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Biomass and Bioenergy 29 (2005) 60–64

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Cultivation of Azolla microphylla biomass onsecondary-treated Delhi municipal effluents

Anju Arora�, Sudhir Saxena

Centre for Conservation of Blue Green Algae, Indian Agricultural Research Institute, New Delhi 110012, India

Received 1 September 2004; received in revised form 4 February 2005; accepted 7 February 2005

Available online 1 April 2005

Abstract

Study was conducted on recycling municipal wastewaters for cultivation of Azolla microphylla biomass, which

is used for inoculation into paddy fields as N biofertiliser and has other applications as green manure, animal

feed and biofilter. Secondary-treated municipal wastewaters were collected from Wazirabad sewage treatment plant in

New Delhi during all four seasons and tested for reactive P and heavy metal content. The reactive P levels in

effluents ranged between 1–2 ppm and levels of heavy metals like Cd, Pb, Ni, Zn, Fe and Mn were well below

permissible limits. A. microphylla was grown in sewage effluents and its dilutions prepared with tapwater. It showed

good growth potential on sewage effluents. Doubling times during September and December months compared well

with those on Espinase and Watanabe (E and W) medium and tapwater. Dried Azolla biomass produced on

sewage waters did not show presence of toxic heavy metals Cd, Cr and Pb. However, levels of P in dried biomass

cultivated on sewage effluents were lower as compared to those from E and W medium and tapwater. The biomass

produced can be used for inoculating paddy fields or for other applications and polished wastewaters can be recycled

for irrigation purposes.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Wastewaters; Recycling; Azolla microphylla; Biomass production; Bioremediation

1. Introduction

The after effects of green revolution have shiftedconcern from increasing productivity to sustain-ability and resource conservation. Due to acute

e front matter r 2005 Elsevier Ltd. All rights reserve

ombioe.2005.02.002

ng author. Tel.: 91 11 25848431;

648.

ss: anjudev@yahoo.com (A. Arora).

scarcity of fresh water resources and ever increas-ing volumes of wastewaters, it has becomeimperative for both developed and developingnations to conserve water and address problems ofwater scarcity and wastewater disposal. The bestway to conserve water is to recycle it. Use oftreated wastewaters for irrigation and othernonresidential purposes is being encouraged.These wastewaters when used for irrigation

d.

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A. Arora, S. Saxena / Biomass and Bioenergy 29 (2005) 60–64 61

increase productivity because they supply manur-ial ingredients also [1]. But depending on theorigin, wastewaters may contain toxic heavymetals, which accumulate in soil and biologicalsystems and prove hazardous. However, waste-waters can be biologically treated to lower the loadof harmful pollutants and safely reused. Biologicaltreatment of wastewaters is a field of intenseinterest for developing and developed countriesand success of these treatment processes dependson the usefulness of the biomass produced/easydisposal [2,3]. Many aquatic plants and algae havebeen used for reclamation of wastewaters [4–6].These include Eichhornia, Lemna, Spirodela, Nas-

turtium, Ipomea, etc. and algae which have beenused for renovation of different types of domesticsewage, dairy and animal wastewaters [7–10].These plants yield protein-rich biomass, whichcan be used to supplement animal feed or used asgreen manure. Many applications for Azolla

biomass produced on wastewaters have beenexplored [11].

Azolla spp., the free floating aquatic fern, hasdistinct advantages as it has high biomassproductivity coupled with high rate of N fix-ation, ability to grow in varied environmentsand multiple applications as biofertiliser, animalfeed, biofilter, bioweedicide, ability to concen-trate nutrients and heavy metals from flood waters[12]. Azolla sp, harboring N fixing symbiontAnabaena azollae, is an established N biofer-tilizer for rice and has been extensively used inAsian countries for N fertilisation of paddycrop and as green manure [13]. The only con-straint in popularising Azolla use is the largeamounts of fresh biomass required for all applica-tions and its maintenance in vegetative form [14].Azolla spp. cultivation per se is simple and cheapas it grows in all aquatic environments from freshto polluted wastewaters ability to concentratenutrients and heavy metals from floodwaters. Itcan grow in nitrogen deficient wastewaters andremoves P even when N is depleted from waste-waters [8]. Present study was conducted toevaluate sewage effluents for cultivation of Azolla

microphylla so that biomass produced can be usedfor different applications and wastewaters re-cycled.

2. Materials and methods

2.1. Collection and characterization of secondary-

treated sewage effluents

The sewage effluents were collected from localsewage treatment plants located at Wazirabadduring all four seasons of the year. Its reactive P,organic N and heavy metal contents were analysedusing APHA methods [15].

2.2. Growth of Azolla microphylla on sewage

waters

A. microphylla was obtained from culture collec-tion at Centre for Conservation and Utilisation ofBlue Green Algae, Indian Agricultural ResearchInstitute, New Delhi. It was vegetatively multipliedin 800ml of Espinase and Watanabe (E and W)medium [16] in plastic trays (25� 20� 5 cm3) toprepare inoculum at 3072 1C in a polyhouse.One gram of fresh fronds were inoculated into

1 l of sewage effluents or suitably diluted sewageeffluents with tapwater in trays and grown underabove-mentioned conditions. On alternate daysthe levels of medium was topped with tapwater tocompensate for evaporation losses. Azolla frondsgrown in tapwater and E and W medium served ascontrols. After 7 days Azolla biomass was har-vested and weighed and dried at 60 1C in an oventill constant weights were recorded.

2.3. Analysis of phosphorus and heavy metal

contents in dry biomass

Known amounts of dried biomass samples weredigested with triacid (nitric: perchloric: sulphuric9:2:1). P in digested samples was assayed by ascorbicacid method as described in APHA handbook [15].The heavy metal content was analysed using AtomicAbsorption Spectrophotometer Model AAS 4141(Electronic Corporation of India).

3. Results and discussion

The characteristics of secondary-treated sewageeffluents collected from Wazirabad plant are listed

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in Table 1. Levels of reactive P, which is theavailable form of P, was 1–2 ppm while toxic heavymetals like Cd, Ni, Cr, Pb were not detected.Concentration of Fe was 0.431–1 ppm, Mn0.3–2 ppm and Zn 0.01–0.06 ppm. All theselevels fall below permissible limits describedby Bureau of Indian Standards for municipaleffluents [17].

A. microphylla was grown on these effluentsduring all four seasons and fresh biomass

Table 1

Characteristics of the sewage effluents collected during different seaso

Season pH Reactive

May 2003 (Summer) 7.3 1

July 2003 (Rainy) 7.4 1

September 2003 (Autumn) 7.4 2

December 2003 (Winter) 7.8 2

Table 2

Growth of A. microphylla on sewage effluents and other media in po

Season Growth medium Initial wt. (g)

May 2003 (Summer) Tapwater 1

E&W 1

SW 1

LSD (0.05)

July 2003 (Rainy) Tapwater 1

E&W 1

SW 1

LSD (0.05)

Sep.2003 (Autumn) Tapwater 1

E&W 1

SW 1

SW (1:1) 1

SW (1:3) 1

LSD(0. 05)

Dec.2003 (Winter) Tapwater 1

E&W 1

SW 1

SW (1:1) 1

SW (1:3) 1

LSD (0.05)

All values are means of three replications.

SW denotes sewage water.

SW 1:1 denotes Sewage water 1part+tapwater 1part.

SW 1:3 denotes Sewage 1part+tapwater 3parts.

harvested (Table 2). During the summer monthsof May and July the biomass production onsewage was much lower (about 50%) as com-pared to E and W medium and tapwater whileduring September and December biomass produc-tion on sewage was higher than on E and Wmedium and tapwater. However, the differencein biomass harvested from E and W mediumand sewage during September and December wasnot statistically significant. The higher biomass

ns

P (ppm) EC (mmho cm�1) Organic N (%)

1.78 1.18

1.74 0.81

2.23 1.26

1.81 1.6

lyhouse at 3072 1C

Final wt. (g) Doubling time (days) P in dry wt. (%)

5.62 2.82 0.54

5.93 2.72 0.54

2.92 4.5 0.16

0.05

3.21 4.16 0.56

3.65 3.76 0.56

2.31 5.83 0.18

0.06

2.92 4.54 0.65

3.30 4.0 0.60

3.58 3.80 0.21

3.65 3.76 0.20

3.35 4.02 0.20

0.05

4.06 3.46 0.58

3.85 3.60 0.57

3.62 3.78 0.18

3.64 3.76 0.18

3.93 3.55 0.20

0.9

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production on sewage in September and Decembermay be due to more available phosphoruspresent during these months than May and July.Thus biomass production on sewage appearedto be affected by level of available P. This strainof A. microphylla produces optimum biomass atavailable P levels from 1 to 20 ppm [18] How-ever, the levels of P incorporated in drybiomass harvested from different sewage mediawere much lower than those from E and Wmedium and tapwater. Toxic heavy metalslike (Cd, Cr and Ni) were not detected indried biomass. This strain of A. microphylla

showed highest biomass production on E andW medium during May as compared to allother seasons. This showed its thermo tolerantnature.In this study A. microphylla strain used, which

has been found to perform best under NorthIndian climatic conditions [19], showed itspotential for growth on secondary-treatedsewage effluents. Earlier studies by Vermat andHanif [5] on comparison of common duck-weed and A. filiculoides for growth in diffe-rent wastewaters and mineral medium showedthat Lemna and Spirodela performed equallywell on both types of growth media while A.

filiculoides yielded less biomass on waste watermedium.This strain of A. microphylla yielded higher

biomass on sewage when 2 ppm P was present.However, overall biomass production in all thefour seasons showed that A. microphylla (speciesalso known for its temperature tolerance) showedshorter doubling times during summer months.Reddy and Debusk [20] while studying P removalby A. caroliniana found that its growth rates wereinfluenced by plant density, temperature, nutrientcomposition and solar radiation. Shiomi andKitoh conducted studies with Azolla spp. fordecontamination of sewage and nutrient absorp-tion [8]. They found that P absorption capacityof Azolla spp. in secondary-treated effluentsdid not differ from that observed in syntheticculture medium and analyses of biomass showedgood crude protein content and amino acidcomposition showed its suitability for use asanimal feed.

4. Conclusion

Azolla microphylla strain can be cultivated onsecondary-treated sewage effluents during all theseasons and biomass produced can be safely used.

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