O.D in Scenedesmus sp CCAP 217 7. The algal growth enhanced by the
addition of nitrogen source in the ten day but the treatment OS+ still the best. These results were matched with the
studies which proved that the algal growth is directly affected by the availability of nutrients [21].
Fig.6: Growth curve of the two promising microalgal strains Chlorella vulgaris strain KCTC AG10191and Scenedesmus
sp CCAP 217 7 growing on (OS) water supplemented with NaNO3
Fig.7: Growth curve of the two promising microalgal strains Chlorella vulgaris strain KCTC AG10191and Scenedesmus
sp CCAP 217 7 growing on (OS) water without NaNO3
0.22 0.661.9 2.1 2.5 2.7 2.75 2.8 3.2
4.7
6.4 6.2
0.084 0.107 0.27 0.61 0.93 1.2 1.7 2.1 2.4 2.5 2.6 2.6
0 2 4 6 8 10 12 14 16 18 20 22
O.D
68
0 n
m
Incubation period (day)
Growgh curve on (os+)
C.V S.S
0.28 0.440.67
1.1 1.3 1.1 1.31.6 1.68 1.8 2
3.2
0.087 0.088 0.11 0.14
0.84 0.78 0.92 11.3 1.5 1.7 1.82
0 2 4 6 8 10 12 14 16 18 20 22
O.D
68
0 n
m
Incubation period (Day)
Growth curve on(OS-)
C.V S.S
J. Algal Biomass Utln. 2016, 7 (1): 78-85 Cultivation of microalgae using municipal wastewater ISSN: 2229 – 6905
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Conclusion
Based on current tools, algal cultivation for biofuel production alone is unlikely to be economically viable or
provide a positive energy yield.
Dual-use microalgae cultivation for wastewater treatment coupled with biofuel generation is therefore an
attractive route in terms of reducing the nutrient (fertilizer) and freshwater resource costs of biofuel generation
from microalgae.
The high biomass productivity of wastewater-grown microalgae suggests that this cultivation method offers
real potential as a viable means for biofuel generation and is likely to be one of many approaches used for the
production of sustainable and renewable energy.
The results show that the supplementation of waste water with nitrogen source is the best treatment give high
biomass production.
The algal growth rate enhanced by the addition of nitrogen source from the starting of cultivation.
For further investigation, the lipid content and fatty acid profile of lipid extracted from Chlorella vulgaris strain
KCTC AG10191and Scenedesmus sp CCAP 217 7 when growing on wastewater will be investigated in the
following research paper.
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