Harvesting Microalgal Biomass Grown in Anaerobic Sewage Treatment Effluent by The Coagulation-Flocculation Method: Effect of PH

Brazilian Archives of Biology and Technology
Harvesting Microalgal Biomass grown in Anaerobic Sewage

Treatment Effluent by the Coagulation-Flocculation Method:
Effect of pH
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Journal:
Manuscript ID
Manuscript Type:
Keyword:

BABT-2016-0174.R1
Original Article
microalgae, anaerobic effluent, coagulation-flocculation
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https://mc04.manuscriptcentral.com/babt-scielo
Page 1 of 24
Figure 1 - Microalgae species found in the anaerobic efluente after 6 days of MBM growth: (A) Scenedesmus
sp. (B) Chlorella sp. (C) Synechocystis sp.
164x53mm (96 x 96 DPI)
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Figure 2 Daily cycle variation of pH and temperature of the MBM batch growth, over 8 days.
165x74mm (96 x 96 DPI)
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Harvesting Microalgal Biomass grown in Anaerobic Sewage

Treatment Effluent by the Coagulation-Flocculation
Method: Effect of pH
Abstract
Harvesting is a critical step in microalgal biomass production process for many reasons. Among the existing
techniques available for harvesting and dewatering microalgal biomass, recovery from aqueous medium by
coagulation-flocculation has been the most economically viable process, although it is highly dependent on pH. This
study aims to assess alternative coagulants compared to the standard coagulant aluminum sulfate for microalgal
biomass recovery from anaerobic effluent of domestic sewage treatment. The effluent quality was also analyzed
after biomass recovery. Coagulants represented by modified tannin, cationic starch and aluminum sulfate recovered
more than 90% of algae biomass, at concentrations greater than 80 mg/L, in the pH range 7-10. Cationic starch
promoted higher microalgal biomass recovery with a wider pH range. Powdered seeds of Moringa oleifera and
Hibiscus esculentus (okra) gum promoted biomass removal of 50%, only in the acidic range of pH. After
sedimentation of the microalgal biomass, the effluents showed a removal of >80% for phosphorus and nitrogen
values and >50% for BOD and COD when using aluminum sulfate, cationic starch and modified tannin as
coagulants. Natural organic coagulants in a wide pH range can replace aluminum sulfate, a reference coagulant in
microalgal biomass recovery, without decreasing microalgal biomass harvesting efficiency and the quality of the
final effluent.
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Keywords: microalgae, anaerobic effluent, coagulation-flocculation, pH
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INTRODUCTION
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Microalgae is a general, rather than taxonomic, designation generally referring to photosynthetic

unicellular microorganisms, with visible growth in water bodies as a major component of phytoplankton.
The wide variety of microalgal species include prokaryotic algae (cyanobacteria) and eukaryotic algae,
such as diatoms (Bacillariophyta), green algae (Chlorophyta) and red algae (Rhodophyta)1.
For microalgal biomass (MBM) production, microalgae rely on sources of inorganic carbon and other
nutrients, which mostly include nitrogen (N) and phosphorus (P). When provided these nutritional
requirements, microalgae can be grown in natural water matrices (fresh and marine), synthetic growth
media and effluent from domestic sewage or industrial treatment systems2,3.
The use of wastewater for microalgal growth provides two significant benefits: the tertiary treatment of
effluent at the end of the biomass growth and recovery process, which incorporates the nutrients required
by microalgae and generates bulky biomass for many purposes4,5. Microalgal biofuels could be an
important alternative to conventional biofuels, as microalgae can be produced at high rates without the
need of arable land or potable water and without having competition with food production2. In addition to
the benefits of microalgal biomass production in the effluent medium, microalgae are able to assimilate
CO2 as a carbon source for growth. Approximately 1.6 to 2.0 g of CO2 can be captured per gram of MBM
produced, thus contributing to a reduction in greenhouse gases6.
The energy-intensive harvesting processes are limiting the commercial production of microalgal biofuels
and other products that could be commercially viable. Harvesting microalgal biomass from the aqueous
media is a critical step for microalgal biomass production because it represents 20 to 50% of the total
cost7,8. The main microalgal harvesting processes include filtration, centrifugation, magnetic or
electromagnetic separation, coagulation-flocculation, flotation and sedimentation or a combination of
these processes. However, despite the abundance of microalgae recovery studies for microalgal biomass
recovery, there is no consensus on the most appropriate methodology for this process9,10. The coagulationflocculation process, followed by sedimentation, has been the most widely adopted method because it has
been shown to be economically viable for large production volumes of biomass11,12.
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The coagulation-flocculation process can promote microalgal aggregation by the addition of coagulants
(electrolytes), which can be metal salts, natural or synthetic polymers, or through pH adjustment,
providing floc formation which facilitates microalgal biomass sedimentation13. According to Duan and
Gregory14, this process is critically dependent on pH values. Inorganic coagulants are very effective for
MBM harvesting, but require a high dosage of coagulant addition, and thus, contaminates the MBM with
aluminum and iron. Unlike inorganic metal salts, biodegradable organic coagulants do not contaminate
the recovered biomass, and generally require low application doses, considering a wide pH range.
Therefore, coagulants obtained from natural polymers are better alternatives for MBM harvesting than
inorganic coagulants15,16,17.
In this work, aluminum sulfate was used as the reference coagulant, and natural organic coagulants were
obtained from Moringa oleifera seeds, Acacia tannin, Hibiscus esculentus gum and cationic starch and
were evaluated for MBM recovery by the coagulation-flocculation-sedimentation method. The optimal
dose and pH values were evaluated, which provide the best efficiency rates for recovery of MBM for each
polymer. Furthermore, the quality of the effluent after sedimentation of the three best MBM recovered
coagulants was assessed.
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MATERIALS AND METHODS
Microalgal Growth Conditions

Microalgal growth was performed in effluent media from the anaerobic domestic experimental sewage
treatment plant, located at Federal University of Esprito Santo (UFES), Goiabeiras campus, Vitoria, ES,
Brazil. The pH, alkalinity, COD, BOD, total phosphorus (TP), and NTK nitrogen (TN) of the anaerobic
effluent were analyzed, according to methodologies proposed by the Standard Methods for the
Examination of Water and Wastewater18, as shown in Table 1.
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Table 1 Averaged values ( ) and coefficients of variation (CV) of the main parameters evaluated for anaerobic
effluent quality (n=3).

Parameter

CV (%)
pH
7.8
0.5
Alkalinity (mg/L CaCO3)
207
2
COD (mg/L O2)
66
9
BOD (mg/L O2)
110
8
Total Phosphorous (mg/L)
9.8
1.4
Total Nitrogen (mg/L)
37.2
6.0
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The anaerobic effluent was previously filtered through a sand-gravel filter to remove large particles. The
effluent was inoculated with a Chlorella sp. strain previously isolated and grown in sewage anaerobic
effluent, at a final concentration of 2.0x107 cells/mL. The production of microalgal biomass was
conducted in an outdoor batch pilot-scale photobioreactor (FBR), which consisted of a 1.0 m plastic tank
with an aeration system programmed to operate for 15 minutes, every three hours, and maintained at a
temperature of approximately 30C during daytime hours.
Optical microscopy images of the main species of microalgae grown in the anaerobic effluent were
obtained through optical microscopy (CARL ZEISS Axioplan), using a digital camera (MC80DX)
coupled to the equipment, on a 40 m scale.
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Coagulation-Flocculation Assays
The coagulation-flocculation process was performed in jar tests under hydraulic parameters according to
Di Bernardo & Di Bernardo19 and Richter20. Four alternative coagulants were evaluated (modified tannin
Tanfloc Pop, Moringa oleifera seed powder, Hibiscus esculentus gum and SUPERION 300 cationic
starch). The coagulant aluminum sulfate (Al2(SO4)3) was used as a reference for the microalgal
coagulation process.
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The SUPERION 300 cationic starch solution was obtained from the mixture of the powder with distilled
water at a concentration of 10 g/L and solubilized at 60C, until the formation of a gelatinous product.
Tanfloc Pop (TANAC) and aluminum sulfate (Dinamica, Al2(SO4)3.(14-18)H2O) solutions were
established in accordance with the guidelines of the product manufacturer. The dry seeds of M. oleifera
were processed in accordance with the recommendations of Teixeira et al.13. Okra (Hibiscus esculentus)
gum prepared from raw okra involved the following macro-steps: (1) Raw pods of H. esculentus, obtained
from a local market, were washed with distilled water, and then, the seeds were removed from the pods,
according to Mishra et al.21. (2) For gum extraction, the pods were triturated for one minute, with distilled
water at 50C, in a ratio 1:7 w/v. After grinding, the contents were filtered as recommended by Pranne &
Tanatcha22. (3) The liquid fraction was mixed with ethanol 3:1 v/v and stirred at 150 rpm for an hour. The
obtained gum was washed in acetone 1:1 v/v, stirred at 150 rpm for 20 minutes, and then filtered through
a muslin fabric vacuum system to remove excess of ethanol and acetone. (4) The solubilized material was
placed in distilled water at 65C, until the formation of a gelatinous product; this product was used as the
okra coagulant.
The coagulation diagrams were created according to the methodology proposed by Paixo23 and Di
Bernardo & Di Bernardo19, and plotted in the software Surfer 11. The microalgae biomass nutrient
removal efficiency was determined by Equation 1, where E is the removal efficiency in percent, C0 is the
value determined for the parameter evaluated, and C is the concentration of the parameter evaluated in the
final effluent after MBM sedimentation.
E%
100
(Eq. 1)
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Microalgal Biomass and Final Effluent Characterization

MBM characterization was determined by the ratio of volatile solids to total solids VSS/TS, according to
Andreoli et al.24. The physical-chemical characterization of the final effluent after the separation process
was determined by measuring pH, alkalinity, COD, BOD, total phosphorus and NTK nitrogen. All assays
were performed in accordance to the Standard Methods for the Examination of Water and Wastewater18.
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Statistical Analysis
Statistical analysis was performed by using an analysis of variance (ANOVA), followed by a Tukeys
post hoc test, to determine the significance in differences among the physical-chemical parameter values,
with a significance level of = 5.0%.
RESULTS AND DISCUSSION
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The dominant genera of microalgae naturally associated with the anaerobic effluent were Scenedesmus
sp. (Fig. 1A), Chlorella sp. (Fig. 1B) and Synechocystis sp. (Fig. 1C). Additionally, other
microorganisms, such as fungi and protozoa, were associated with the anaerobic effluent due to natural
growth in the non-sterile medium.
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Figure 1 - Microalgae species found in the anaerobic effluent after 6 days of MBM growth: (A) Scenedesmus sp.,
(B) Chlorella sp., and (C) Synechocystis sp.
In study reported by Torres25, six microalgae, identified as Desmodesmus sp., Chlorococcum sp.,
Coccomyxa sp., Chlorella sp., Scenedesmus sp. and Tetradesmus sp., were isolated from a Domestic
Sewage Anaerobic Experimental Treatment Plant. Unlike the results of this study, Torres25 observed
abundant growth of Chlorella sp. in the anaerobic effluent, which showed a survival greater than 90%
compared to other species. The evaluation of the microalgal growth in a photobioreactor, using previously
primary- and secondary-treated effluent from a meat processing industry as the growth medium, was
performed in the study by Tango26. Similar to the present study, Scenedesmus sp. was the dominant
microalgae in both evaluated effluents.
The growth of microalgae was dependent on pH and temperature of the medium, characterized as a daily
variation cycle, as shown in Figure 2.
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Figure 2 Daily cycle variation in pH and temperature of the MBM batch growth over 8 days.
The microalgal biomass growing in culture showed a large variation in pH values varying from
approximately 8 to a peak of 11 over the course of the daily diurnal cycle. Initial pH values were less
variable and after stable growth was achieved by day 4, the diurnal cycle was fully operating. This could
be associated with the CO2/ CO32- balance, as seen in Equation 2.
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CO2(aq) H2CO3 H+ + HCO3- H+ + CO32-
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(Eq. 2)
The inorganic carbon species normally used by microalgae are CO2 and HCO3. The assimilation of
HCO3, assisted by the enzyme carbonic anhydrase, likely represents a true H+ removal from water
medium and consequently results in an increase in pH values. At higher pH values, e.g., pH >9, most of
the inorganic carbon is in form of carbonate (CO32) which is also used by microalgae in high alkalinity
values, resulting in H+ removal from water medium and consequently results in an increase in pH. Night
respiration and other metabolic processes replenish the CO2 in the medium, lowering the pH values and
restoring the pH cycle27.
Addition or removal of CO2 have no direct effect on the alkalinity, but these processes do affect the pH
and are responsible for the significant diurnal changes in pH. Because of the pH values observed in the
daily cycle of microalgal growth, the choice in coagulant type became a critical factor. Thus, we propose
optimized microalgal biomass recovery through a coagulation-flocculation mechanism based on the pH
range of the medium. The results of coagulation assays performed with jar tests and plotted by diagrams
are shown in Figure 3.
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Figure 3 - Coagulation diagrams based on Jar tests data for different coagulants. The lightest areas represent lower
turbidity values, while the darker areas have the greatest remaining turbidity values.
Based on the results obtained from the diagrams, it was possible to determine the microalgal biomass
(MBM) removal efficiency values, as a function of dose and pH, as recorded in Table 2.
Table 2. Dose, optimum pH values and MBM removal efficiencies for each coagulant when evaluated by means of
the coagulation-flocculation process followed by sedimentation.
Coagulant
Aluminum sulfate
SUPERION 300 cationic starch
TANFLOC POP
Moringa oleifera seed
Hibiscus esculentus (okra) gum
pH
7.0
10.0
9.0
5.0
5.0
Dose (mg/L)
50.0
80.0
100.0
150.0
100.0
Removal efficiency (%)

95.9
92.5
95.6
85.8
80.3
From the analysis of the results presented in this study and in the related literature, it could be inferred
that aluminum sulfate is an effective coagulant in the recovery of MBM for neutral and acidic pH ranges,
in doses less than 80 mg/L. At basic pH values, aluminum sulfate reached a biomass recovery of 60 to
70% with doses higher than 80 mg/L. Efficiencies greater than 90% were obtained by applying doses
greater than 50 mg/L, at neutral pH; however, according to this study, neutral pH is not within the
operating pH range for microalgal growth. In a study by Teixeira et al.13, C. vulgaris biomass recovery
was 93.8%, applying 1000 mg/L of aluminum sulfate at pH 9.24. Gerde et al.16 applied aluminum sulfate
to recover Scenedesmus sp. grown in synthetic medium at an adjusted pH between 8.5 and 9.5. Doses of
250 mg/L of aluminum sulfate were required to achieve 90% of Scenedesmus sp. removal by means of
coagulation-flocculation. The data presented in the literature corroborate the findings of this study
because a high MBM efficiency removal requires higher doses of aluminum sulfate by coagulationflocculation at high pH.
For doses greater than 80 mg/L of modified tannin, recovery efficiencies were greater than 80%, with pH
ranging from 7 to 10. Similar results were obtained to remove Microcystis aeruginosa cultured in BG-11,
using a modified tannin coagulant (Q-TN)28. Recovery efficiencies obtained were greater than 90% in a
pH range from 6 to 928. In a recent study, Chlorella vulgaris biomass cultivated in swine effluent treated
by phytoremediation had a 97% removal efficiency rate using 11 mg/L of commercial modified tannin at
neutral pH29. In a study to recover C. vulgaris and Nannochloropsis oculata, grown in synthetic medium
with pH of 8, the removal efficiencies for C. vulgaris were 99, 100 and 100% for concentrations of 5, 15
and 45 mg/L, respectively. In contrast, N. oculata removal efficiencies were 92, 96 and 99% for
concentrations of 5, 15 and 45 mg/L, respectively30. In this study, the effect of pH at the same low
concentrations of modified tannin (5, 15 and 45 mg/L), the removal efficiencies were lower, between 30
and 60%. For pH values of approximately 8, the MBM recovery from a raceway pond, which treats
wastewater, were greater than 90% by using 50 mg/L of Tanfloc SG30 as the coagulant. In contrast, the
same study conditions of Gutierrez et al.31 were established in this study efficiency of 96.7% using
Tanfloc Pop modified tannin as coagulant.
When cationic starch is used as a coagulant in MBM recovery, with doses between 80 and 150 mg/L, the
removal efficiencies were above 80% for a wide range between pH 7and 11. Two types of cationic starch
(GREENFLOC 120 and CARGILL C*BOND RH 35 849) were tested for Parachlorella sp. and
Scenedesmus sp. recovery in a study by Vandamme et al.32,33. With a pH range between 7 and 8, than
80% at cationic GREENFLOC 120 was more efficient in biomass recovery than Cargill C*BOND RH
35 849 for both genera of microalgae. The removal efficiency was greater than 80% for starch doses
ranging from 20 to 40 mg/L (Parachlorella sp.) and 5 mg/L (Scenedesmus sp.); these results corroborate
those observed in the current study for MBM removal efficiency using the same dosage and pH ranges.
Gerde et al.16 used two types of cationic starch (DS02 and DS05) to harvest Scenedesmus sp. and
Chlamydomonas reinhardtii in synthetic medium at pH adjusted between 8.5 and 9.5. The specie
Scenedesmus sp. required 20 mg/L of DS5 to remove 90% of the biomass. The specie C. reinhardtii
required approximately 40 mg/L of DS2 and 20 mg/L of DS5 to obtain 85 and 90% of removal,
respectively. Hansel et al.34 used modified cationic potato starches for flocculation of Scenedesmus
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dimorphus cultures. The results showed that all cationic starches were highly effective and the best
removal rate used 10 mg/L of the tested starches, resulting in recovery efficiency above 95%.
According to Vandamme et al.32,33, the cationic starch positive charge is due to quaternary ammonium
salts introduced in the manufacturing process, which are able to maintain their positive charge even at
high pH values and can contribute for MBM autoflocculation in higher pH; this may have promoted an
increase in the recovery efficiency of BMA at high pH. Furthermore, according to the authors, the
efficiency of flocculation by cationic starches and modified tannins can also be related to the degree of
substitution of the quaternary groups, the site of substitution and the molecular weight of the polymers.
The use of M. oleifera seed coagulant was not the best option for MBM recovery in this study based on
alkaline pH range. A maximum removal efficiency of approximately 80% was achieved by adjusting the
pH to the acidic range (4 to 5) and applying doses of 100 to 150 mg/L of the M. oleifera seed coagulant.
Teixeira et al.13 achieved an efficiency of 88.6% in C. vulgaris biomass removal, using WC medium and
adjusting the pH to 9.2 and applying 1000 mg/L of M. oleifera powdered seeds, although the long 2 hours
settling time. It should be noted that the selected concentration and settling time in the study by Teixeira
et al.13 were significantly higher than those observed in the present work. Hamid et al.35 obtained
efficiencies greater than 95% for Chlorella sp. removal using a M. oleifera coagulant, but using Bold
basal medium, at an approximate pH value of 6, and the coagulation process with 30 mg/L M. oleifera
powdered seeds with a settling time of 2 hours.
Using polymers extracted from okra (H. esculentus) as a coagulant was not adequate for biomass growth
in FBR alkaline pH range as observed in the MBM coagulation diagram charts. Low removal rates were
observed in acidic pH ranges, approximately 4 and 5, reaching efficiencies of up to 80%. There is no
sufficient literature data for MBM harvesting studies with microalgae grown in anaerobic sewage
treatment effluent, but several studies investigate turbidity removal pH range domestic sewage effluent
treated with polymers extracted from okra. Anastasakis et al.36 presented data of turbidity removals of 70
to 74% from biologically treated effluent, applying 2.5 mg/L of polymers extracted from okra seeds in pH
6.09. In contrast, Agarwal et al.37 achieved a suspended solids removal efficiency of approximately 86%
for raw wastewater treatment, with only 0.12 mg/L of extracted okra gum at neutral pH.
Modified tannin, aluminum sulfate and cationic starch as coagulants were the most efficient for MBM
recovery after the sedimentation process. The consolidated results for three best coagulants for MBM
recovery for pH range of microalgal growth are presented in Table 3.
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Table 3 - Means ( ) and coefficients of variation (CV%) of the main parameters for the evaluation of anaerobic
effluent used for MBM growth (EUASB) and of the effluents after MBM removal by aluminum sulfate (ESAL),
modified tannin (ETFP) and cationic starch (EACS). The different letters on each line indicate significant
differences (p <0.05) by Turkeys test.
EUASB
ESAL
ETFP
EACS
Parameter
CV(%)
CV(%)
CV(%)
CV(%)

pH
7.8
0.5
3.4b
4.0
4.1b
14.8
7.1
1.2
207
2
15b
12
15b
25
21b
9
BOD (mg/L O2)
66a
10
43b
2
45b
7
21c
5
a
b
b
b
COD (mg/L O2)
110
8
21
29
22
8
11
13
TP (mg/L)
9.8
1.4
1.3b
22.5
1.4b
7.5
0.9b
5.1
TN (mg/L)
37.2
6.0
3.6bc
20.8
5.4b
15.8
1.8c
15.8
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From the analysis of the main physical-chemical parameters, the results suggest that the effluent pH
values varied significantly after biomass removal and were generally more acidic, except for the effluent
after biomass removal with the cationic starch, which provided an effluent with a nearly neutral pH.
The results presented in Table 3 illustrate one of the major advantages of MBM growth in anaerobic
sewage treatment effluents: the growth of MBM represents a tertiary sewage treatment, because of the
removal of phosphorus and nitrogen from the aqueous phase. The total P and total N decrease were more
evident after using cationic starch as a coagulant, showing removal efficiencies of 90.64% and 95.14%,
respectively. Nutrient removal could be related to the MBM assimilation and the other associated
biomass, such as fungi and bacteria as microalgal growth was not performed in sterile medium.
Interestingly, a decrease in chemical and biochemical oxygen demands (BOD and COD) of 44% and
84%, respectively, were observed (Table 3). The removal of BOD and COD may be related to the
removal of the suspended and dissolved organic material with negative charges present in anaerobic
sewage treatment effluent (EUASB), microalgal cells and other microorganisms associated with EUASB;
therefore, this process decreases the oxygen demand, particularly the chemical oxygen demand (Table 3).
Alkalinity is another parameter that showed the same trend with decreasing values as compared to the
anaerobic sewage treatment effluent, taking on average by 91%. As mentioned previously, growth
medium pH and alkalinity increasingly coincide with the exponential MBM growth phase; this is a
phenomenon that can be explained by the consumption of CO2 for photosynthesis, increasing the
concentration of HCO3- and CO32- and consequent rising in pH and alkalinity from carbonates and
bicarbonates. As the culture moves to a lower stage of growth, during which stage the samples were
collected for biomass removal, the medium alkalinity slightly reduced due to a reduction in dissolved CO2
consumption.
CONCLUSIONS
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It was possible to verify polymer dosage, the optimum pH values and MBM removal efficiencies through
the process of coagulation-flocculation followed by sedimentation. It is worth noting that, for high pH
values from 8 to 10, only the cationic starch and modified tannin coagulants provided optimal MBM
removal efficiencies with doses of 80 and 100 mg/L of the polymers, respectively.
From the analysis of the coagulation diagrams and the biomass removal efficiency data, cationic starch
had the lowest dose required with highest recovery efficiency in the target pH range observed for the
MBM growth; however, the pH range of application and recovery efficiencies were similar to those
reached using the modified tannin. Aluminum sulfate showed a smaller pH range for application than the
range of modified tannin and cationic starch polymers, and required larger doses to achieve the same
MBM recovery efficiency.
M. oleifera seeds and H. esculentus (okra) gum promoted MBM removals of only approximately 50%, in
the acidic pH range. With aluminum sulfate, modified tannin and cationic starch as coagulants, the
effluents generated after MBM floc settling revealed a total P removal of greater than 80%, and total N,
BOD and COD removals were all greater than 50%.
Although aluminum sulfate is commonly used as a reference coagulant in microalgal biomass recovery,
this work verified that it can be replaced by natural coagulants (e.g., modified tannin and cationic starch)
under the conditions presented here, without decreasing the biomass removal efficiency and causing
negative impacts to the final effluent and, consequently, to the recipient water bodies.
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ACKNOWLEDGMENTS
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This work was completed under the Biogas Project. Authors are thankful to Department of Environmental
Engineering Master's program for their support. The authors also gratefully acknowledge the financial
support and scholarship of FINEP/UFES and CNPq, respectively.
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biomass production. Trends Biotechnol. 2013 apr; 31(4): 233-239.
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by bio-flocculation by Moringa oleifera seed derivatives from aquaculture wastewater phytoremediation. Int
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Page 13 of 24
Harvesting Microalgal Biomass grown in Anaerobic Effluent

from Sewage Treatment by Coagulation-Flocculation: Effect
of pH
Abstract
Harvesting is a critical step in microalgae biomass production process for many purposes. Among the existing
techniques available for harvesting and dewatering microalgae biomass, recovery from aqueous medium by
coagulation-flocculation has been the most economically viable process, although highly dependent on pH. This
study aims to assess alternative coagulants for microalgae biomass recovery as compared with the standard
coagulant aluminium sulphate from anaerobic effluent of domestic sewage treatment. The effluent quality was also
analysed after biomass recovery. Coagulants represented by modified tannin, cationic starch and aluminium
sulphate recovered more than 90% of algae biomass, at concentrations greater than 80 mg/L, in the pH range 7-10.
Cationic starch promoted higher microalgae biomass recovery with the wider pH range. Seeds of Moringa Oleifera
and Hibiscus esculentus (okra) gum promoted biomass removal of 50%, only in the acidic range of pH. The
effluents after sedimentation of the microalgae biomass showed removal >80% for phosphorus and nitrogen and
>50% for BOD and COD, using aluminium sulphate, cationic starch and modified tannin. Natural organic
coagulants in a wide pH range can replace aluminium sulphate, which is a reference in microalgae biomass
recovery, without decreasing microalgae biomass harvesting efficiency and the quality of the final effluent.
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Keywords: microalgae, anaerobic effluent, coagulation-flocculation, pH
INTRODUCTION
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Microalgae is a general designation, not taxonomic, generally referring to photosynthetic unicellular

microorganisms, with visible growth in water bodies, as a major component of phytoplankton. Among the
wide variety of microalgae, can be highlighted prokaryotic algae (cyanobacteria) and eukaryotic algae
such as diatoms (Bacillariophyta), green algae (Chlorophyta) and red algae (Rhodophyta) (BICUDO &
MENEZES, 2005).
For microalgae Biomass (MBM) production, microalgae rely on sources of inorganic carbon and other
nutrients, which include mostly nitrogen (N) and phosphorus (P). Providing these nutritional
requirements, microalgae can be grown in other media, including natural water matrices (fresh and
marine), synthetic growth media and even effluent from sewage or industrial treatment systems (MATA
et al., 2010; FAKIR et al., 2011).
The use of wastewater for microalgae growth provides two significant benefits: the tertiary treatment of
effluent at the end of the process, through the incorporation of nutrients by microalgae and the generation
of bulky amount of biomass for many other purposes. (KUNJAPUR & ELDRIDGE, 2010;
MAHAPATRA et al., 2015). Microalgae biofuels could be important alternative to conventional biofuels
since microalgae could be produced at high rates without the need of neither arable land, potable water or
competition with food. (MATA et al., 2010). Besides the benefits of microalgae biomass production in
effluent medium, microalgae are able to assimilate CO2 as a carbon source for growth. About 1.6 to 2.0 g
of CO2 can be captured, per gram of MBM produced, thus contributing to greenhouse gases reduction
(ZENG et al., 2011).
The energy intensive harvesting processes are limiting the commercial production of microalgae biofuels
and other products to be commercially viable. Since it can represent 20 to 50% of the total costs, harvest
microalgae biomass from the aqueous media is a critical step for microalgae biomass production (GRIMA
et al., 2003; GREENWELL et al., 2010). The main microalgae harvesting processes comprises filtration,
centrifugation, magnetic or electromagnetic separation, coagulation-flocculation, flotation and
sedimentation or even a combination of these. However, despite the abundance of microalgae recovery
studies for microalgae biomass recovery, there is no consensus on the most appropriate methodology for
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this process. (MILLEDGE & HEAVEN, 2013; CASTRILLO et al., 2013). The coagulation-flocculation
mechanism, followed by sedimentation, has been the most widely adopted method, since this has been
shown to be economically viable for large volumes of biomass production (UDUMAN et al., 2010;
GRANADOS et al., 2012).
Coagulation-flocculation process can promote microalgae aggregation by the addition of coagulants
(electrolytes) which can be metal salts, natural or synthetic polymers, or by pH adjustment, providing floc
formation facilitates microalgae biomass sedimentation (TEIXEIRA et al., 2012). According to Duan and
Gregory (2003), this process is critically dependent on pH values. Inorganic coagulants are very effective
for MBM harvesting but requires high dosage and thus contaminating MBM with aluminum and iron.
Instead inorganic metal salts, biodegradable organic coagulants do not contaminate the recovered biomass
and generally require low application doses, although highly dependent on pH range. Thus, coagulants
obtained from natural polymers are therefore a better alternative (WAN et al., 2014; GERDE et al., 2014;
BAPTISTA et al., 2015).
In this work, aluminum sulfate was assumed as reference coagulant, and natural organic coagulants
obtained from Moringa oleifeira seed, Acacia tannin, Hibiscus esculentus gum and cationic starch were
evaluated for MBM recovery by coagulation-flocculation-sedimentation and evaluate the optimal dose
and pH values, which provide the best recovery of MBM for each polymer. Furthermore, the quality of
the effluent after sedimentation of the three best MBM recovered coagulants.
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MATERIALS AND METHODS
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Microalgae Growth Conditions

Microalgae growth was performed in effluent media from the anaerobic domestic experimental sewage
treatment plant, located at Federal University of Esprito Santo (UFES), Goiabeiras campus, Vitoria, ES,
Brazil. The anaerobic effluent was analysed by pH, alkalinity, COD, BOD, total phosphorus (TP), NTK
nitrogen (TN), according to methodologies proposed by the Standard Methods for the Examination of
Water and wastewater (APHA, 2013), as shown in Table 1.
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Table 1 Averaged values ( ) and coefficients of variation (CV) of the main parameters evaluated for anaerobic
effluent quality (n=3).

Parameter

CV (%)
pH
7.75
0.47
206.80
2.44
COD (mg/L O2)
66.00
9.09
BOD (mg/L O2)
110.00
7.58
TP (mg/L)
9.79
1.39
TN (mg/L)
37.16
5.96
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The anaerobic effluent was previously filtered through a sand-gravel filter for large particles removal and
possibly regulate the bacteria and protozoa population and inoculated with Chlorella sp. strain previously
isolated and grown in anaerobic effluent, at final concentrations of the order of 2.0x107 cells/mL. The
production of microalgae biomass was carried out in an outdoor batch pilot photobioreactor (FBR), which
is consisted of a 1.03 m plastic tank with aeration system programmed for 15 minutes, every three hours,
at kept at temperatures around 30C during daytime.
Optical microscopy images of the main species of microalgae grown in the anaerobic effluent obtained by
optical microscopy CARL ZEISS Axioplan using digital camera (MC80DX) coupled to the equipment,
scale of 40 m.
Coagulation-Flocculation Assays
Coagulation-flocculation process was performed in jar tests using hydraulic parameters according to Di
Bernardo & Di Bernardo (2002) and Richter (2009). Four alternative coagulants were evaluated
(noodified tannin Tanfloc Pop, Moringa olifera seeds powder, Hibiscus esculentus gum and
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Page 15 of 24
SUPERION 300 cationic starch). The coagulant of aluminum sulfate (Al2(SO4)3) was used as a reference
for microalgae coagulation process.
SUPERION 300 cationic starch solution obtained from the mixture of the powder with distilled water at
a concentration of 10 g/L and solubilized at 60C, until the formation of a gelatinous product. Tanfloc
Pop (TANAC) and aluminum sulfate (Dinamica, Al2(SO4)3.(14-18)H2O) solutions and were established
in accordance with guidelines of the product manufacturer. The dry seeds of M. oleifera processed in
accordance with recommendations of Teixeira et al. (2012). Okra (Hibiscus esculentus) gum prepared
from raw okra, involving the following macro-steps: (1) Raw pods of H. esculentus, obtained from local
Market, washed with distilled water and then removed cups of the seeds from the pods according to
Mishra et al. (2001). (2) For okra extraction, the pods were triturated for one minute, with distilled water
at 50C, in ratio 1: 7 w/v. After grinding, the contents were filtered under and Pranne & Tanatcha (2011)
recommendations. (3) The extracted fraction mixed with ethanol 3:1 v/v and stirred at 150 rpm for three
hours. The gum obtained was washed in acetone 1:1 v/v stirred at 150 rpm for an hour and then filtered
through a muslin fabric vacuum system to remove ethanol and acetone excess. (4) The solubilized
material in distilled water at 65C, until the formation of a gelatinous product used as main okra
coagulant.
The Coagulation diagrams performed according to the methodology proposed by Paixo (1996) and Di
Bernardo & Di Bernardo (2002), and plotted in software Surfer 11. Microalgae biomass nutrient
removal efficiency was determined by the Equation 1, where E is the removal efficiency in percent, C0
the value found for the parameter evaluated and C the concentration of the parameter evaluated in the
final effluent after MBM sedimentation.
E% =
100
(Eq.
1)
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Microalgae Biomass MBM and Final Effluent Characterization

MBM characterization was determined by the ratio VSS/TS, according to Andreoli et al. (2001). The
physicochemical characterization of the final effluent after the separation process was determined by pH,
alkalinity, COD, BOD, total phosphorus and NTK nitrogen. All assays were performed in accordance
with the methods proposed by Standard Methods for the Examination of Water and Wastewater (APHA,
2013).
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Statistical Analysis
Statistical analysis corresponded to ANOVA associated with Tukey test to determine the existence of
differences between the values of physical-chemical parameters, with a significance level of = 5.0%.
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RESULTS AND DISCUSSION
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The main genera of microalgae naturally associated with the anaerobic effluent, were Scenedesmus sp.
(Fig. 1A), Chlorella sp. (Fig. 1B) and Synechocystis sp. (Fig. 1C). Also, associated with the anaerobic
effluent were also found other microorganisms such as fungi and protozoa due to natural growth of nonsterile medium.
Figure 1 - Microalgae species found in the anaerobic efluente after 6 days of MBM growth: (A) Scenedesmus sp. (B)
Chlorella sp. (C) Synechocystis sp.
In study reported by Torres (2014), it was isolated from a Domestic Sewage Anaerobic Experimental
Treatment Plant six microalgae identified as Desmodesmus sp., Chlorococcum sp., Coccomyxa sp.,
Chlorella sp., Scenedesmus sp. and Tetradesmus sp. Unlike the findings of this study, Torres (2014)
found abundant growth of Chlorella sp. in anaerobic effluent, which showed a survival greater than 90%,
in relation to other species. The evaluation of the microalgae growth in photobioreactor, with the use of
meat processing industry efluente previously primary and secondary treated as the growth medium, was
performed in study of Tango (2015). As in the present study, Scenedesmus sp. was the dominant
microalgae in both evaluated effluents.
The growth of microalgae was dependent on the pH and temperature of the medium, characterized as a
daily variation cycle, represented by Figure 2.
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Figure 2 Daily cycle variation of pH and temperature of the MBM batch growth, over 8 days.
The microalgae biomass growing culture showed a large variation in pH values varying from around 8 to
peak 11 during daily diurnal cycle. Initial values with less variability and after the stable growth of day 4th
the diurnal cycle was fully operating. This could be associated with the CO2/ CO32 balance as seen in
Equation 2.
CO2(aq) H2CO3 H+ + HCO3- H+ + CO32(Eq. 2)
The inorganic carbon species normally used by microalgae are CO2 and HCO3. Assuming that
assimilation of HCO3,assisted by enzyme carbonic anhydrase, represents a true H+ removal from water
medium and consequently a raise in pH values. At higher pH values, e.g. at pH greater than 9, most of the
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inorganic carbon is in form of carbonate (CO32) which cannot be assimilated by the algae. Night
respiration and other metabolism replenish the CO2 to the medium lowering the pH values and restoring
the cycle (SUTHERLAND et al., 2015).
Due to the pH values observed in the daily cycle of microalgae growth, the choice of coagulant type turn
out a critical factor. Some authors (MENNAA et al., 2015; KUNJAPUR & ELDRIDGE, 2010) reports a
low efficiency coagulant for harvesting microalgae studies and could be associated with the pH effect as
the coagulant was employed at incorrect pH range. Thus, we are proposing an optimized microalgae
biomass recovery through coagulation-flocculation mechanism based on pH range of the medium. Results
of coagulation assays performed with jar tests and plotted by diagrams are shown in Figure 3.
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Figure 3 - Coagulation diagrams established from jar tests for the different coagulants. The brightest bands represent
lower turbidity values, while the darker bands have the greatest remaining turbidity values.
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Based on the results obtained from the diagrams, it was possible to determine the MBM removal
efficiency values, as a function of dose and pH, according to Table 2.
Table 2 - Dose and optimum pH values and MBM removal efficiencies for each coagulant evaluated by means of
coagulation-flocculation followed by sedimentation process.
Coagulant
Aluminum sulfate
SUPERION 300 cationic starch
TANFLOC POP
Moringa olefera seed
Hibiscus esculentus (okra) gum
pH
7.0
10.0
9.0
5.0
5.0
Dose (mg/L)
50.0
80.0
100.0
150.0
100.0
Removal efficience (%)

95.9
92.5
95.6
85.8
80.3
From the analysis of the results presented in this study and in the relevant literature, it can be inferred that
aluminum sulfate is an effective coagulant in the recovery of MBM for neutral and acidic pH ranges, for
doses less than 80 mg/L. For pH values in basic range, aluminum sulfate reached biomass recovery of 60
to 70% with doses higher than 80 mg/L, Efficiencies greater than 90% were obtained by applying doses
greater than 50 mg/L, at neutral pH, which is not within the operating pH range for microalgae growth,
according to this study. In study of Teixeira et al. (2012) C. vulgaris biomass recoveries were 93.8%,
applying 1000 mg/L of aluminum sulphate at pH 9.24. Gerde et al. (2014) applied aluminum sulfate to
recover Scenedesmus sp. grown in synthetic medium at pH adjusted between 8.5 and 9.5. Doses of 250
mg/L were required to achieve 90% of Scenedesmus sp. removal by means of coagulation-flocculation.
The data presented in the literature corroborate the findings of this study, since a high MBM efficiency
removal requires higher dosages of aluminum sulphate by coagulation-flocculation at high pH.
For doses greater than 80 mg/L modified tannin, recovery efficiencies were greater than 80%, with pH
range from 7 to 10. Similar results were obtained to remove Microcystis aeruginosa cultured in BG-11,
using modified tannin coagulant (Q-TN) (WANG et al., 2013). Recovery efficiencies obtained were
greater than 90% in a pH range from 6 to 9 (WANG et al., 2013). In a recent study, were obtained 97%
removal of Chlorella vulgaris cultivated in swine effluent treated by phytoremediation using 11 mg/L of
commercial modified tannin at neutral pH (MEZZARI et al., 2014). In a study to recover C. vulgaris and
Nannochloropsis oculata, grown in synthetic medium with pH of 8, the removal efficiencies for C.
vulgaris was 99, 100 and 100% for concentrations of 5, 15 and 45 mg/L, respectively. In contrast, N.
oculata removal efficiencies were 92, 96 and 99% for concentrations of 5, 15 and 45 mg/L, respectively
(ROSELET et al., 2015). In this study, the effect of pH at the same low concentrations of modied tannin,
5, 15 and 45 mg/L, the removal efficiencies were lower, between 30 and 60%. For pH values of about 8,
MBM recovery from a raceway pond, which treats wastewater, biomass recoveries were greater than 90%
at 50 mg/L of Tanfloc SG (GUIRREZ et al., 2015). In contrast, in the same study conditions of
Gutierrez et al. (2015) were established in this study efficiency of 96.7% using Tanfloc Pop modified
tannin as coagulant.
Regarding the effect of pH on the MBM recovery using cationic starch, data show that for doses between
80 and 150 mg/L, the removal efficiencies of cationic starch were above 80% for a wide range pH,
between 7 and 11. Two types of cationic starch (GREENFLOC 120 and CARGILL C * BOND RH 35
849) were tested for Parachlorella sp. and Scenedesmus sp. recovery in study of Vandamme et al. (2010,
2013). Considering pH range between 7 and 8, GREENFLOC 120 was more efficient than Cargill C *
BOND RH 35 849, for both genera of microalgae. The removal efficiency was greater than 80% at doses
ranging from 20 to 40 mg/L and 5 mg/L for Parachlorella sp. and Scenedesmus sp., respectively, which
corroborates the results for MBM removal efficiency in this study for the same range of dosage and pH.
Gerde et al. (2014) used two types of cationic starch (DS02 and DS05) to harvest Scenedesmus sp. and
Chlamydomonas reinhardtii, in synthetic medium at pH adjusted between 8.5 and 9.5. The specie
Scenedesmus sp. required 20 mg/L of DS5 to obtain 90% of removal. The specie C. reinhardtii demanded
approximately 40 mg/L of DS2 and 20 mg/L of DS5 to obtain 85 and 90% of removal, respectively.
Modified cationic potato starches were used for flocculation of Scenedesmus dimorphus cultures in study
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of Hansel et al. (2014). The results showed that all cationic starches were highly effective and best
efficiency was above 95% for the starches tested at dosages of 10 mg/L.
According to Vandamme et al. (2010, 2013), the cationic starch positive charge is due to quaternary
ammonium salts introduced in the manufacturing process, which are able to maintain their positive charge
even at high pH values, and can contribute for MBM autoflocculation of higher pH, which may have
promoted an increase in the recovery efficiency of BMA at high pH. Furthermore, according to the
authors, the efficiency of flocculation by cationic starches and modified tannins can also be related to the
degree of substitution of the quaternary groups, the site of substitution and the molecular weight of the
polymers.
The MBM recovery with the use of M. oleifera was not satisfactory in this study in relation to the alkaline
pH range founded in MBM growth. The maximum removal efficiency of approximately 80% was
achieved by adjusting the pH in acidic range (4 to 5), applying doses of 100 to 150 mg/L of M. oleifera
seeds coagulant. Teixeira et al. (2012) achieved efficiencies of 88.6% C. vulgaris removal from WC
medium, adjusted pH of 9.2, applying 1000 mg/L of M. oleifera seeds powder, although the settling time
was set to 2 hours. It should be noticed that the selected concentration and settling time were significantly
higher than those observed in present work. Hamid et al. (2014) obtained efficiencies greater than 95%
for Chlorella sp. removal using M. oleifera coagulant but it should be noted that microalgae was cultured
in Bold basal medium, at pH values of approximately 6, and coagulation process was carried out using M.
oleifera seeds powder concentrations of 30 mg/L and settling time of 2 hours.
MBM removal efficiency by making use of polymers extracted from okra (H. esculentus), as can be seen
by MBM coagulation diagram charts, was not satisfactory for biomass growth in FBR alkaline pH range.
Low removals can be noted in acidic pH ranges, around 4 and 5, reaching efficiencies of up to 80%.
There is no sufficient literature data for MBM harvesting studies with microalgae grown in domestic
sewage of anaerobic effluent, but there is some studies relating turbidity removal of domestic sewage
effluent treated with polymers extracted from okra. Anastasakis et al. (2009), presented some data of
turbidity removals of 70 to 74% was achieved from biologically treated effluent, applying 2.5 mg/L of
polymers extracted from okra seeds in pH of 6.09. In contrast, Agarwal et.al. (2001) achieved removal
efficiency of suspended solids around 86% for raw wastewater treatment, with only 0.12 mg/L of
extracted okra gum, at neutral pH.
After MBM sedimentation process, using modified tannin, aluminum sulfate and cationic starch as
coagulants were the most efficient for MBM recovery. Thus, the consolidated results for three best
coagulants for MBM recovery for pH microalgae growth range is presented in Table 3.
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Table 3 - Means ( ) and coefficients of variation (CV%) of the main parameters for the evaluation of anaerobic
effluent used for MBM growth (EUASB) and of the effluents after MBM removal by aluminum sulfate (ESAL),
modified tanin (ETFP) and cationic starch (EACS). Different letters on the line indicate significant differences (p
<0.05) by Tukey test.
EUASB
ESAL
ETFP
EACS
Parameter
CV(%)
CV(%)
CV(%)
CV(%)

pH
7,75
0,47
3,36b
3,97
4,10b
14,84
7,05
1,22
206,80
2,44
15,30b
11,93
15,40b
24,74
20,90b
9,12
BOD (mg/L O2)
66,00a
9,09
43,40b
2,44
44,83b
6,98
20,83c
4,76
a
b
b
b
COD (mg/L O2)
110,00
7,58
20,50
29,03
21,50
8,37
10,50
13,22
TP (mg/L)
9,79
1,39
1,33b
22,55
1,36b
7,53
0,92b
5,16
TN (mg/L)
37,16
5,96
3,61bc
20,83
5,41b
15,75
1,80c
15,75
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From the analysis of the main physical-chemical parameters, it can be inferred that the effluent pH values
varied significantly after biomass removal, which are found in the more acidic range except for the
removal from the cationic starch, which provided an effluent with a pH around neutrality. The results
presented in Table 3 illustrate one of the major advantages of MBM growth in anaerobic sewage
treatment effluents, since the growth of MBM represents a tertiary sewage treatment, with the removal of
phosphorus and nitrogen from the aqueous phase.
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Page 21 of 24
The removal of nutrients such as nitrogen (N) and phosphorus (P) achieved significant values when
compared to anaerobic effluent, although not significantly different among the treatments. The total
phosphorus and total nitrogen removal were more efficient using cationic starch, which were 90.64% and
95.14%, respectively. Nutrient removal is related to the MBM assimilation and others associated biomass,
like fungi and bacteria.
Interestingly observed in Table 3 is the decrease in chemical and biochemical oxygen demands (BOD and
COD) of 44% and 84%, respectively. The removal of BOD and COD seen in Table 3 may be related to
the removal of suspended and dissolved organic material with negative charges present in EUASB,
microalgae cells and other microorganisms associated with EUASB, thus decreasing the oxygen demand,
particularly chemical oxygen demand.
Another parameter that diverts from the anaerobic effluent was alkalinity, which decreased on average of
91%. As discussed earlier, growth medium pH and alkalinity increasing coincides with the exponential
MBM growth phase, a phenomenon which is explained by the consumption of CO2 for photosynthesis,
increasing the concentration of HCO3- and CO32- and consequent rising in pH and alkalinity of carbonates
and bicarbonates. As the culture moves to a lower stage of growth, at which time the samples were
collected for biomass removal, the medium alkalinity slightly reduced due to reduction of dissolved CO2
consumption, to achieve an equilibrium.
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CONCLUSIONS
It was possible to verify polymer dosage, the optimum pH values and their MBM removal efficiencies
through the process of coagulation-flocculation followed by sedimentation, for microalgae biomass
effluent grown. It is worth noting that, for high pH values of 8 to 10, only the cationic starch or modified
tannin provided good MBM removal efficiencies with dosages of 80 and 100 mg/L of polymer,
respectively.
From the analysis of coagulation diagrams and biomass removal efficiency data, it can be demonstrated
that cationic starch showed the lowest dose with highest recovery efficiency in the pH range observed for
the MBM growth, although the pH range of application and recovery efficiencies were similar to those
reached using the modified tannin. Aluminum sulfate showed a smaller pH range of application than the
range of modified tannin and cationic starch polymers, and required larger doses to achieve the same
MBM recovery efficiency.
Moringa oleifera seeds and Hibiscus esculentus (okra) gum promoted MBM removals of only about 50%,
in the acidic pH range. With aluminum sulfate, modified tannin and cationic starch as coagulants, the
effluents generated after MBM flocs settling showed total phosphorus removal greater than 80% and total
nitrogen, BOD and COD removals greater than 50%.
While aluminum sulfate is a reference coagulant in microalgae recovery, it can be verified in this work
that it can be replaced by natural coagulants, for example modified tannin and cationic starch, under the
conditions of this study, without decreasing the biomass removal efficiency and causing damage to the
final effluent, and consequently to the environment.
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ACKNOWLEDGMENTS
This work was done under the Biogas Project. Authors are thankful to Department of Environmental
Engineering master's program for their support. The authors also gratefully acknowledge the financial
support and scholarship of FINEP/UFES and CNPq, respectively. We are thankful to Anne Mountier and
Landon Shanks for critical reading and improvement of the manuscript.
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Figure 3 - Coagulation diagrams based on Jar tests data for different coagulants. The lightest areas
represent lower turbidity values, while the darker areas have the greatest remaining turbidity values.
311x438mm (96 x 96 DPI)
Page 24 of 24

Harvesting Microalgal Biomass Grown in Anaerobic Sewage Treatment Effluent by The Coagulation-Flocculation Method: Effect of PH

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