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Biomass and Bioenergy 24 (2003) 175–178

Comparison of biomass productivity and nitrogen xingpotential of Azolla SPP

Anju Arora∗, P.K. SinghNational Centre for Conservation and Utilization of Blue Green Algae, Indian Agricultural Research Institute,

New Delhi 110012, India

Received 19 March 2001; received in revised form 25 September 2002; accepted 27 September 2002

Abstract

Study was conducted on six di0erent Azolla species, available in the germplasm collection of NCCUBGA, IARI,New Delhi namely A. !liculoides, A. mexicana, A. microphylla, A. pinnata, A. rubra and A. caroliniana in a polyhouseto assess their growth potential by determining their maximal biomass productivity, doubling time and relative growth rates.Their nitrogen xing potential was assessed by acetylene reduction assay. Among them Azolla microphylla gave highestbiomass production and relative growth rate followed by Azolla caroliniana. Both these had high nitrogenase activity also.Peak nitrogenase activity of these strains was found on 14th day of growth and it declined on further incubation. Azolla mi-crophylla and Azolla rubra were more tolerant to salinity than others. On the other hand Azolla pinnata, which is endemicspecies found in India, exhibited low biomass production, relative growth rate and lower nitrogenase activity compared toother species. It was unable to sustain growth in saline medium. Under polyhouse conditions, A. microphylla was found toperform better than other cultures in terms of biomass productivity, N xing ability and salt tolerance. Hence it is taken upfor mass production.? 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Azolla sp.; Biomass productivity; Nitrogenase activity; Salinity tolerance

1. Introduction

The free <oating aquatic fern Azolla is distributedworldwide in tropic and temperate fresh water ecosys-tems. It has symbiotic association with nitrogen xingalga Anabaena azollae. Both the partners are photo-synthetically competent but the fern provides photo-synthates to the alga while the cyanobiont provides forthe N requirement of the association. This associationis outstanding because of its high productivity coupledwith high rate of nitrogen xation. It compares well

∗ Corresponding author. Fax: +91-11-5766420.E-mail address: anjudev@yahoo.com (A. Arora).

with legume—Rhizobium symbiosis in its eEciencyto x atmospheric nitrogen and is rich in N and pro-tein. It can serve as an excellent N biofertilizer for ricecrop due to the fact that paddy elds form an ideal en-vironment for its growth. It can be employed as greenmanure or dual crop to provide N nutrition to rice crop[1,2]. One or more species of Azolla are found natu-rally in almost every geographical area where lowlandrice is grown. Therefore, in most of the earlier stud-ies, endemic species of Azolla have been used [3].Besides N biofertilizer, Azolla has multiple uses likebiological herbicide, animal feed and as water puri- er. Azolla is also known for accumulating nutrientslike P, K and minerals from the water and makes them

0961-9534/03/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved.PII: S0961 -9534(02)00133 -2

176 A. Arora, P.K. Singh / Biomass and Bioenergy 24 (2003) 175–178

available to plants as it decomposes. Research is beingdone to put Azolla to agricultural use under dry landconditions also in nutrient poor soils [4]. Possibility ofusing Azolla as a slow release biofertilizer for essen-tial elements like Fe for nutrition of vegetable cropsunder de cient conditions is also seen. It is by virtueof the ability of dry biomass of Azolla to adsorb metalions [5].Popularization of use of Azolla as biofertilizer has

limitations because the application rate is very high(0.5–1 ton ha−1). Also biomass is highly perishableand cannot be stored. Therefore, all these applicationsof Azolla require its fresh biomass to be produced inbulk amounts. Azolla farming, per se, is inexpensiveand it can be multiplied in natural water bodies forproduction of biomass. Biomass productivity is depen-dent on doubling time and relative growth rate and forits eEciency as N biofertilizer, its high nitrogen xingpotential is highly desirable. Therefore, this study re-ports on the biomass productivity and nitrogen xingpotential of six Azolla species so that rapidly multi-plying species can be taken up for mass productionfor further applications.

2. Materials and methods

2.1. Growth of Azolla spp. and its measurement

The study was conducted on six Azolla speciesnamely A. !liculoides, A. microphylla, A. pinnata,A. rubra, A. mexicana and A. caroliniana that arebeing maintained in germplasm collection of NationalCentre for Conservation and Utilization of Blue GreenAlgae, Indian Agricultural Research Institute, NewDelhi. The in vitro experiment was carried out in apoly house (3–5 klux and 10=14 h light/dark cycle)and temperature was maintained at 30 ± 2◦C. TheAzolla species were grown in 200 ml of N free Es-pinase and Watanabe medium [6] dispensed in 500 mlcalibrated glass beakers. Each beaker was inoculatedwith 0:5 g of fresh Azolla fronds and three replica-tions of each species were maintained. Throughout theincubation period the level of medium in beakers wasmaintained by adding fresh medium. For determin-ing biomass productivity, fresh biomass of Azolla washarvested after 14 days of incubation, blot dried andtheir fresh weight was measured. The relative growth

rate and doubling time were calculated using standardformulae [7].The nitrogen xing potential of Azolla species was

measured by Acetylene Reduction Assay. 0:1 g offresh Azolla fronds were put in 5 ml of medium inCorning glass tube (15 mm dia and 150 mm length)made airtight with Subaseal stoppers and incubated at2–3 klux at 30± 2◦C for 1 h under 10% acetylene inair atmosphere. The ethylene formed was measuredby injecting 1 ml sample drawn from these tubes intoNucon gas chromatographwith 2 m long stainless steelcolumn packed with Porapak N/R (80–100 mesh).Nitrogen was used as carrier and hydrogen and airmixture (<ow rate 35 ml=min) was used for pro-ducing <ame for detection of samples. Nitrogenaseactivity was expressed as nmol g−1 (fresh wt.) h−1.

2.2. Nitrogenase activity pro!les

In a separate experiment, the six Azolla spp. weregrown in E & W medium as described above andsmall amount of fronds of each species were harvestedon 3rd, 7th, 10th, 14th, 17th and 21st day, blot driedand 0:1 g sample of each was taken for estimatingnitrogenase activity by above described procedure.

2.3. Salinity tolerance

Salinity tolerance of the Azolla species was stud-ied by growing them in salinized growth medium byadding 0.32% NaCl. Incubation was carried out inpolyhouse for 28 days and growth was compared withthat in normal medium.

3. Results and discussion

Fresh biomass of Azolla spp was harvested andweighed after 14 days (Table 1). A. microphyllaproduced signi cantly more biomass than all otherspecies studied. It showed lowest doubling time of5.4 days under polyhouse conditions. The biomassproductivity and doubling times of A. !liculoides, A.rubra, A. mexicana and A. caroliniana did not dif-fer signi cantly under polyhouse conditions where asA. pinnata showed poor biomass productivity andlonger doubling time. All the species tested showedgood nitrogenase activity. A. caroliniana showed the

A. Arora, P.K. Singh / Biomass and Bioenergy 24 (2003) 175–178 177

Table 1Biomass, doubling time, relative growth rate (RGR) and nitrogenase activity of Azolla spp. after 14 days growth in polyhouse at 30±2◦C

Azolla sps. Biomass (g) (fresh wt.) Doubling time (days) RGR (g−1 d−1) Nitrogenase nmol (g−1 h−1)

A. !liculoides 4.65 6.3 0.11 240A. microphylla 5.86 5.4 0.13 336A. pinnata 2.41 11.1 0.06 232A. rubra 4.93 6.1 0.11 219A. mexicana 4.29 6.6 0.10 327A. caroliniana 4.89 6.1 0.11 445S:Em±a 0.43 — — —CD (0.05) 1.19 — — —

aS.Em denotes standard error while CD is critical di0erence.

Table 2Biomass and doubling time of Azolla sps. grown in saline medium (0.32% NaCl) after 28 days growth in polyhouse at 30± 2◦C

Azolla sps. Biomass (g) (fresh wt.) Doubling time (days)

Control Saline Control Saline

A. !liculoides 9.8 2.2 (22.4) 8.9 90.6A. microphylla 11.1 4.8 (43.4) 8.3 14.6A. pinnata 4.78 0.4a 11.8 —A. rubra 11.1 3.3 (30) 8.3 23.3A. mexicana 10.9 2.6 (23.8) 8.5 38.8A. caroliniana 10.3 2.7 (26.2) 8.7 36.9S:Em:±b 0.6CD (0.05) 1.4

Values in parentheses are percent of control biomass.aThe fresh weight is lesser than the inoculum.bS.E denotes standard error and CD is critical di0erence.

highest activity (445 n mol g fresh biomass in 1 h fol-lowed byA. microphylla (336 nmol g−1) andA. mex-icana (327 nmol g−1). Gopalaswamy and Kannaiyanalso reported doubling time of 5.8 days at 14 daysincubation in IRRI medium for A. microphylla [8](Table 2).Nitrogenase activity pro les along incubation pe-

riod were developed for A. microphylla, A. carolini-ana and A. pinnata. As evident from Fig. 1, the nitro-genase activity increased with incubation period andpeaked at 14th day. It started decreasing graduallyon further incubation. It almost diminished after 21days of incubation in all the species. This is in agree-ment with earlier observations by Manna and Singh[9,10], where A. caroliniana showed higher nitroge-nase activity than A. pinnata and both showed peak Fig. 1. Nitrogenase pro les of Azolla spp.

178 A. Arora, P.K. Singh / Biomass and Bioenergy 24 (2003) 175–178

nitrogenase activity on 14th day of growth under eldconditions. Kannaiyan [11] observed that di0erentspecies of Azolla showed higher nitrogenase activityon 14th day than on 27th day under di0erent growthconditions like high temperature, salinity.Salinity considerably decreased the biomass pro-

ductivity in all the species. A. microphylla showedhighest tolerance to salinity producing 43% of the con-trol biomass in 28 days of incubation under salineconditions followed by A. rubra. A. pinnata was notable to tolerate salinity and decomposed on long in-cubation. A. microphylla was found to be more tol-erant to salinity than other species like A. pinnata,A. !liculoides and A. caroliniana by Majumdar et al.[12]. Kannaiyan observed decreased relative growthrates of di0erent Azolla cultures in presence of 0.32%NaCl but Azolla pinnata BR-M, was found to be in-hibited to a lesser extent than Azolla microphylla andA. !liculoides [10].

4. Conclusion

Thus, in this poly house study A. microphylla hasbeen seen to perform better than other species in its ca-pacity to produce biomass and x free nitrogen. Thisspecies has been taken up for outdoor mass multipli-cation for further application as inoculum in paddy elds in northern region of India.

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