Proceeding of the

International Conference on Waste Water Management

17,18 and 19th 2017

3.
A study on scale up process in phycoremediation of Rice mill
effluent by Scenedesmus Abundans
J. Umamaheswari
Environmental Engineering Laboratory, School of Civil and Chemical Engineering
VIT University, Vellore-632014, India
[email protected]
S. Shanthakumar*
Department of Environmental and Water Resources Engineering
School of Civil and Chemical Engineering
VIT University, Vellore-632014, India
[email protected]
*Corresponding author
ABSTRACT

Phycoremediation is the economic as well as eco-friendly technique in the wastewater treatment, mostly employed in
treating the agro-based industrial effluent. Rice mills are the agro-produce processing units which discharge huge as
well as nutrient rich effluent, without proper treatment, causes threat to the environment (eutrophication in
water/land/soil and soil infertility). Most of the rice mill industries in the country, fit in small and medium scale
industries where the knowledge on the treatment system is comparatively less. Phycoremediation is the technique in
which algae assimilate the nutrients and pollutants in the wastewater for their growth and metabolism thereby treating
the wastewater effluent. Also, the biomass accumulated in the treatment system is the source of valuable by-products,
bio-fuel, aquatic feed and the raw materials for medicine, cosmetics etc. The treatment efficiency and biomass
production of phycoremediation varies based on the operating conditions such as scale and mode of cultivation, light,
temperature etc. In this experimental study, the growth potential and pollutant removal efficiencies of algal sp.
scenedesmus abundans have been investigated in lab scale (100ml culture in 250ml conical flasks) as well as in scale
up condition (1.5l culture in 1-2 litres polybags) under constant temperature (27ºC), inoculum size (20% v/v) and light
conditions (1200 lux). Growth indicators of maximum biomass dry weight, cell concentration or no of cells and
chlorophyll-a content in lab scale are measured as 1.95 g/l, 9.36x10^04 cells/ml and 11.04 mg/l respectively which
are 14%, 25% and 18% higher than that of 1.5l culture. The maximum ammonical nitrogen (NH3-N) removal of
80.67% has been achieved in lab scale whereas it was 74.32% in 1.5l culture. Removal percentages of phosphorous
(PO43-) in lab scale and poly bags are reported as 99.45% and 94.96% respectively. This is mainly due to faster growth

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International Conference on Waste Water Management
17,18 and 19th 2017

of microalgae in lab scale (conical flask) due to its occurrence of interaction with the nutrients of growth medium
(RME). The treated wastewater was checked for its suitability for reuse, after harvesting microalgae by centrifugation

Key words: Phycoremediation, rice mill effluent, lab scale, scale-up process

1. INTRODUCTION

Disposal of untreated industrial effluent to the environment is a major concern of

developing countries. Now a days, government policies, rules and regulations enforce the
industries to follow the effluent standards for the safe disposal of waste. Hence, there is an increase
in capital borne by industries which results in the requirement of economic and efficient treatment.
Phycoremediation is one such technique which utilizes the algae to treat the wastes or wastewater
[1]. Microalgae are small photosynthetic organisms, adopted to any kind of possible environment
and utilizes the nutrients or pollutants in wastewater for their growth metabolism in the presence
of light energy. The biotransformation of pollutants occurring in the microalgae cell, leads to the
accumulation of lipids and proteins and is thereby useful in bio energy production [2, 3].

Rice mill industries are agro-based production industries. They play a vital role in the

Also, India is the second largest rice producer in the world next to China. Processing of rice in
milling industries involves various processes including soaking of paddy in water. This is the
foremost process of rice production and requires 1.3 times of water per kg of paddy [4]. The
soaking process releases huge quantities of effluent to the environment. Rice Mill Effluent (RME)
does not contain any toxic substances but is rich in organic contents. It pollutes the medium on
which it is disposed (water/land). Further, the country holds mostly small and medium scale
industries and the knowledge on the treatment system is negligible among them. The treatment
efficiency of phycoremediation of industrial wastewater is mainly dependent on operating
parameters such as light, temperature, scale of operation, mode of cultivation and types of aeration
provided for the system and most of the studies were conducted in lab scale (conical flasks) [5-9].
The application of lab scale to the field is the challenging task and the present study intended to
provide the assessment on growth parameters and pollutant removal efficiency of scale up process
(1-2 litres polybags) in comparison with lab scale culture.

2. MATERIALS AND METHODS

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International Conference on Waste Water Management
17,18 and 19th 2017

The micro algae Scenedesmus abundans (Figure 1b) was purchased from National Centre of
Industrial Microorganism (NCIM), Pune and cultured in Blue Green (BG 11) medium which
contains NaNO3(1.5g), K2HPO4(0.04g), MgSO4.7H20(0.075g), CaCl2.2H20(0.036g), Citric
acid(0.006g), Ammonium ferric citrate (0.006g), EDTA disodium salt(0.001g), Na2CO3(0.02g),
H3BO3(2.86mg), MnCl2.4H20(1.81mg), ZnSO4.7H20(0.22mg), Na2MoO4.2H20(0.39mg),
CuSO4.5H20(0.079mg) and CO(NO3)2.6H20(0.049mg) per litre of distilled water and pH of the
medium is adjusted to 7.1 by using 1M NaOH or HCl. Further, the culture is incubated at 25ºC
under the light intensity of 1200 lux. After the growth period of 14 days, the cultured algae are
inoculated in the Rice mill effluent (RME), collected from the rice mill industries located in
Vellore district of Tamil Nadu. The various physico-chemical parameters of collected RME is
presented in table 1. The study was conducted in 100ml conical flasks (lab scale) as well as 1 to 2
litres capacity poly bags (scale-up process) (Figure 1a) with an inoculum size of 20% (20ml of
culture in 100ml RME v/v (volume /volume)) with an initial cell concertation of 1.45 x 10^5 cells
ml-1.

Table 1. Physico-chemical characteristics of RME

Rice Mill Effluent

Parameters
(RME)
Colour Dark yellow
Odour unpleasant
pH 7.1
Turbidity (NTU) 17
Acidity (mg/l) 140
Total alkalinity mg/l 520
Total hardness mg/l 350
Total solids, mg/l 1800
Suspended solids, mg/l 400
Dissolved solids, mg/l 1400
Chloride mg/l 190
Sulphate mg/l 32
Phosphorous mg P/l 43
COD mg/l 1280
BOD (3 days at 27ºC) mg/l 473

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Ammonical Nitrogen (NH4- N) mg/l 274

(b)

(a) (c)

Figure 1. (a) Experimental set-up (b) Microalgae Scenedesmus abundans (c) RME before
and after phycoremediation

Growth indicators such as chlorophyll-content, cell concentration (cells per ml of culture) and
biomass dry weight were measured in alternate days of the growth period to understand the growth
potential of Scenedesmus abundans in RME.

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The chlorophyll-a content is determined by measuring the optical densities (OD) at 660nm and
642nm by UV spectrophotometer (Model: Hach DR6000). The formula used to calculate the
chlorophyll content is given in equation 1 [10].

-------------------- (1)

The number of cells per ml of culture is determined by haemocytometer (Neubauer: improved)

in which the average number of viable cells per square has been measured by considering five
squares in a checkerboard pattern in the consecutive three rows. Further, the concentration or cell
density is measured by using the below equation 2.

--------------------- (2)

Where the dilution factor is one when there is no dilution has been taken place.

Growth curve of a typical algal batch culture consisting of five phases namely, (i) lag phase
(ii) exponential phase (iii) declining growth phase (iv) stationary growth phase and (v) death phase.
During the exponential phase, the cell concentration increases as the function of time and it can be
represented as the equation (3) given below.
----------------------------------------
(3)

Where, Ct Initial concentration at time t; C0 Initial concentration at time 0 & m = specific

using Equation (4)

----------------------------------------
(4)

Where, is the log10 value of cell number at time zero (t0) & is the log10 value
of cell number at specific time (t1).

The biomass dry weight is calculated by the difference in weight between the weight of an
aliquoted vacuum filtered algal sample and the constant weight obtained after keeping the sample
at 95ºC in a hot-air oven.

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International Conference on Waste Water Management
17,18 and 19th 2017

The excess nutrients or pollutants such as ammonical nitrogen and phosphorous as

phosphates have been measured by the procedure followed in American Public Health Association
(APHA) standard.

3. RESULTS AND DISCUSSION

3.1.Growth indicators
3.1.1. Chlorophyll a

Chlorophyll content of algae plays a key role in their growth metabolism as they are
photosynthetic organisms [11]. The maximum chlorophyll content of 11.04mg l-1 has been
achieved in lab scale whereas it is 9.968mg l-1 in the case of scale up process which is 10.76%
lesser than the former one (Figure. 1).

12

10
Chlorophyll-a (mg/l)

4 1.5l culture
2

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (days)
Figure 1. Comparison of chlorophyll-a content of Scenedesmus abundans in lab scale
(100ml) and scale-up (1.5l) culture
3.1.2. Cell concentration or cell count per ml of culture

The growth curves of Scenedesmus abundans in 100ml and 1.5l RME are shown in figure
2. The initial concentrations of inoculated culture are measured as 3.32 x 10^04 and 2.68 x 10^04
respectively for the lab scale and scale up processes. This is mainly due to the dilution of RME as
the inoculum is added on a volume basis. Initially, the growth curve has the short induction or lag
phase as the liquid culture is used for the inoculum and reached its exponential phase in four days.

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International Conference on Waste Water Management
17,18 and 19th 2017

The specific growth rate is mainly dependent on algae species used, temperature and light
intensity. Further, the phase of declining relative growth occurs in between four to seven days of
culture period where the nutrients (nitrogen and phosphorous), pH, light, carbon dioxide and other
physical and chemical parameters began to limit the growth of algae. There is no stagnant growth
phase in 1.5l culture and the slight declination was identified in between nine to eleven days
however it occurs in 100ml culture during the period. Finally, the algal growth reaches its death
phase where the nutrients are depleted to the minimum value that will not support the algal growth
and hence the cell density decreases rapidly and started collapsing. The specific growth rate
constant of lab scale and 1.5l culture have been calculated as 0.279 and 0.272 respectively
indicated that there is not the much significant difference between their growth rates.

5.1
5
log10 (Cell numbers)

4.9 100 ml culture

4.8 1.5l culture
4.7
4.6
4.5
4.4
4.3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (Days)

Figure 2. Growth curve of Scenedesmus abundans in lab scale RME and scale-up
(1.5l) RME culture
3.1.3. Biomass dry weight

Biomass dry weight is another predominant indicator of algal growth. The biomass dry
weight of lab scale increases with increase in time but there is a declination in between seven to
nine days culture of 1.5l RME. Other factors such as light density, temperature and improper
mixing of culture may inhibit the biomass value. The maximum biomass dry weight of 1.71g l -1
has been obtained in 11 days from a 1.5l culture which is merely 88% of the biomass obtained

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from the 100ml culture in lab scale (Figure 3). Further, the biomass value started decreasing due
to the fact that the algal growth reaches its death phase.

2.50

2.00

1.50

1.00 100ml culture

1.5l culture
0.50

-
0 2 4 6 8 10 12 14 16
Time (days)
Figure 3. Biomass dry weight of Scenedesmus abundans in lab scale RME and scale-
up (1.5l) RME culture
3.2. Pollutant / Nutrient removal

The optimum nitrogen to phosphorous ratio to enhance the growth of green algae is suggested
to be in the range of 6.8-10.0 [12 - 14]. In this study, the N/P ratio (6.37) of RME (Table 1) supports
the growth of microalgae, Scenedesmus abundans thereby acclimatize the nutrients from the
wastewater.

3.2.1. Ammonical Nitrogen

Nitrogen is an important nutrient for all kind of photosynthetic organisms. Microalgae utilize
the inorganic nitrogen in the form of nitrogen (NO2-), nitrate (NO3-) and ammonia (NH4+) in
wastewater and converts into organic form by assimilation process. The accumulation of lipids
and protein in the micro cell has been taken place during this process [15]. The ammonical nitrogen
of 100ml and 1.5l inoculated RME are measured periodically to understand the nutrient uptake
potential of Scenedesmus abundans. It was noted that both the cultures follow the same pattern of
removal percentage nevertheless the maximum removal efficiency of 80.67% has been obtained

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in lab scale culture. Further, the scale up process (1.5l RME) utilized 74.32% of ammonical
nitrogen in the treatment system (Figure. 4).

90%
80%
70%
Percentage Removal

60%
50%
100 ml culture
40%
1.5l culture
30%
20%
10%
0%
0 2 4 6 8 10 12 14 16
Time (Days)

Figure 4. Percentage removal of ammonical nitrogen by Scenedesmus abundans in

lab scale RME and scale-up (1.5l) RME culture

3.2.1. Phosphorous as phosphates

Phosphorous plays a vital role in the growth metabolism of micro algae. Phosphates in the
form of mono hydrogen phosphate and di hydrogen phosphate in the treatment system are the main
source of energy producers in the micro algal cell metabolism. The process, phosphorylation
releases the high energy exchange medium in the cell, adenosine tri phosphate (ATP) by adding
the third phosphate group to adenosine di phosphate (ADP) [15, 16]. Initially, 43.36% removal of
phosphates has been achieved in 1.5l RME culture then increased gradually and reached the
optimum value of 94.96% in 14 days. The phosphates removal efficiency of Scenedesmus
abundans in 100ml RME culture increases suspiciously with the increase in culture period and
reaches the maximum value of 99.45% at the end of period (Figure. 5).

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International Conference on Waste Water Management
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3.3.Reuse potential of treated RME

The reuse potential of treated RME (Figure 1c) has been checked after harvesting the cultivated
microalgae by centrifugation. It was identified that 95% of the reduction in phosphates has been
achieved in the treatment. Further, 80 90% reduction in turbidity, alkalinity and BOD of the
effluent have noticed in the system which may suggest the treated water can be reusable. Total
hardness, ammonical nitrogen and COD of the RME have reduced to 66%, 74% and 75%
respectively after the phycoremediation. Also, more than 30% reduction in other parameters has
been noticed in the treated RME. Furthermore, the slight alkalinity has been noticed in the
treatment system as the algae consume carbon dioxide during its photosynthesis, and this
consumption is responsible for an increase in pH [17].

120%

100%
Percentage Removal

80%

60% 100 ml culture

1.5l culture
40%

20%

0%
0 2 4 6 8 10 12 14 16
Time (Days)

Figure 5. Percentage removal of phosphorous (PO4) by Scenedesmus abundans in

lab scale RME and scale-up (1.5l) RME culture

Table 2. Characteristics of RME after phycoremediation by Scenedesmus Abundans

Percentage reduction in
Parameters Value
physicochemical parameters
Colour Nil Clear water
Odour Nil Removes odour
pH 7.8 Slightly alkaline in nature

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Turbidity (NTU) 3 82%

Acidity (mg/l) 20 86%
Total alkalinity mg/l 540 -4%
Total hardness mg/l 120 66%
Total solids, mg/l 1307 27%
Suspended solids, mg/l 220 45%
Dissolved solids, mg/l 1087 22%
Chloride mg/l 120 37%
Sulphate mg/l 21 34%
Phosphorous mg P/l 2 95%
COD mg/l 320 75%
BOD (3 days at 27ºC) 89 81%
mg/l
Ammonical Nitrogen 70 74%

4. CONCLUSION

It is evident that the treatment efficiency decalins when go for scale up process. The
phycoremediation of RME with Scenedesmus abundans in lab scale (100 ml conical flasks) given
the maximum growth which was identified by measuring the growth indicators of chlorophyll-a
(11.04 mg/l), cell concentration (9.36x10^04 cells/ml) and biomass dry weight (2.15 g/l). Further,
the optimum removal efficiencies of 80.67% for ammonical nitrogen and 99.45% for phosphorous
have been achieved in lab scale which is 8.54% and 4.73% more than the removal efficiencies of
1.5l RME culture in poly bags. Nevertheless, the specific growth constant of lab scale and scale
up process not having a significant difference as it mainly depends on the cell concentration in the
exponential phase of micro algal culture. The treated RME after phycoremediation has the
considerable reduction in excess nutrients or pollutants which could recommend the water can be
reusable in the industry. To conclude this study, even though the efficiency will reduce in case of
scale up process, there is always in need of process optimization and improvement in employed
technology to carry forward the lab scale to pilot scale, which is mostly applicable in industries.

ACKNOWLEDGMENT

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The authors acknowledge the financial support provided by the Science and Engineering Research
Board (SERB), Department of Science and Technology (DST), Government of India under grant:
YSS/2015/000527.

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