Journal of Environmental Chemical Engineering 5 (2017) 635643

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Journal of Environmental Chemical Engineering

journal homepage: www.elsevier.com/locate/jece

Microalgae growth in polluted efuents from the dairy industry for

biomass production and phytoremediation
Jos Ignacio Labba , Juan Luis Ramos-Surezb,* , Alexis Hernndez-Preza , Andrea Baezaa ,
Felipe Hansena
a
ProCycla SPA, Gral Eugenio Garzn 6561, Vitacura, 7640274, Regin Metropolitana, Chile
b
ProCycla SL, Pau Casals 18, 4-4, 08240 Manresa, Spain

A R T I C L E I N F O A B S T R A C T

Article history:
Received 7 October 2016 A link can be established in both the dairy and microalgae industries through the recycling of nutrients
Received in revised form 15 December 2016 from dairy efuents (DE) in order to cultivate microalgae, using biomass on the farm or for commercial
Accepted 26 December 2016 endeavors. This study assesses the development of Chlorella and Scenedesmus mixed cultures (CMC and
Available online 29 December 2016 SMC) in dairy farms using four different DE as culture media under ambient and laboratory conditions.
Viability of the cultures for scaling up was determined based on biomass growth characteristics.
Keywords: CMC and SMC were able to grow in DE. Growth pattern was mainly affected by the type of efuent used,
Scenedesmus with almost no inuence from culture conditions. CMC grew satisfactorily in efuents with high organic
Microalgae
and ammonium loads such as cattle standing yard efuents (CSYE), reaching cell concentrations of
Wastewater treatment
1.70  108 and 1.67  108 cel ml 1 outdoors and indoors, respectively. Contrarily, SMC grew better on
Dairy efuents
Phytoremediation efuents with high chemical and detergent loads such as milking parlor efuents (MPE), reaching cell
concentrations of 3.20  107 and 5.61 107 cel ml 1 outdoors and indoors, respectively. Despite
microalgal growth, there is a need of advance analysis for determining precisely nutrient reduction,
since electrical conductivity could not be established as a remediation efciency indicator.
Overall, results show that there is a potential to treat DE through microalgae culture.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Dairy farms produce large quantities of wastewater due to the
cleaning of their facilities [1]. Dairy efuents (DE) consists mainly
of milking parlors and dairy wash water, milk spillages, runoff from
soiled yard areas, drainage water from roofs of buildings, silage
efuent and occasionally leachate from manure heaps [1,2]. These
efuents are characterized by high biological oxygen demand
(BOD) and chemical oxygen demand (COD), high pH, high
ammonia nitrogen and residues of cleaning and disinfectant
chemicals [1,3,4]. Moreover, they are highly variable in composi-
tion depending on the season and the cattle farm. An appropriate
treatment of dairy wastewater is necessary to avoid eutrophication
of surface and groundwater, aquatic life affection and to minimize
Abbreviations: DE, dairy efuents; CMC, Chlorella mixed culture; SMC,
Scenedesmus mixed culture; CSYE, cattle standing yard efuent; MPE, milking
parlor efuent; WW, well water; CCW, Cholqui Canal water; vvm, gas volume ow
per unit of liquid volume per minute.
* Corresponding author.
E-mail addresses: jlramos@procycla.com, jlramossuarez@gmail.com
(J.L. Ramos-Surez).
the effect of efuent addition to land. The latter can lead to
degradation of soil structure, salinization, waterlogging, chemical
contamination and/or erosion [5].
Composition of dairy wastewater allows for biological treat-
ment [2]. Therefore, they are treated both by aerobic and anaerobic
methods. Biological methods are often followed by physical-
chemical methods such as coagulation/occulation and membrane
processes in order to completely remove inorganic nutrients from
the water efuent [3,6]. Aerobic treatments are generally energy-
intensive, whereas anaerobic technologies such as UASB and AF
require highly trained staff. Therefore, the implementation of the
abovementioned treatment methods in small and medium scale
farms is difcult. Microalgal wastewater treatment systems could
be a solution in remote areas and for small and medium scale
farmers [7]. Thus, they have been studied at laboratory and pilot
scale and used to treat different types of agro-industrial waste-
waters [6,811]. Within these systems, naturally occurring bacteria
are in charge of degrading organic matter to CO2, ammonia and
phosphate, whereas microalgae retain most of these nutrients
during their growth [6], being the latter the dominant organisms.
Other complex pollutants such as organic compounds, heavy

http://dx.doi.org/10.1016/j.jece.2016.12.040
2213-3437/ 2016 Elsevier Ltd. All rights reserved.
636 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643

metals and pathogens are also removed by microalgal treatment Table 1

Detergents and chemical products used for cleaning the milking parlor/dairy
systems [7].
facilities and present in MPE.
Microalgae have many potential uses: pharmaceuticals, food,
animal feed, chemicals, cosmetics, biofuels and biofertilizers can Commercial name (Brand) Chemical product
be produced from microalgae [1214]. However, the production of CIRCUITOX LQUIDO (Ecolab) Sodium hydroxide
sustainable and economical microalgae cultures requires a Sodium hypochlorite
Sodium metasilicate
decrease in energy, water and fertilizer consumption [15]. Focusing
OXONIA ACTIVO 150 (Ecolab) Peracetic acid at 15% concentration
on water and fertilizers, culture media could make up to 35% of the NITRO PLUS (Ecolab) Nitric acid
costs associated with raw materials [16], whereas water is a scarce Phosphoric acid
resource that is not readily available and supposes an important
cost. The link seems clear: dairy wastewaters can be used as
culture media for microalgae due to their rich nutrient content, 2.2. Microalgal cultures
decreasing their COD and nutrient load, whereas harvested
biomass could have different commercialization purposes or uses Two microalgae cultures were used to assess the viability of
on farm for animal feed, biofertilizer or bioenergy production, their growth in the different media under study. The rst culture
improving farm nances and sustainability. was a mixed culture in which Chlorella sp. was the dominant genus
Although, as described above, several research works about DE with over 99% of the total cells (from now on referred as CMC). The
treatment by microalgae have been reported, the application of second microalgae culture was dominated by Scenedesmus sp.,
this system in raw DE (without pretreatments) has been barely with over 98% of the total cells (from now on referred as SMC). Both
studied in depth and more research is necessary to develop reliable genuses were isolated from pond water nearby the dairy farm Los
and efcient systems [17,18]. In this study, two series of experi- Nogales by serial dilutions and repeated subcultures according to
ments were performed in order to determine the effect of different Torrentera & Tacn [19]. The organisms were identied as
dairy efuents on the growth curve, growth rate and maximum described by Parra et al. [20].
biomass concentration of two cultures of microalgae. The main
goal is to assess through small scale batch cultures the technical 2.3. Culture conditions
viability of developing microalgae cultures in dairy farms using
dairy wastewaters as culture media in order to produce biomass The experiment was carried out in the laboratories of ProCycla
and treat the dairy efuents. located in Melipilla next to the dairy farm Los Nogales
This paper is the rst of a series of three papers of a research (33.7066 S71.162 W) between June and July 2014.
that tries to develop microalgae cultures in dairy farming in order For each microalgae culture, all the efuent and water samples
to improve the nances and sustainability of this industry, were studied under ambient and laboratory conditions. Therefore,
especially for small and medium scale (1501000 cows) farms the experiment was divided into two different experimental sets,
in Chile where most farms are this size. Hence, this research hopes each set corresponding to each microalgae culture and experi-
to have a wide applicability. mental condition (see Table 2). Sets corresponding to the same
condition were done at the same time in parallel, i.e. set 1 and 3
2. Materials and methods were performed at the same time outdoors, and afterwards, sets 2
and 4 were performed indoors.
2.1. Culture media Therefore, 24 asks with a total volume of 1 l were used each
time for the experiment. 12 of them were used for the cultivation of
The dairy farm Los Nogales, located in Melipilla (Chile) CMC and the other 12 for the cultivation of SMC, each culture
(33.7066 S71.162 W), kindly provided the efuents used as having three replicates for each culture media (WW; CCW; MPE;
culture media. Four different culture media were used in this CSYE). For each experimental unit the experimental procedure was
study: efuents produced in the milking parlors (milking parlor the same: 400 ml of inoculum (CMC or SMC) was added to an
efuent MPE), in the cattle standing yards (cattle standing yard empty ask and then lled with 400 ml of the culture media under
efuent CSYE), water from the Cholqui Canal (CCW) and water study. Each ask was vigorously and continuously agitated by air
from the well (WW). The Cholqui Canal and the well feed the dairy injection in order to distribute light uniformly and to provide the
farm, the rst being used to clean the cattle standing yards while CO2 necessary for microalgae growth, all the while desorbing the
the second was used to clean the milking parlor. Therefore, using O2 generated by photosynthesis. All the experiments were carried
WW and CCW as culture media serve for determining whether out during 14 days.
inhibitors or nutrients are consequence of dairy farms activities or Solar irradiation during the experiment under ambient con-
are already present in the water sources. ditions was calculated based on the data provided by the closest
It should be noted that the Cholqui Canal is used for irrigation meteorological station (San Pedro de Melipilla) that belongs to the
and livestock in the area. During some months this canal is closed meteorological station network of INIA (Agricultural Research
and dried for cleaning and maintenance, limiting the productivity Institute, Government of Chile). Average solar irradiation during
of the primary sector and making the reuse of water necessary. the experimental period corresponded to 2.01 kW h m 2 d 1 and
For cleaning the milking parlor different detergents and 2.296 kW h m 2 d 1 (21,403 lx and 24,448 lx) for June and July,
chemical products were used (see Table 1), whereas the cattle
standing yard was cleaned only with water. Both, the CSYE and the Table 2
MPE were settled for 24 h and ltered through 1 mm mesh size Experimental sets in which the experiment was divided.
prior to use in the experiment in order to remove settled and large Set Culture Conditions Growth media
solids. CSYE and MPE ltrates showed coffee and whitish color,
1 CMC Ambient WW; CCW; MPE; CSYE
respectively. No pretreatment was done to the WW and the CCW. 2 CMC Laboratory WW; CCW; MPE; CSYE
Chemical and physical composition of the culture media were 3 SMC Ambient WW; CCW; MPE; CSYE
measured. 4 SMC Laboratory WW; CCW; MPE; CSYE

WW = well water; CCW = Cholqui canal water; MPE = milking parlor efuent;
CSYE = cattle standing yard efuent.
 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643 637

respectively. Under laboratory conditions light was provided by ten Table 3

Composition of CSYE, MPE, WW and CCW used as culture media. (values shown are
white light (5000  K), 20 W uorescent tubes, placed at 20 cm from
the mean results of triplicates, with less than 5% standard error for macroelements
the culture asks under a 12/12 light/dark cycle, providing and 1% for minor elements).
10.000 lx. Mean temperature for experimental sets 1 and 3 was
Parameter WW CCW MPE CSYE
around 8.3  C ( 2.7  C min and 20.8  C max). Under laboratory
conditions the cultures were maintained at temperatures between pH 6.88 7.35 7.26 7.82
EC (dS m 1) 1.7 1.2 2.0 2.0
13  C and 20  C.
DO (mg L 1) 39.43 65.23 51.78 13.27
NNH4+ (mg L 1) 1.1 1.2 3.8 28.4
2.4. Analytical methods NNO3 (mg L 1) 4.2 13.1 2.9 14.8
P (mg L 1) 0.08 0.19 7.9 5.1
A multi-parameter probe HI9828 (Hanna Instruments, USA) was K+ (mg L 1) 2 4 34 78
Ratio N/P 66.3 75.3 0.9 8.5
used for measuring the pH, electrical conductivity (EC) and
Ratio P/K 0.04 0.05 0.23 0.07
dissolved oxygen (DO) in days 0, 2, 4, 7, 9, 11 and 14 between 10:00 Ca2+ (mg L 1) 166 144 168 174
and 11:00 AM Cell density was determined on the same days via Mg2+ (mg L 1) 51 22 4.9 43
cell count of an aliquot (200 ml) of the culture in a Neubahuer Fe (mg L 1) 0.03 0.08 1.1 0.57
Mn (mg L 1) <0.01 0.02 0.40 0.23
chamber with a depth of 0.1 mm (Marienfeld, Germany) and an
Zn (mg L 1) <0.01 <0.01 0.04 0.1
optical microscope OMAX 40-1600X (OMAX, South Korea). Dilu- SO42 (mg L 1) 312 245 302 384
tions were performed when necessary in order to avoid cell Na+ 69 71 150 136
number in each check box to increase over 200 cells. HCO3 323 183 439 458
Culture media composition was determined in a certied Cl (mg L 1) 170 142 248 163
Cu (mg L 1) 0.01 <0.01 0.03 0.03
laboratory. pH, EC, Cl , NO3 and NH4+ was determined by
B (mg L 1) 0.17 0.3 1.00 1.5
potentiometry. Ca, Mg, Na, K, Fe, Mn, Zn, Cu and other metals were As (mg L 1) <0.01 <0.01 <0.01 <0.01
analyzed by atomic absorption. P, B and SO4 2 were determined Pb (mg L 1) 0.01 0.01 0.04 0.04
using colorimetric methods. Finally, HCO3 was determined by Cd (mg L 1) <0.01 <0.01 <0.01 <0.01
titration. Methods and certication ensure that the results are WW = well water; CCW = Cholqui canal water; MPE = milking parlor efuent;
expressed with less than 5% standard error for macroelements and CSYE = cattle standing yard efuent.
1% for minor elements.
The specic growth rate (m) in the exponential growth phase the culture media showed pollution due to heavy metals. CSYE was
was calculated according to Lu et al. [18] as m = ln (Xt/X0)/(tt0), slightly alkaline, probably as a consequence of the high ammonium
where m is the specic growth rate (d 1), Xt is the cell density at load.
time t (cel ml 1) and X0 is the cell density at time t0 (cel ml 1)
3.2. Biomass growth
2.5. Statistical analyses
The biomass growth analysis is the main parameter dening the
Experimental results were analyzed with the statistical development of a culture [23], in which the growth phase, cell
software Infostat v.2015, using heterogeneous mixed models density and specic growth rate are the most relevant data. Results
[21]. Results were compared and graphed based on a DGC test related to biomass growth will be discussed separately for CMC
(exclusive groups formation test) (p < 0.05) [22]. Furthermore, and SMC to facilitate reading and understanding.
Pearson correlation analyses were performed between EC and the
biomass concentration, both throughout the whole experimental 3.2.1. Chlorella mixed cultures
period (days 014) and between day 0 and the day of maximum Biomass growth patterns are shown in Fig. 1 for CMC under
biomass concentration. Correlation was considered signicant ambient (a) and laboratory conditions (b).
when p < 0.05. Under ambient conditions, CMC showed all growth phases only
when using CSYE, CCW and MPE as culture media (Fig. 1a). On the
3. Results and discussion other hand, using WW the cell concentration showed a constant
decrease. Cultures grown on CSYE and CCW reached the stationary
In this section the composition of the culture media, the phase in day seven with 1.7  108 cel ml 1 and 1.2  108 cel ml 1,
biomass growth, the treatment capacity of microalgae, the respectively. CMC grown on MPE reached the stationary phase on
technical viability of each culture and the correlation analysis day nine with 9.1 107 cel ml 1. Based on growth pattern and
between biomass concentration and EC are described and maximum cell concentration, CSYE and CCW were adequate
discussed. culture media for CMC, especially CSYE, which showed a 41%
higher cell concentration than CCW. On the other hand, CMC
3.1. Culture media composition showed little biomass growth on MPE and no growth at all on WW,
indicating that these were not adequate culture media for CMC.
The chemical and physical composition of the four culture Under laboratory conditions, cultures using CSYE and CCW as
media used during this study is shown in Table 3. growth media showed all growth phases (Fig. 1b). In this case the
It has to be stressed that CCW, MPE and CSYE showed a high stationary phase was reached on day nine for CCW with a similar
nutrient load, based on N and P concentrations. MPE stood out for concentration (1.8  108 cel ml 1) to CSYE, which reached its
its high P concentration consequence of the detergents used for stationary phase in day seven with a cell concentration of
cleaning the milking parlor. CSYE showed a high N concentration, 1.7  108 cel ml 1. Cultures grown on MPE and WW showed a
consequence of the manure content. CCW showed also a high N poor development. Cultures grown on MPE showed a constant
load although it came from a clean canal. This was consequence of decrease of cell concentration, whereas cultures grown on WW
runoff from crop elds and the discharge of efuents from other showed a longer lag phase with loss of biomass followed by a slight
farms upstream. Finally, WW showed the lowest nutrient content, increase in cell concentration (Fig. 1b). Results suggest that CSYE
as expected, especially low for P (0.08 mg L 1) which could limit and CCW were adequate culture mediums for CMC under
microalgal growth. Despite the main nutrients (N and P), neither of laboratory conditions, whereas MPE and WW were not.
638 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643

Fig. 1. Growth pattern of CMC (a) under ambient conditions and (b) under laboratory conditions for the different growth media used [CSYE (&); WW(*); MPE(~); CCW(^)].
Signicant differences (p < 0.05) are shown with capital letters. Bars indicate standard error. Data from day 0 was used as covariate in the statistical analysis.

The growth pattern was similar in both culture conditions for
each culture media tested, with slight differences in growth phases
and cell concentration between ambient and lab conditions.
Therefore, results suggest that the differences observed in the
growth pattern are consequence of culture media composition.
CSYE and CCW were rich in nutrients and had no toxic components
for CMC growth (see Table 3). Other authors have already noted the
potential of efuents similar to CSYE for the development of
Chlorella cultures [18,24,25]. In this study there was no pretreat-
ment of CSYE, which is very important for the scalability and
economics of the process. WW had extremely low nutrient content
(Table 3) that limited algal growth.
Finally, it is important to highlight the lack of adaptation of CMC
to MPE. Chlorella is considered a genus very tolerant to organic
pollutants in culture media [26]. There are several reasons for the
lack of adaptation. On the one hand, MPE components are toxic to
aquatic life and nature, mainly chlorine, sodium hydroxide and
phosphoric acid. For instance, sodium hydroxide, used to clean the
milking parlor, is known to affect negatively Chlorella cultures [27].
On the other, N/P ratio of MPE was 0.9, suggesting that the nitrogen
deciency inhibited CMC growth. Microalgae are able to adapt to
nutrient concentration in its culture media [28]. However, the
Redeld ratio (N/P) [29] is used as an index to determine the
suitability of the culture media for microalgal growth. The Reeld
ratio is 7.23 (w/w) being much higher that N/P ratio found in MPE.
In this study, similar yields were obtained under ambient and
laboratory conditions, whereas other authors observed better
growth when culturing microalgae indoors on raw dairy waste-
water [18]. Ambient conditions are better due to energy savings
and therefore are herein suggested.
3.2.2. Scenedesmus mixed cultures
Biomass growth patterns for SMC are shown under ambient and
laboratory conditions in Fig. 2a and b, respectively.
Under ambient conditions SMC showed all growth phases when
using MPE and CCW as culture media. On the contrary, when using
CSYE and WW biomass concentration showed no increase (see
Fig. 2a). Cultures grown on MPE and CCW reached the stationary
phase on day 7 (3.2  107 cel ml 1) and day 9 (3.8  107 cel ml 1),
respectively. SMC growing on CSYE and WW showed uctuations
in cell concentration nishing with lower cell concentrations than
at the beginning of the culture period. Results suggest that MPE
and CCW were adequate culture mediums for SMC, whereas CSYE
and WW did not support SMC growth.
Under laboratory conditions (Fig. 2b) SMC showed a good
adaptation to MPE and CCW reaching the stationary phase on day 9
in both culture mediums with maximum biomass concentrations
of 5.6  107 cel ml 1 and 5.0  107 cel ml 1, respectively. When
growing on WW, SMC showed an initial decrease of biomass
concentration until day 2, thereafter increasing biomass concen-
tration for a maximum of 2.2  107 cel ml 1 on day 11. CSYE caused
a slight increase at the beginning of the culture period followed by
a constant decrease in cell number. Results suggest again that MPE
and CCW were adequate culture media for culturing SMC indoors,

Fig. 2. Growth pattern of SMC (a) under ambient conditions and (b) under laboratory conditions for the different growth media used [CSYE (&); WW(*); MPE(~); CCW(^)].
Signicant differences (p < 0.05) are shown with capital letters. Bars indicate standard error. Data from day 0 was used as covariate in the statistical analysis.
 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643 639

whereas CSYE and WW were not. As described above for CMC, Table 4
Specic growth rate for each experimental set. Mean value of triplicates  Standard
there were slight differences between growth patterns and
error.
biomass concentration of SMC under ambient and laboratory
conditions. These were probably caused by small changes in Culture Condition Growth Exp. phase m (d 1)
media (didf)
culture media composition and irradiation and temperature levels.
The comparison of the growth pattern of CMC and SMC led to CMC Ambient CSYE 47 0.339  0.019
MPE ND
some interesting observations. First, when using CCW as culture
CCW 24 0.389  0.164
media all phases of growth and high biomass concentration of CMC WW ND
and SMC were observed. These ndings suggest that there is an
important risk of eutrophication in this canal, highlighting the CMC Lab CSYE 27 0.221  0.012
need to develop an appropriate treatment systems for dairy MPE ND
CCW 04 0.245  0.007
efuents in this area, which are dumped now without any
WW ND
treatment.
Second, SMC and CMC showed a similar behavior when growing SMC Ambient CSYE ND
on WW. WW is treated with low concentrations of chlorine for MPE 24 0.293  0.096
CCW 49 0.166  0.051
human use. Chlorine is known to be toxic for microalgal growth at
WW ND
high concentrations [30,31]. However, Cl concentration found in
WW was similar to other culture mediums, which proved to be SMC Lab CSYE ND
suitable for microalgal growth. Therefore, the poor microalgae MPE 24 0.156  0.002
growth on WW is only explained by the extremely low nutrient CCW 24 0.239  0.026
WW ND
level of WW, especially P (see Table 3). In some cases (SMC under
ambient and laboratory conditions and CMC under laboratory didf: initial and nal day, respectively, for the exponential growth phase; ND:
Exponential growth phase non-detectable.
conditions) microalgae showed growth only after 911 days. This
can be explained by the partial microalgal death and cell
components mineralization that released nutrients that the rest
of microalgae used for survival and growth. on CCW m was higher under laboratory than under ambient
Comparing SMC and CMC, they showed opposite behaviors conditions (Table 4). These ndings support outdoors cultures in
when growing on CSYE and MPE. CSYE was an excellent culture order to save inputs in microalgae production, since ambient
media for CMC, but unsuitable for SMC. On the other hand, SMC conditions led to higher m, except for SMC on CCW.
showed an appropriate growth on MPE, whereas CMC showed In the scientic literature information about specic growth
little or no growth at all. Despite the high chemical load in MPE rates of Chlorella and Scenedesmus cultures is diverse. Zhu et al. [38]
(Table 1), SMC was able to grow, forming a healthy and stable observed m for C. zongiensis grown on diluted, sterilized piggery
population according to its growth curve. Biedlingmaier et al. [32] wastewater between 0.320 and 0.340 d 1 with similar m for
showed that the resistance of microalgae to detergents depends on cultures grown indoors and outdoors. However, N and P
the cell wall composition, specically to the presence of thick walls concentrations were higher in the culture media used by these
and sporopollenin components. Scenedesmus has both character- authors than in the dairy efuents used in this study. Travieso et al.
istics [33,34]; thus, its higher adaptability to MPE compared to [39] used mixed culture of C. vulgaris to treat piggery wastewater
CMC. It is important to highlight that no research has been found with different dilution rates observing growth rates between 0.192
using culture media similar to MPE, with such high chemical and and 0.576 d 1 depending on the culture media concentration, the
detergent loads. highest being obtained for N concentrations slightly higher than
Similarly, the poor growth of SMC in CSYE has not been that found in CSYE but with P concentration much higher than in
described before [24,35]. It is possible that the high ammonium any efuent used in this study. Ruiz Martin et al. [35] observed m of
concentration led to inhibition, which is increased at high pH levels 0.186 and 0.285 d 1 for C. vulgaris and S. obliquus, respectively,
due to the shift towards the most inhibiting form of free ammonia grown on urban wastewater with similar N concentration to CSYE.
[36]. Chlorella is more tolerant to high ammonia concentrations Finally, Martinez et al. [40] observed m from 0.458 to 0.768 d 1 for
than Scenedesmus [36]; however, free ammonia concentrations S. obliquus grown on sterilized efuent from a conventional
were below the inhibiting threshold observed by other authors for secondary-treatment facility of urban wastewater, being nutrient
Scenedesmus [36,37]. Another explanation for the limited growth is concentration similar to CSYE.
that the combination of the coffee-colored CSYE and SMC led to a According to the results above, provided close to optimal
decrease in the Photosynthetically Active Radiation (PAR) available environmental conditions, differences in m are mainly due to the
in culture asks as a consequence of the higher cell size of different compositions of the culture media with little inuence of
Scenedesmus. In fact, it is known that Scenedesmus sp. has a much pretreatments such as sterilization or chlorination. Furthermore,
higher cell size than Chlorella sp. [20], and therefore, in high results obtained in this study are in the normal range for
concentrated cultures of Scenedesmus sp. light limitation becomes Scenedesmus and Chlorella cultures growing in wastewaters.
stronger at lower cell concentrations.
3.3. Viability of using dairy efuents as growth media
3.2.3. Specic growth rate
According to data shown in Figs. 1 and 2, the exponential The culture systems studied (including microalgal cultures,
growth phase was identied and the specic growth rate (m) was culture media and conditions) can be classied as viable cultures,
calculated for the different cultures according to culture media and cultures with intermediate viability and non-viable cultures
conditions. Results are shown in Table 4. according to maximum cell concentration, specic growth rate and
Results showed differences in the CMC and SMC growth for the growth pattern. As described above, this classication system
different culture media. CMC showed higher m for ambient depends mainly on growth media, with little inuence of culture
conditions than for laboratory conditions and slightly higher for conditions (outdoors/indoors), and it determines which efuents
CCW than for CSYE (Table 4). SMC showed higher m in MPE under are capable of sustaining microalgal growth for commercial
ambient than under laboratory conditions, whereas when growing purposes. Although savings related to the use of wastewaters as
640 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643
culture media are very important [41,42], production of microalgae
will not be economically viable if biomass productivity is low.
CMC grown on CSYE and CCW are classied as viable cultures
both for ambient and laboratory conditions due to their high cell
concentration and specic growth rates. Both growth mediums are
characterized by a high nitrogen concentration (see Table 3), which
facilitates microalgal growth. On the other hand, SMC viable
cultures grew on CCW and MPE, both for ambient and laboratory
conditions.
Cultures with intermediate viability showed a lower cell
concentration and specic growth rate than viable cultures.
Although they adapted well to the culture media, their commercial
production is not recommended due to the low expected biomass
productivity. CMC grown outdoors on MPE belongs to this group.
Cultures not mentioned before are considered non-viable
cultures. Neither CMC nor SMC were able to grow on WW due to
the extremely low nutrient concentration in WW. Negative results
of CMC on MPE were up to some point expected due to the high
chemical content of MPE. However, the case of SMC on CSYE was
surprising, since other authors have observed a good development
of Scenedesmus cultures on efuents similar as CSYE [35,41].
Besides the latter case, this classication agrees with other authors
who used Chlorella and/or Scenedesmus for wastewater treatment
with very good results [35,41,42]. Beuckels et al. [28] showed that
Chlorella adapts better to wastewaters with high nitrogen
concentration, whereas Scenedesmus is more suitable for waste-
water with high P content. Results obtained in this research
conrmed results obtained by Beuckels et al. [28]. However, more
research is needed in order to elucidate the effects of the
components of each wastewater on SMC and CMC.
According to Park et al. [43] a viable culture for biomass
production and phytoremediation should comply with three
conditions: high growth rate, high tolerance to seasonal variations
and good harvesting properties. In this case, both genus showed
high growth rates and cell concentration in some culture media.
Moreover, both, Chlorella and Scenedesmus, are tolerant to climatic
and nutrient variations [26,44]. However, only Scenedesmus stands
out for its capacity to form colonies that could ease harvesting [45].
Additionally, a fourth condition could be added in which the
commercial use of the biomass produced is taken into account. For
this condition, Chlorella stands out for having many commercial
uses which could give a higher price to its biomass [23,46]. The
culture of microalgae in wastewater possibly limits its commercial
nal use but the extent of limitation should be studied case by case
according to the specic wastewater composition. In this study, the
efuents used as culture media do not show heavy metals
(Table 3), important factor as these can be absorbed, adsorbed
Fig. 3. EC variation during culture of CMC (a) under ambient conditions; (b) under lab conditions for the different growth media used [CSYE (&); WW(*); MPE(~); CCW(^)].
Signicant differences (p < 0.05) are shown with capital letters. Bars indicate standard error. Data from day 0 was used as covariate in the statistical analysis.
and accumulated by microalgae [47]. Therefore, harvested biomass
in this study could be used for any application, but prospects for
human consumption are low since consumers will probably reject
the product due to the source of the culture media used.
Conversely, the lack of heavy metals, a proper sanitization process
and a rich protein content would allow for the biomass to be used
as animal feed in farms without much problems, since its
legislation is more lax than for human food. Moreover, no
problems at all are expected if the biomass is intended for use
as biofertiliser, biofuels or as source for other valuable chemicals,
increasing commercialization potential of algal biomass cultivated
on farm.
Both cultures used in this study deserve more research in order
to scale up culture systems classied as viable, since they showed
high specic growth rates, high cell concentrations and expected
HRT around 47 days.
3.4. Treatment capacity
The use of wastewater as a nutrient source for microalgae
cultures has been widely studied because of the decrease in
microalgae production costs and in the pollutant load of the
wastewaters [6,23]. Martnez-Suller et al. [48] characterized
composition of dairy wastewater in Spain, Italy and Ireland,
nding a strong correlation between nutrient content and EC. This
relationship is very important, since it facilitates the determination
of the degree of purication of the culture media without analyzing
specic nutrient contents daily, avoiding the costs associated to
these analyses. Consequently in this study, EC was used as
parameter to evaluate nutrient load reduction. Furthermore,
correlation analyses between the biomass concentration and EC
were performed in order to determine if EC was a good indicator of
wastewater treatment and nutrient consumption by microalgae.
On the other hand, pH and DO are control parameters in
microalgae cultures [23], they are modied by microalgae growth
all the while inuencing water quality. These parameters were also
measured during the experiment.
3.4.1. Chlorella mixed cultures
Fig. 3a shows EC for CMC grown outdoors. EC decreased in all
culture media showing a reduction in nutrient load. CSYE was the
medium with the strongest EC drop that was correlated (p < 0.05)
to the biomass growth in this efuent and therefore, to a high
consumption of nutrients [49]. Despite the poor development of
CMC grown on MPE and WW there was a decrease in the EC. This
was probably due to volatilization and precipitation of nutrients
occurring as a consequence of the continuous aeration of the
 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643 641

culture media and pH changes [50]. In fact, volatilization and 3.4.3. Electrical conductivity as parameter to evaluate treatment
precipitation could be the main processes for nutrient removal in capacity
wastewater treatment by microalgae [6]. In practical experiences, where the use of agricultural
The EC trend in CMC under laboratory conditions (Fig. 3b) was wastewater for microalgal production is feasible, the easiness of
slightly different compared to cultures under ambient conditions. operation and performance will be a plus, thus, the need for
Only CSYE showed a strong EC drop, related to a high biomass avoiding costly and complicated analyses that could hinder the
growth and nutrient consumption. In the other cultures there was development of the technology in the rural environment, where
a slight EC drop the rst two days of culturing, thereafter EC values resources are scarce and no specialization in this eld is expected.
were stable. In CCW there was an EC increase at the end of the Therefore, in this study EC was used as parameter for determining
experiment (day 11) which could be related to cell death and remediation capacity of microalgae. However, it was necessary to
release of intracellular compounds. It is important to note that the evaluate the ability of the EC to determine reliably the reduction in
largest decrease in EC occurred always between days 0 and 2, nutrient content. Consequently, correlation analyses were per-
whereas the highest biomass growth occurred normally between formed between EC and cell concentration. The rst analysis
days 2 and 4. Therefore, most of EC reduction was probably due to included data of the entire duration of the experiment. The second
phenomena not related to microalgae growth, such as the two was performed only with the cultures that proved to be viable and
mentioned above (volatilization and precipitation). included only the data until the maximum biomass concentration
was observed, since these data are related to the highest biomass
3.4.2. Scenedesmus mixed cultures growth rate and therefore, highest nutrient consumption rate
EC variation during SMC growth is shown in Fig. 4. Under (see Table 5).
ambient conditions (Fig. 4a) the highest drop occurred in cultures In experiments with CMC under ambient conditions, biomass
growing on CSYE, which at the same time showed a poor growth. concentration and EC were negatively correlated in all treatments
This clearly indicates that the decrease of the nutrient load was not for the whole experimental period, except in WW for which a
only due to microalgae growth but also due to physical-chemical positive correlation was observed (Table 5). The negative correla-
phenomena such as volatilization and precipitation. It is well tion supports the remediation capacity attributed to microalgae
known that in microalgal wastewater treatment systems not only
microalgae acted as a remediation factor, but also naturally
occurring bacteria, light intensity, pH and DO played an important Table 5
Pearson correlation coefcient (r) between biomass concentration and electrical
role for increasing the treatment capacity of the system [46]. Under conductivity for CMC and SMC for the whole experimental period and until the day
ambient conditions the decrease of EC was higher for all growth with max biomass concentration. * No correlation (p < 0.05).
mediums than under lab conditions except for CSYE.
Culture Culture media Ambient conditions Lab conditions
For both SMC and CMC, efuents with a high organic load (e.g.
CSYE) showed the lowest DO values at the beginning, with an Period r Period r
increase during the experiment indicating a reduction in the CMC CSYE 014 0.54 014 *
organic load (data not shown). 07 * 07 0.68
MPE 014 0.83 014 *
The pH showed no clear trend during the experiment (data not

shown). However, pH was stable around 7.2 and 9.5. According to CCW 014 0.72 014 *
Park et al. [51] pH 8 is recommended for Chlorella and Scenedesmus. 07 0.87 09 *
As mentioned above, pH depends on efuent characteristics (e.g. WW 014 0.57 014 *
composition, alkalinity), but during the experiment it was

modied by photosynthesis (CO2 consumption and O2 release) SMC CSYE 014 * 014 0.5
and aeration rates. pH changes during the culture period due to
changes in wastewater composition (e.g. fermentation of milk MPE 014 0.6 014 *
sugars to lactic acid) were probably buffered by the CO2 system 07 * 09 0.59
CCW 014 0.67 014 0.5
(CO2, HCO3 , CO3 2) [52], and therefore, would not affect
09 0.67 09 0.54
microalgal growth. Most of the cultures showed a relatively high WW 014 * 0 14 *
pH at the end of the experiment that might limit efuent
discharges in waterbodies.

Fig. 4. EC variation during culture of SMC (a) under ambient conditions; (b) under lab conditions for the different growth media used [CSYE (&); WW(*); MPE(~); CCW(^)].
Signicant differences (p < 0.05) are shown with capital letters. Bars indicate standard error. Data from day 0 was used as covariate in the statistical analysis.
642 J.I. Labb et al. / Journal of Environmental Chemical Engineering 5 (2017) 635643
[41]. Data of CMC grown outdoors on CCW and CSYE were analyzed
also until the end the exponential phase. With this analysis, CCW
maintained the negative correlation observed in the entire
experimental period. However, the culture with CSYE was not
correlated in the exponential phase with microalgae growth,
opposing to the observations in the entire experimental period. In
CMC grown under laboratory conditions no correlation was found
for the entire experimental period and the only negative
correlation was found for data obtained until the highest biomass
concentration for CMC grown on CSYE.
For SMC growing outdoors, negative correlation between
biomass concentration and EC for the entire experimental period
was only observed in MPE and CCW (see Table 5), classied above
as viable cultures. The negative correlation was maintained only
in the case of CCW when analyzing the data obtained until the
maximum cell concentration was observed. For SMC under
laboratory conditions a negative correlation was observed only
in the case of CCW, whereas a positive correlation was observed in
the case of CSYE, maybe as a consequence of the poor growth of the
culture and microalgae death. In SMC grown indoors, the
correlation study for data obtained until the day of maximum
cell concentration showed a negative correlation in MPE and CCW.
The lack of correlation between EC and biomass concentration
in all viable cultures hinders the possibility of relating directly
nutrient depletion to EC measurements in wastewater treatment
and point at the need of advanced analysis to determine the
purication degree of wastewaters by microalgae. EC could be used
as control parameter only once a regression model could be
established between nutrient consumption and EC reduction, but
more research is needed in this direction. Microalgae cultures have
previously shown their ability to reduce nutrients and inorganic
salts present in dairy waste. In the case of MPErich in casein,
lactose, fats and inorganic salts- other authors have shown that
microalgae can reduce BOD and COD between 70 and 90%,
ammonia nitrogen and nitrates more than 90% and other nutrients
around 80% [5356]. The same applies for efuents similar to CSYE,
rich in ammonia nitrogen and phosphorus as a consequence of the
high manure content [8,9]. Therefore, although in our study EC
could not be correlated to microalgal biomass concentration,
remediation was taken place due to microalgal growth.
4. Conclusions
Chlorella mixed cultures (CMC) and Scenedesmus mixed cultures
(SMC) were capable of growing in different dairy farms efuents,
being the response to efuent composition different for each
culture but independent of culture conditions.
Viable cultures for commercial purposes were identied based
on biomass growth pattern, cell concentration and specic growth
rate. CMC grown on cattle standing yard efuents (CSYE) and
Cholqui canal water (CCW); and SMC grown on milking parlor
efuents (MPE) and CCW were viable cultures. Results showed that
Chlorella grew better in wastewaters with high organic and
ammonium loads such as CSYE with 1.70  108 and 1.67  108 cel
ml 1 outdoors and indoors, respectively; whereas Scenedesmus
grew better on wastewaters with high chemical and detergent load
such as MPE, reaching 3.20  107 and 5.61 107 cel ml 1 outdoors
and indoors, respectively. By growing microalgae outdoors it is
certainly possible to decrease microalgal production costs by using
dairy efuents. Correlation analyses showed that more research is
needed before using EC as an indicator of the treatment capacity of
microalgae.
This work shows that there is potential in using dairy efuents
for microalgae growth in order to treat these efuents and improve
the nances of small and medium dairy farms. Further research is
needed to optimize these systems before upscaling.
Acknowledgements
This paper present research results funded by Procycla SpA
(Chile), privately held organization. Authors would like to thank Dr.
Emky H. Valdebenito Rolack for his contribution in this paper.
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