development of integrated bioremediation and anaerobic digestion process using

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

57

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 ºC and 76 % at

30 ± 2 ºC 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

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ISSN 0976 - 6480 (Print)

ISSN 0976 - 6499 (Online)

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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 m

3

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 4ºC. 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 121ºC for 20 minutes for the removal

of foreign contaminants.



59

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 ± 3ºC 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):

�

�� µmax x

��

µmax 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.



60

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

Parameter Values 19 ± 2ºC Values 30 ± 2

ºC

µmax (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

1 19 ºc ± 2ºc 87 6.19 92.8 %

2 30 ºc ± 2ºc 84 20.16 76 %



61

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

organic carbon sources. 12:979-985, 2002.

[2] 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.



62

[3] 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.

[4] Xiaoqiang Wang, Eva Nordlander, Eva Thorin, and Jinyue Yan. Microalgal biomethane

production integrated with an existing biogas plant: A case study in Sweden. 2012.

[5] 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.

[6] Ayodhya D Kshirsagar. Bioremedation of waste water by using microalgae: An experimental

study. IJLBPR, 2:339-346, 2013.

[7] 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.

[8] 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.

[9] Seema Dwivedi. Bioremedation of heavy metal by algae: Current and future perspective

biotechnology. Journal of advanced laboratory research in biology, III: 229-233, 2012.

[10] Dwivedi, S.: Bioremedation of heavy metal by algae: Current and future perspective

biotechnology. Journal of advanced laboratory research in biology 3, 229-233 (2012).

[11] 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).

[12] 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.

[13] 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.