International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976

6480(Print), ISSN 0976 6499(Online) Volume 5, Issue 7, July (2014), pp. 57-62 IAEME

INTERNATIONAL JOURNAL OF ADVANCED RESEARCH

IN ENGINEERING AND TECHNOLOGY (IJARET)

ISSN 0976 - 6480 (Print)

ISSN 0976 - 6499 (Online)
Volume 5, Issue 7, July (2014), pp. 57-62
IAEME: http://www.iaeme.com/IJARET.asp
Journal Impact Factor (2014): 7.8273 (Calculated by GISI)
www.jifactor.com

IJARET
IAEME

DEVELOPMENT OF INTEGRATED BIOREMEDIATION AND ANAEROBIC

DIGESTION PROCESS USING 3rd GENERATION FEEDSTOCK
Rohit Sharma[1],

Avanish K Tiwari[2],

G Sanjay Kumar[1],

Bhawna Yadav Lamba[1]

[1]

College of Engineering Studies, University of Petroleum and Energy Studies, Bidholi,

Dehradun, India
[2]
Senior Principal Scientist, Center for Alternate Energy Research, University of Petroleum &
Energy Studies, New Delhi, India

ABSTRACT
The possibility of microalgae nitrogen treatment was tested in biogas digester wastewater. In
this work, Chlorella pyrenoidosa was cultivated in biogas digester wastewater as a nutrient source.
The growth kinetics of the algae as well as the bioremediation effect on the waste water was studied
at different environmental conditions. The microalgae, Chlorella pyrenoidosa can utilize the nitrogen
content present in biogas digester wastewater as a substrate for its growth. The growth of microalgae
was found to follow the Monod growth model satisfactorily. Under the different condition in biogas
waste water medium of microalgae, a maximum biomass of 3.75 gm/l and 1.5 gm/l was obtained in
fifteen days. The net specific growth rate of microalgae Chlorella pyrenoidosa was found to be 0.1
D-1. The growing algae also removed 92.8 % of nitrate nitrogen (NO3 -N) at 19 2 C and 76 % at
30 2 C from the biogas wastewater. Treated Biogas waste water can be further used for the
anaerobic digestion of algal biomass for the production of biogas. Anaerobic co-digestion of cultured
microalgae and cow dung is to be done with treated water to maintain C\N ratio and to optimize the
yield of biogas. This suggests that the cultivation of C. pyrenoidosa in biogas wastewater would be
efficient, saving water as well as producing digestible biomass. Thus, on one hand the biogas waste
water is being treated and on the other, the alga is showing substantial growth.
Keywords: Anaerobic Digestion, Biogas, Biogas Waste Water, Chlorella Pyrenoidosa, Cultivation,
Nitrate;
1. INTRODUCTION
The world is facing problems with a wide variety of pollutants and contaminates from
various developmental activities. Microalgae have vast industrial and economic potential as valuable
57

International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976
6480(Print), ISSN 0976 6499(Online) Volume 5, Issue 7, July (2014), pp. 57-62 IAEME

sources for pharmaceuticals, health foods, carotenoids, dyes, ne chemicals, bio fuels, and others [1].
Bioremediation of wastewater by microalgae can provide the microalgae feedstock for their biomass
energy, as well as reduce the material cost of the bio fuel [2]. The algal cells were able to consume
high concentrations of nitrate ion and, therefore, can possibly contribute to purification of industrial
and domestic wastewater [3]. The coupled process of algae cultivation and succeeding biogas
production is a better option compared to algal biodiesel production [4].
A possible solution for overcoming the high cost of production is to integrate algae
cultivation with an existing biogas plant, where algae can be cultivated using the discharges of CO2
and digestate as nutrient input, and then the attained biomass can be converted directly to biogas or
bio methane by the existing infrastructures[4]. Energy and GHGs balances of algal bio methane
production were assessed in the perspective of life cycle, and comparison with ley crop was
conducted [4].
The growth of green algae Chlorella sp. on wastewaters sampled from four different points of
the treatment process flow of a local municipal wastewater treatment plant (MWTP) was done. They
investigate how well the algal growth removed nitrogen, phosphorus, chemical oxygen demand
(COD), and metal ions from the wastewaters [5].
The domestic wastewater samples were collected from sewage wastewater treatment plant
Bopodi from Pune city was used to study the role of microalgae in wastewater treatment. Chlorella
sp. shows the best removal capacity of nitrate and phosphate reduction [6].
Microalgae have been used for the bioremediation of textile dyes in wastewater from
industrial textile processes [7]. Microalgae such as Chlorella and Scenedesmus have shown tolerance
and bioremediation capabilities to certain heavy metals [7]. Both nitrogen and phosphorus are the
major sources of eutrophication, therefore, high concentrations of nitrogen or phosphorus can cause
algal blooms and other hazardous environmental problems [1]. Nitrate in wastewater is generally
produced as an intermediate of nitrogen metabolism by microorganisms, beginning with
ammonification of proteins or other nitrogen-containing compounds, followed by nitrification of
ammonia into nitrite, and later, oxidation of nitrite into nitrate. Based on the understanding nitrate
accumulation becomes a concern in water quality management [8].
Algae can capture carbon dioxide in the flue gas from coal red power plants thereby reducing
greenhouse gas and also producing algal biomass, which can be converted into bio-fuel. Chlorella,
Scenedesmus and Spirulina are the most widely used algae for nutrient removal [9].
This study is focused on applications for nitrogen removal in biogas digester wastewaters,
exploiting the photosynthetic ability of microalgae. When microalgal cells are cultured under
photoautotrophic conditions, these cells can utilize nitrate nitrogen from waste water.
2. MATERIAL & METHODS
2.1 Sludge Collection
Biogas digester outlet slurry used as substrate for this research was collected from the 3 m3
cow dung based biogas plant in UPES, Dehradun. The sludge obtained from the gravity thickener at
the facility was filtered on-site with a mesh and transferred into a polymer container for storage.
2.2 Substrate Preparation
Sludge was stored in a polymer container following collection and stored at 4C. For use as
substrate, the collected sludge was transferred to 1 lit conical flask (mini reactors) for incubation.
The substrate containing mini reactors were then autoclaved at 121C for 20 minutes for the removal
of foreign contaminants.

58

International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976
6480(Print), ISSN 0976 6499(Online) Volume 5, Issue 7, July (2014), pp. 57-62 IAEME

2.3 Algal Strains Culture

Chlorella pyrenoidosa were purchased from National Collection of Industrial
Microorganisms (NCIM) for this research. It was cultivated in the fog medium at room temperature
which is about 25 3C each day before the microalgae was inoculated to the biogas digester
wastewater.
2.4 Incubation
0.1 gm of microalgae strain was aseptically transferred into the sterilized 1 litre flask of waste
water. The flask was incubated at the different room temperatures with an electric bulb as the light
source till the stationary phase of monod kinetics started. 750 ml of biogas wastewater was
inoculated with C. pyrenoidosa in a 1 litre flask.
2.5 Data Collection and Analysis
UV-VIS spectrophotometer was used for nitrate content analysis as well as for the
concentration of microalgae. In order to study the growth of C. pyrenoidosa in the biogas
wastewater, absorbance of sample was checked at 680 nm [11]. To check the bioremediation, nitrate
content was analyzed by calculating the concentration of nitrates in the initial and the final day of
inoculation. The nitrate nitrogen (NO3 -N) content consumption kinetics may be expressed as
substrate conversion to product (1).


1 


 / 
Where - dS/dt is the total consumption rate of nitrate nitrogen; t is total time for microalgae growth
and substrate consumption; Yx/s is the maximum microalgae Yield coefficient; S is nitrate nitrogen
concentration. Microalgae growth process can be explained Monod equation. This is based on mass
balance. There are various terms associated with Monod kinetics with the contribution of biomass
and substrate to the environment. S represents the substrate concentration and X represents the
biomass concentration. The rate of substrate and biomass growth is ds/dt and dx/dt. Rate equation for
algal growth is (2):

max x


  
max is the maximum specific growth rate of the microalgae; x is the conc. of microalgae in
the medium; s is the substrate concentration.
3. RESULTS & DISCUSSION
3.1 Growth of C. Pyrenoidosa in Biogas waste water
Algal growths in terms of optical density OD680 in the Biogas wastewaters under axenic
condition were plotted in Fig 1. C. pyrenoidosa could grow well in the biogas wastewater. Their
growth increased rapidly in the third to fifteenth day, then slowed down in the next seven days, and
almost stopped in the last five days. This could be due to the gradual consumption of certain nutrient
elements like nitrogen in the biogas wastewater. Wet 3.71 gm and 1.40 gm of microalga C.
pyrenoidosa was cultivated in 750 ml of medium in fifteen days of cultivation period in biogas waste
water. This also proved that the microalga C. pyrenoidosa could grow well in biogas wastewater and
might be used for the treatment of the wastewater. Table1 shows the substrate limited Monod growth
kinetic variables.
59

International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976
6480(Print), ISSN 0976 6499(Online) Volume 5, Issue 7, July (2014), pp. 57-62 IAEME

Table 1: Model state variables and initial and final concentration of nitrogen source
Parameter

Values 19 2C

Values 30 2C

max (d 1)

0.1002

0.100

x0 (gm/750 mL)

0.1

0.35

xf (gm/750 mL)

3.71

1.40

Initial nitrogen nitrate So (mg/l)

87

84

Final nitrogen nitrate Sf (mg/l)

6.19

20.16

Figure 1: Growth of C. pyrenoidosa in the biogas wastewater at room temperatures

3.2 Bioremediation of the biogas wastewater by C. pyrenoidosa
With the growth of C. pyrenoidosa, the concentrations of nitrate nitrogen decreases. The NO3
-N in the biogas wastewater was eliminated by 92.8 % and 76 % respectively, within fifteen (15)
days. The nitrate concentration in the biogas wastewater was tested initially, during the introduction
of the inoculum, and subsequently after fifteen days. This clearly indicated a 92.8 percent reduction
in the nitrate concentration, proving its consumption by the microalgae. Table 2 shows the utilization
of nitrate by microalgae in the biogas waste water slurry.
Table 2: Elimination of nitrate nitrogen in the biogas wastewater by C. pyrenoidosa
Sr.
No.

Conditions

Initial nitrogen nitrate So

(mg/l)

Final nitrogen nitrate Sf

(mg/l)

%
Removal

19 c 2c

87

6.19

92.8 %

30 c 2c

84

20.16

76 %

60

International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976
6480(Print), ISSN 0976 6499(Online) Volume 5, Issue 7, July (2014), pp. 57-62 IAEME

3.3 Anaerobic co-digestion of cultured microalgae using treated water

The compositions of the gas were analyzed via gas chromatography (Nucon 5765) by a
thermal conductivity detector (TCD) and helium as the carrier gas. It is shown in Table 2.
Table 2: Co digestion analysis of microalgae and cow dung
Substrate

HRT (d)

Biogas yield (ml)

Methane (%)

Cow dung (50 %) + Algae (50 %)

20

430

67

Cow dung (100 %)

20

490

61

Algae (98 %)

20

455

62

It was clear that the set that contained algae alone can effectively digested compared to the
cow dung alone. In order to keep the nitrogen balance within the system, the amount of nitrogen
leaving the system must also enter the system, either through the co-digestion material or as fertilizer
[10]. By co-digesting cow dung-microalgae and treated water mixture, the C\N can be maintained.
4. CONCLUSION
The cultivation of the microalga, C. pyrenoidosa in the biogas wastewater was studied and
analyzed. The results from this study demonstrated the feasibility of cultivating Chlorella sp. in
biogas wastewaters outlet slurry. Chlorella sp. could adapt well in biogas wastewaters outlet slurry
with small lag phases observed. Algal growth was significantly enhanced in the centrate because of
its much higher levels of nitrogen content in biogas wastewaters outlet slurry. The microalgae C.
pyrenoidosa had good growth in the biogas wastewater and its wet weight reached 3.71 gm in 750 ml
after cultivation for fifteen (15) days. Although, the concentrations of nitrate nitrogen in the
wastewater were extremely high, the microalga could still grow well in the wastewater. Biogas waste
water is the suitable method for cultivation of microalgae. Microalgae C. pyrenoidosa removes
around 92.8 percent of the concentration of nitrate in the biogas waste water in the 30 days of
inoculation. This work treat the biogas waste water for further use and produced microalgae may be
further undergo an-aerobic digestion in a lab scale batch type anaerobic digester for the production of
bio gas as a renewable source of energy. This concludes that the cultivation of C. pyrenoidosa in
biogas wastewater would be efficient, economic and saving water for anaerobic digestion as well as
producing biogas.
5. ACKNOWLEDGEMENT
The authors thank the Ministry of New and Renewable Energy for research grants. They also
thank the Centre for Alternate Energy Research, UPES for support.
REFERENCES
[1]
[2]

Choul-gyun Lee. Nitrogen removal from wastewaters by microalgae without consuming

organic carbon sources. 12:979-985, 2002.
Lu Yinghua Tang Xuemin Lu Bin Li Yuanyue Lu Zhiqiang Lin Yaojiang Zheng Jiang, Li
Zhongbao and Zhou Jixin. Cultivation of the microalga, chlorella pyrenoidosa, in biogas
wastewater. African Journal of Biotechnology, 10:13115-13120, 2011.
61

International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976
6480(Print), ISSN 0976 6499(Online) Volume 5, Issue 7, July (2014), pp. 57-62 IAEME

[3]

[4]
[5]

[6]
[7]

[8]

[9]
[10]
[11]
[12]

[13]

Laila Al-balushi, Nitin Rout, Sahar Talebi, Ahmed Al Darmaki, and Maryam Al-qasmi.
Removal of Nitrate from Wastewater Using Trentepohlia Aurea Microalgae. Engineering, I:
8-10, 2012.
Xiaoqiang Wang, Eva Nordlander, Eva Thorin, and Jinyue Yan. Microalgal biomethane
production integrated with an existing biogas plant: A case study in Sweden. 2012.
Liang Wang & Min Min & Yecong Li & Paul Chen & Yifeng Chen & Yuhuan Liu &
Yingkuan Wang & Roger Ruan. Cultivation of green algae chlorella sp. in di erent
wastewaters from municipal wastewater treatment plant. Appl Biochem Biotechnol, 10-13,
2009.
Ayodhya D Kshirsagar. Bioremedation of waste water by using microalgae: An experimental
study. IJLBPR, 2:339-346, 2013.
Asif Rahman, Joshua T Ellis, and Charles D Miller. Bioremediation of Domestic Wastewater
and Production of Bioproducts from Microalgae Using Waste Stabilization Ponds. J
Bioremed Biodeg, 3(6):6199, 2012.
Jalan Sultan, Ahmad Shah, Bandar Indera Mahkota, Kuantan Pahang, Jalan Gombak, and
Kuala Lumpur. Removal of nitrate and phosphate from municipal waste water sludge by
chlorella vulgaris, spirulina plantesis. Biotechnology, 12(4):125-132, 2011.
Seema Dwivedi. Bioremedation of heavy metal by algae: Current and future perspective
biotechnology. Journal of advanced laboratory research in biology, III: 229-233, 2012.
Dwivedi, S.: Bioremedation of heavy metal by algae: Current and future perspective
biotechnology. Journal of advanced laboratory research in biology 3, 229-233 (2012).
Nigam, S., Rai, M.P., Sharma, R.: Effect of nitrogen on growth and lipid content of chlorella
pyrenoidosa. American Journal of Biochemistry and Biotechnology 7, 124-129 (2011).
R Radhakrishanan and A Praveen, Sustainability Perceptions on Wastewater Treatment
Operations in Urban Areas of Developing World, International Journal of Civil Engineering
& Technology (IJCIET), Volume 3, Issue 1, 2012, pp. 45 - 61, ISSN Print: 0976 6308,
ISSN Online: 0976 6316.
Bharati S. Shete and Dr. N. P. Shinkar, Kinetic Modeling for Anaerobic Digestion: A
Review, International Journal of Civil Engineering & Technology (IJCIET), Volume 5,
Issue 2, 2014, pp. 127 - 136, ISSN Print: 0976 6308, ISSN Online: 0976 6316.

62