96_nov3

Mamta Awasthi and Rajiv Kumar Singh, 2011

INT J CURR SCI 2011, 1: 14-23

REVIEW ARTICLE

Development of algae for the production of bioethanol, biomethane, biohydrogen and biodiesel

Mamta Awasthia* and Rajiv Kumar Singhb

aCentre for Energy and Environment, National Institute of Technology, Hamirpur

Himachal Pradesh-177 005, India

bDepartment of Botany, Rajiv Gandhi University, Itanagar, Arunachal Pradesh-791 112, India

*Corresponding author: E-mail: [email protected]

Abstract

Microalgal biofuels are a viable alternative for clean, economical and sustainable sources of energy. The research and development of

micro-algae needs a massive boost to ease the technical difficulties to overcome the large cost advantage of other biofuel feed stocks. It is a

large source of biomass on non-arable lands and capture of CO2. Lipids produced from algae contain saturated and polar lipids, which are

suitable for use as a fuel feedstock and it exceeds the best producing oil crops. It has been found that representatives of green and diatom

microalgae are the most promising producers of TAGs, the highest content of which (4060%) has been found in diatom microalgae. This

paper discusses current knowledge on different techniques for microalgal biomass production, harvesting and lipid extraction methods. The

paper also discusses biodiesel production via transesterification of the lipids and other biofuels like biomethane, biodiesel, bioethanol and

biohydrogen depending on the species as well as the biorefinery approach. Besides lipid extraction, the byproducts of processed algae can

be utilized for many other purposes.

Key words: biofuel, bioethanol, biodiesel, bioenergy, microalgae cultivation, harvesting, lipids

Received: 02nd September; Revised: 25th September; Accepted: 19th October; IJCS New Liberty Group 2011

Introduction

The concept of using micro algae for energy production is not

new. Algae require much less water than the traditional cereals,

produce more biomass (Table 1) as well as it can be grown in

salt water or in sewage water. Microalgae do not belong to the

category of traditional raw food materials. One of the possible

ways in which the cost of biofuel obtained from microalgae can

be reduced is by adopting biorefinery approach and using wastes

from other production processes for cultivating them. With the

latest technologies it has become possible to cultivate the

biomass of algae on a large scale all year round, not only under

the conditions of tropical and subtropical climate, but also in

zones with moderate climate, even at negative temperatures of

outdoor air in winter. There is potential to effectively reduce the

amount of carbon dioxide and nitrogen oxides released into the

atmosphere from many stationary emitters by feeding the

carbon-rich flue gas to the algae (Ackman et al., 1968; Adey and

Loveland, 2007). Algae are able to fix approximately 1.8 kg of

CO2 for every 1 kg of algae biomass produced (Antoni et al.,

2007). Approximately 40 ha of algae ponds are required to fix

the carbon emitted from one MW of power generated from a

coal plant (Becker, 1994). Treated wastewater rich in nitrogen

and phosphorus can be utilized by algae, thereby providing the

co-benefit of producing biofuels and removing nitrogen and

phosphorus (Bilanovic et al., 1988; Benemann and Oswald,

1996; Bigogno et al., 2002).

Algae cultivation

The necessary technology for developing profitable algae-based

fuel is still in various states of development and the final

configuration is yet to be determined and demonstrated at the

industrial scale. The high capital cost associated with


producing microalgae in closed culture systems is the main

challenge for commercialization of such systems.

Table 1. Biomass yields of microalgae versus conventional

crops. Source: Richmond (1986)

Two methods of cultivation are used for realizing the

biosynthesis abilities of natural and modified strains of

autotrophic microalgae: in photobioreactors (PBRs) and in open

cultivators. Cultivation can be conducted in batch, semi-batch,

and continuous systems. In a continuous system, two types can

be used: turbidostat and chemostat culture. Algae can be

produced in closely-controlled laboratory methods to less

predictable methods in outdoor tanks.

Open pond systems

Open pond systems are shallow ponds in which algae

are cultivated. Nutrients can be provided through runoff water

from nearby land areas or by channelling the water from

sewage/water treatment plants. The water is typically kept in

motion by paddle wheels or rotating structures, and some mixing

can be accomplished by appropriately designed guides. Algal

cultures can be defined (one or more selected strains), or are

made up of an undefined mixture of strains (Borowitzka, 1988;

Chaumont, 1993; Boichenko and Hoffmann, 1994).

Indoor culture / Closed culture

Vessels such as tubes, flasks, carboys, bags, etc. or

ponds covered with green house or usually a photobioreactor

which allows control over illumination, temperature, nutrient

level, contamination with predators and other competing algae

can be used. Researchers also took the path of creating

heterotrophic strains of algae from obligate photoautotrophs due

to inadequate illuminance. Heterotrophic cultivation of micro

algae for lipids production does not involve CO2 mitigation and

wastewater treatment programme along with production of algal

biofuel.

A photo bioreactor is equipment that is used to harvest

algae. Photo bioreactors can be set up to be continually

harvested (the majority of the larger cultivation systems), or by

harvesting a batch at a time (like polyethylene bag cultivation).

Some photo bioreactors types include: tubular photo bioreactors,

flat-plated photo bioreactors, an inclined triangular tubular photo

bioreactor, rectangular tanks, continuous stirred tank reactors

(CSTR), helical coils made of plastic tubing placed across a

column-like structure, square tubular reactors consist of plastic

tubing arranged in a series of squares.

Algal Turf Scrubber (ATS)

In addition to the open ponds or photo bioreactors, an

attached algal culture system such as an algal turf scrubber

(ATS) can be used in which benthic algae grow on the surface of

solid support for removing nutrients from animal wastewater

(Bridgwater et al., 1999; Bosma et al., 2003).

Harvesting Algae

Conventional processes used to harvest micro-algae

include concentration through centrifugation (Briens et al.,

2008), foam fractionation (Brown et al., 1997), flocculation

(Chaumont 1993; Canakci and Van Gerpen, 2001), membrane

filtration (Chisti, 2007) and ultrasonic separation (Chynoweth

et al., 1993). However, most harvesting methods still involve

economic or technical drawbacks, such as a high energy cost,

flocculant toxicity, or non-feasibility of scaling-up. The

harvesting of algal cells by flocculation is more convenient than

centrifugation or gravity filtration, because it allows large

quantities of culture to be treated. A variety of chemicals have

been tested as flocculants and the most effective was found to be

aluminum sulfate followed by certain cationic polyelectrolyte

(Conover, 1975). The flocculating reactions of an algal biomass

are particularly sensitive to the pH, properties of the cellular

surface, concentrations of the flocculants and divalent cations,

ionic strength of the culture solution and other factors (Craggs

et al., 1996; Csordas and Wang, 2004). High-density algal

cultures can be concentrated by either chemical flocculation or

centrifugation.

Crops Annual yield (tons/ha)

Sugar cane 54-125

Sweet sorghum 35-70

Soybean 1.1-4.0

Sweet potato 10-40

Trees 20-50

Microalgae 800-1600


Froth flotation is a method of separating algae from

the medium by adjusting pH and bubbling air through a column

to create a froth of algae that accumulates above liquid level.

Ultrasound based methods of algae harvesting are currently

under development, and other, additional methods are currently

being developed. Dissolved Air Flotation (DAF) separates algae

from its culture using features of both froth flotation and

flocculation. It uses alum to flocculate an algae/air mixture, with

fine bubbles supplied by an air compressor. Alum is a common

name for several trivalent sulfates of metal such as aluminum,

chromium, or iron and a univalent metal such as potassium or

sodium, for example AlK(SO4)2.

Algal drying

Dewatering/drying process reduces the water content

of the algae prior to oil extraction process. The algae paste

obtained from filtration/centrifugation contains as much as ca.

90% water content. Drying algae to ca. 50% water content is

necessary to produce a solid material that can be easily handled.

Solar drying, a popular and inexpensive method, is used

commercially in grains and timber drying. However, it requires

a considerable area of land. A more efficient method would

make use of the low grade waste heat from the power plant to

dry the algae contained in a vessel. The biomass harvested from

the attached culture system is paste-like pulpy slurry having a

water content to that of the cell pellet centrifuged from the

suspension culture system. This implies a great advantage of the

attached algal culture system in terms of ease of biomass

harvesting (Das and Veziroglu, 2001).

Fig. 1. Energy production pathway

Energy production from algae

Algal lipids (so-called pseudo vegetable oil or PVO)

are much more heterogeneous than the seed-oil lipid. Therefore

makes it difficult to characterize algae-derived fuel products and

treated analogous to vegetable oil. Energy production pathway

can be expressed diagrammatically as shown in figure 1.

Microalgae as biological sources of lipids and hydrocarbons

Algae are far more oil-rich and offer a higher yield of

oil per unit of land in a year compared to terrestrial crops (Table

2). Lipids are one of the main components of micro algae (2-

60% of total cell dry matter) depending on the species and

growth conditions (Dote et al., 1994). Microalgae contain lipids

and fatty acids as membrane components, storage products,

metabolites and sources of energy. Microalgal strains with high

oil or lipid content are of great interest in the search for a

sustainable feedstock for biodiesel. A few micro algal species,

including some Chlorella species (FAO, 1997; Fukuda et al.,

2001) Dunaliella species (Gaffron and Rubin, 1942; Gerpen,

2005) Nannochloris sp. (Gordillo et al., 1998; Ghirardi et al.,

2000), Parietochloris incisa (Haesman et al., 2000) and

Botryococcus braunii (Harris, 1989; Johnson and Wen, 2010),

have been reported to have the capacity of accumulating large

quantities of lipids in cells under favorable conditions. Lipid

content also varies with season as tested in Chlorella vulgaris,

Euglena gracilis and Chlamydomonas sp. (figure 2 Unpublished

data).

Table 2. Oil output of different biofuel feed stocks Source:

Khan et al. (2009)

Crop Oil yield (gal/acre-yr)

Corn 18

Cotton 35

Soybean 48

Canola 127

Jatropha 202

Oil palm 635

Microalgae (15%

oil)

1,200

Microalgae (50%

oil)

10,000

Lipids can be used as a liquid fuel in adapted engines

as Straight Vegetable Oil (SVO). In comparison with SVO, algal

oil is unsaturated to a larger degree making it less appropriate

for direct combustion in sensitive engines (Table 3), however,


some members of these algal group like Chlorella is known for

containing shorter-chain fatty acids (16 to 18 carbon length)

which are ideally suited for biodiesel production (Kadam, 2002;

Khan et al., 2009). Neochloris oleoabundans is a freshwater

species that produces up to 80% triglycerides of its total lipids,

and most of its fatty acids are saturated fatty acid in the range of

16-20 carbons (Gordillo et al., 1998), ideal for biodiesel

production. However the triglycerides produced by P.incisa is

rich of polyunsaturated fatty acid (Haesman et al., 2000),

making them less desirable for biodiesel production.

Table 3. Fatty acid composition of microalgal oil [Source: Meng

et al. (2009)]

Fatty acid

Chain

length:no.

of double

bonds

Oil

composition

(w/total lipid)

Palmitic acid 16:0 121

Palmitoleic acid 16:1 557

Stearic acid 18:0 12

Oleic acid 18:1 5860

Linoleic acid 18:2 420

Linolenic acid 18:3 1430

Factors affecting oil production in algae

Taxonomic groupings, stage of growth, and

environmental factors (e.g., seasonal changes such as

temperature and light intensity, and media composition) affect

the quantity and types of lipids as well as fatty acids

compositions (Klass, 1998; Lee et al., 1998; Knuckey et al.,

2006) as shown in figure 2. The effects of growth conditions,

growth stage of algal cultures and the taxonomic position of

algae, on both lipid content and lipid type, are discussed

(Piorreck and Pohl, 1984; Li et al., 2008). Lipid accumulation in

algae typically occurs during periods of environmental stress,

including growth under nutrient-deficient conditions. The

average lipid content of algal cells varies between 1% and 70 %

but can reach 90% of dry weight under certain conditions

(Livansky, 2005; Liu et al., 2008). Under unfavorable conditions

of growth, Botryococcus enters a stage in which its

unsaponifiable lipid content increases to a level of 90%.

Fig. 2. Changes in lipid content in different algae

Time Period

April May June July August September

Lip

id C

ont

en

t (%

of

DW

)

8

10

12

14

16

18

20Chlorella vulgaris

Chlamydomonas sp

Euglena gracilis

The total amount and relative proportion of fatty acids

can be affected by nutritional and environmental factors e.g.

nitrogen limitation as in case of Neochloris oleoabundans

(Maeda et al., 1995). The effect of nitrogen on the lipid fraction

and on cell growth of the strain Nannochloris cultured under

saline conditions is summarized in Table 4. At the lower

temperature ranges, an increase in fatty acid unsaturation has

been observed. Light enhances the formation of polyunsaturated

C16 and C18 fatty acids as well as mono- and di-galactosyl-

diglycerides, sphingolipids and phosphoglycerides in Euglena

gracilis and Chlorella vulgaris (Melis et al., 2000).

Figure 3. Energy conversion processes from microalgae

Extraction of algal oil

While more efficient processes are emerging as shown in figure

3, a simple process is to use a press to extract a large percentage

(70-75%) of the oils out of algae. The remaining pulp can be

mixed with cyclo-hexane to extract the remaining oil content.

Chemical methods: Algal oil can also be extracted using

chemicals. Benzene and ether have been used, oil can also be


separated by hexane extraction, which is widely used in the food

industry and is relatively inexpensive.

Three chemical methods are hexane solvent method, soxhlet

extraction and supercritical fluid extraction method.

Hexane solvent method: Hexane solvent extraction can be used

in isolation or it can be used along with the oil press/expeller

method. After the oil has been extracted using an expeller, the

remaining pulp can be mixed with cyclo-hexane to extract the

remaining oil content. The oil dissolves in the cyclohexane, and

the pulp is filtered out from the solution. The oil and

cyclohexane are separated by distillation. These two stages (cold

press and hexane solvent) together may derive more than 95% of

the total oil present in the algae.

Table 4. Effect of nitrogen concentration (as KNO3) on lipid

production [Source: FAO (1997)]

Soxhlet extraction: Soxhlet extraction is an extraction method

that uses chemical solvents. Oils from the algae are extracted

through repeated washing, or percolation, with an organic

solvent such as hexane or petroleum ether, under reflux in

special glassware.

Supercritical fluid extraction: In supercritical fluid / CO2

extraction, CO2 is liquefied under pressure and heated to the

point that it has the properties of both a liquid and a gas, this

liquified fluid then acts as the solvent in extracting the oil.

Other lesser-known extraction methods

Enzymatic extraction - Enzymatic extraction uses enzymes to

degrade the cell walls with water acting as the solvent, this

makes fractionation of the oil much easier. However, the cost of

this extraction process is estimated to be much greater than

hexane extraction.

Osmotic shock - Osmotic Shock is a sudden reduction in

osmotic pressure, causing pressure in a solution to rupture.

Osmotic shock is sometimes used to release cellular

components, such as oil.

Ultrasonic-assisted extraction - Ultrasonic extraction can greatly

accelerate extraction processes. Using an ultrasonic reactor,

ultrasonic waves are used to create bubbles in a solvent material,

when these bubbles collapse near the cell walls, it creates shock

waves and liquid jets that cause those cells walls to break and

release their contents into the solvent. The need to subject

biomass to filtration and chemical extraction of oil from it can

be avoided using the method which consists of treating algal

suspension in an alternating electromagnetic field with varying

the pH value by adding CO2 resulting in the cell wall destruction

of algae thereby causing the floating up of oil. The method

developed on the basis of acid catalysis allows the processes of

extracting lipids from microalgae and obtaining biodiesel fuel to

be combined in a single stage (Meng et al., 2009). The culture

can be kept alive during the extraction of lipids from it by using

mesoporous nanoparticles that extract oils from live cells of

algae.

Biodiesel from algal oil

Raw microalgal oil is high in viscosity, thus requiring

conversion to lower molecular weight constituents in the form of

fatty acid alkyl esters. Transesterfication is the process of

converting raw microalgal lipid (triacyleglycerols / free fatty

acids) to give renewable, non-toxic and biodegradable biodiesel.

Transesterification is a reaction of the parent oil with a short

chain alcohol, usually methanol, in the presence of a catalyst.

Products of the reaction are fatty acid methyl esters (FAME) and

glycerol. The use of acid catalyst has found to be useful but the

reaction rates for converting triglycerides to methyl esters are

too slow (Meng et al., 2009). Acid catalysis is suitable for

transesterification of oils containing high levels of free fatty

acids (Metzger and Largeau, 2005). Alkali-catalyzed

transesterification is about 4000 times faster than the acid

catalyzed reaction and hence most frequently used commercially

(Miao and Wu, 2006).

Biochemical conversion

Biomethane production: The production of biogas from biomass

is gaining increasing importance worldwide. An anaerobic

digester contains synergistic microbial populations to convert a

variety of organic substrates to methane and carbon dioxide.

Thus algal organic compounds (lipid, protein, or carbohydrate)

KNO3 con.

(mM)

Cell growth

(g/L)

Internal lipid

content (g/L)

0.9 0.39 42.4

9.9 2.5 32.9

9.9 + feeding 2.6 33.6
http://www.oilgae.com/ref/glos/osmotic_shock.htmlhttp://www.oilgae.com/ref/glos/ultrasonic_assisted_extraction.html


can be converted to methane. Methane is widely used as both a

fuel and a chemical feedstock however under normal conditions

it is a gas and therefore, bulky to handle, its use as a

transportation fuel is limited. Chynoweth et al. (1993) compared

the technical potential of different biomass sources (marine

algae, wood and grass species, municipal solid waste) to be used

in energy farms, and concluded that marine biomass offered the

highest potential for biomethanation. The growth rates of marine

macroalgae exceed those of land plants; however, growth is

often limited by the availability of nutrients. Conversion of

methane to methanol through photochemical conversion is

possible.

Table 5. Carbohydrate and Lipid content of some commonly

found algae

Organism

Carbo

hydrat

e (%

of

DW)

Lipid (% of DW)

Min. Mean

SD

Ma

x.

Mi

n.

Mean

SD

M

ax.

S.

quadricauda 20.2

27.76

5.79

34.

5

4.2

5

6.85

2.12

9.6

9

Pleurotonium

sp. 25.1

28.02

2.09

30.

2

4.9

2

8.29

2.82

11.

23

Anabaena sp. 22.9 25.56

3.03

30.

1

8.6

8

11.33

2.46

14.

82

Ethanol production: Algae are the optimal source for bioethanol

due to high content of carbohydrates/polysaccharides (Table 5)

and thin cellulose walls. Generally two methods are employed

for the production of bioethanol from microalgal biomass,

namely fermentation (biochemical process) and gasification

(thermo-chemical process) (Minowa and Sawayama, 1999).

Fermentation is used commercially on a large scale in various

countries to produce ethanol from sugar crops and starch crops.

The biomass is grounded, and the starch is converted by

enzymes to sugar. The sugar is converted to ethanol by yeast.

The purification process of ethanol by distillation is an energy

intensive step (Minowa et al., 1995). The starch of microalgae is

released from the cells with the aid of mechanical equipment or

an enzyme and yeast, Saccharomycess cerevisiae is added when

the biomass starts degrading to initiate fermentation. The

product of fermentation i.e ethanol is drained from the tank and

pumped to a holding tank to be fed to a distillation unit. Ethanol

production by dark fermentation in the marine green alga

Chlorococcum littorale was also investigated (Ueno et al.,

1998). Under dark anaerobic conditions, 27% of the cellular

starch was consumed within 24 h at 250C, the cellular starch

decomposition being accelerated at higher temperature. Ethanol,

acetate, hydrogen and carbon dioxide were obtained as

fermentation products. Some prominent strains of algae that

have a high carbohydrate content and hence, promising

candidates for ethanol production are Sargassum, Gracilaria,

Prymnesium parvum, Euglena gracilis (Piorreck and Pohl,

1984).

Thermochemical conversion

Thermal decomposition of algal biomass can yield different

types of energy fuels depending on temperature used for

conversion. Pyrolysis is a phenomenon related to decomposition

of biomass under the condition of oxygen deficiency and high

temperature. Pyrolysis of biomass produces charcoal,

condensable organic liquids, acetic acid, acetone, methanol and

non-condensable gaseous products by a simple, effective,

wasteless and pollution free process. This technology may be

more suitable for microalgae because of the lower temperature

required for pyrolysis and the higher-quality of oils obtained

(Olson and Ingram, 1975). Compared to lignocellulose,

microalgae contain high content of cellular lipids, resolvable

polysaccharides and proteins, which are easier to be pyrolyzed

to bio-oils and bio-gases.

Gasification is a term that describes a chemical process by

which carbonaceous materials (hydrocarbon) are converted to a

synthesis gas (syngas) by means of partial oxidation with air,

oxygen and/or steam at high temperatures, typically in the range

800-9000C. Syngas can be burned directly or used as fuel for

diesel or gas turbine engines. A novel energy production system

using microalgae with nitrogen cycling combined with low

temperature catalytic gasification of the microalgae has been

proposed (Sebnem Aslan, 2006). Other conversions are also

possible, most notably a thermochernical conversion to produce

higher alcohols, which have octane-enhancing properties of


ethanol and methanol but pose fewer water solubility and phase

separation problems.

Thermochemical liquefaction

High content of water often exists in micro algae after

harvesting which requires a great deal of energy to remove

moisture from algal cells during pretreatment. Liquefaction has

been developed to produce bio-fuel directly without the need of

drying micro algae (Singh and Gu, 2010). Moreover, wet

microalgae can provide hydrogen for hydrogenolysis. It was

reported that Dunaliella tortiolecta cells with 78.4% water

content converts to oils directly. The yield of oils reached 37%

of the total organic matters (Singh and Gu, 2010). Dote et al.

(1994) reported that B. braunii produced liquid oils at 57-64%

of dry weight under the conditions of a N2 pressure of 10 MPa at

300C in warm water and catalyzed by NaCO3. Liquefaction of

algae uses moderate temperature and pressure and, most

interesting, wet material can be used in the process.

Thermochemical liquefaction requires temperature around 350

and 395C in order to have the optimal amount of extracted oil.

Nevertheless, its composition depends on the working

temperature. Among the technologies used, the thermochemical

liquefaction seems to be more efficient than the extraction.

Hydrogenation

Hydrogenation is a reductive chemical reaction that

results addition of hydrogen (H2), usually to saturate organic

compounds. The process consists of the addition of hydrogen

atoms to the double bonds of a molecule through the use of a

catalyst (Gaffron and Rubin, 1942). Algal hydrogenation is

performed by using an autoclave under high temperature and

pressure conditions in the presence of a catalyst and a solvent.

Algal hydrogenation is a three-phase operation in which contact

must be established between the gaseous phase (hydrogen and

hydrocarbon phase), liquid phase (mixture of solvent and liquid

product), and solid particle phase (algal and catalyst) in order to

achieve algal conversion and to promote the transfer of

momentum, heat and mass.

Algal biohydrogen production

Over the years microalgae for photo-biological

hydrogen production from water are being developed into a

promising and a potentially emission-free fuel stream for the

future, which could also be coupled to atmospheric CO2-

sequestration. Bio-hydrogen production from micro algae has

been known for more than 65 years and was first observed in the

green alga Scenedesmus obliquus and in many other

photosynthetic species (Boichenko and Hoffmann, 1994). Most

studies on algal hydrogen production have been performed using

the green alga Chlamydomonas reinhardtii, a model organism

for photosynthesis research via an aerobic-anaerobic cycle

developed by Melis and coworkers (2000). Earlier, algae biofuel

projects were focused at obtaining biodiesel fuel, but at present,

owing to innovative technologies, producers are becoming

interested in the possibility of obtaining other kinds of fuel from

algae that are close in composition to fuel products obtained by

petroleum distillation e.g. aviation fuel, which is obtained by

subjecting algae oil to hydroprocessing.

Integrated biodiesel production from microalgae (IBPCS)

IBPCS enables the overall system to be operated at a

profit and the design requires the combination and optimization

of several factors such as biomass culture, growth management,

transport to conversion plants, drying, product separation,

recycling, waste (liquid and solid) management, transport of

saleable products and marketing. These factors can be simplified

and reduced to three main groups; culturing of microalgae,

harvesting and processing of biomass. In the idealized case, the

conversion plants are located in or near the biomass growth

areas to minimize the cost of transporting biomass to the plants,

of which all the non-fuel effluents are recycled to the growth

areas (Pushparaj et al., 1993). Integration approaches for

sustainable micro algal biodiesel production use hydrothermal

technology for direct liquefaction of algal biomass.

Bio-refinery approach and other biofuels

The economics of biodiesel production can be significantly

improved by using the bio-refinery based production strategy

where all the components of the biomass raw material are used

to produce useful products (Antoni et al., 2007). Furthermore, it

is recommended that a bio-refinery approach is the best solution

to combine and integrate various processes to maximize

economic and environmental benefits, while minimizing waste

and pollution (Pushparaj et al., 1993).


Acknowledgement

The first and second authors thank their respective organization

viz. Centre of Energy and Environment, National Institute of

Technology, Hamirpur, Himachal Pradesh, India and

Department of Botany, Rajiv Gandhi University, Itanagar, India

for support.

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