review of literature -...

REVIEW OF LITERATURE

2.8 A brief review on Opuntia and Other Cacti: Applications and Biotechnological Insights

A part of this section has been published as:


Phytoremediation approach for removal of environmental pollutants 7

2. REVIEW OF LITERATURE

Based on a study by N. G. Kimani at Dandora municipal dumping site in Nairobi,

Kenya, a report summary has been published (United Nations Environment Programme,

UNEP). As stated in the report summary, over the last three decades there has been an

increasing global concern over the public health impacts attributed to environmental

pollution. The World Health Organization (WHO) has estimated that about a quarter of the

diseases facing humanity today occur due to the prolonged exposure to environmental

pollution. Most of these environment-related diseases are however not easily detected and

may be acquired during childhood and manifested later in the adulthood.

Improper management of solid and liquid waste is one of the main causes of

environmental pollution and degradation in many cities and industrial sectors across the

globe, especially in developing countries. A glance at waste-generation data (OECD

Factbook, 2009) revealed that compared to developed countries, developing countries

generate much less waste. Many of the developing countries lack waste regulations and

proper disposal facilities, including the harmful waste that may be infectious or toxic.

Flow Chart 2.1: Public Health Effects of Environmental Pollution

WASTE DUMPING SITE•Industrial Waste e.g., falloff or unused chemicalsand raw materials, expired products andsubstandard goods•Agricultural Waste e.g., pesticides (herbicides andfungicides)•Hospital Waste e.g., packaging materials andcontainers, used syringes and sharps, biologicalwaste and pharmaceuticals•Textile industrial waste e.g. dyes and effluents•Paint industrial waste e.g. residues of paints andpaint preservatives•Metal electroplating , mining industries, leatherprocessing

ENVIRONMENTAL POLLUTANTS•Heavy Metals e.g., lead, mercury, cadmium, arsenic,chromium, zinc, nickel and copper•Persistent Organic Pollutants e.g., aldrin, dieldrin,dichlorodiphenyl-trichloroethane (DDT), endrin,heptachlor, toxaphene, chlordane, hexachlorobenzene,mirex (organochlorines, organophosphates, carbamates)and polychlorinated biphenyls (PCBs)•Synthetic dyes e.g. Red7B, Malachite green, Green4BD, Navy blue2R, Orange M2R, Methyl Orange,Golden yellow•Paint formulations, paint preservatives e.g. Troysan S89(carbendazim, diuron, octhilinone)

ROUTES OF EXPOSUREThese toxicants can be found in air, water and soiland could find their way into the human bodythrough:•Inhalation – movement of air from the externalenvironment through the airways during breathing•Ingestion – the consumption of a substance by anorganism either man or animals•Absorption – the movement and uptake ofsubstances into cells or across tissues such as skinby way of diffusion or osmosis

PUBLIC HEALTH EFFECTSSkin Disorders – Fungal infection, allergic dermatitis, pruritisand skin cancerRespiratory Abnormalities – bacterial upper respiratory tractinfections (pharyngitis, laryngitis and rhinitis), chronicbronchitis and asthmaAbdominal and Intestinal Problems – bacterial enteritis,helminthiasis, amoebiasis, liver cancer, kidney and renal failureDental Disorders – dental carries and dental painEar Infections – otitis media and bacterial infectionsSkeletal Muscular Systems – back painCentral Nervous System – impairment of neurologicaldevelopment, peripheral nerve damage and headachesEye Infections – allergic conjunctivitis, bacterial eye infectionsBlood Disorders – Iron deficiency anaemiaOthers – malaria, chicken pox, septic wounds and congenitalabnormalities, cardiovascular diseases and lung cancer

Part of the figure has been adapted from UNEP.



Municipal waste dumping sites and water reservoirs are designated places where

the waste disposal is stored. Depending on a regional level of waste management, such

waste may be dumped in an uncontrolled manner, segregated for recycling purposes, or

simply burnt. Poor waste management poses a great challenge to the well-being of city

residents, particularly those living adjacent to the dumpsites due to the potential of the

waste to pollute water, food sources, land, air and vegetation. The poor disposal and

handling of waste thus leads to environmental degradation, destruction of the ecosystem

and poses great risks to public health (Flow Chart 2.1).

2.1 Health Effects of Pollution on Human

An overview of main health effects on human from some common types of

pollution has been recorded (Figure 2.1) (Kallman, 2009; Meinhardt 2009 and Lorenz,

2007). Adverse air quality can kill many organisms including humans. Ozone pollution can

cause respiratory disease, cardiovascular disease, throat inflammation, chest pain, and

congestion. Water pollution causes approximately 14,000 deaths per day, mostly due to the

contamination of drinking water by untreated sewage in the developing countries. An

estimated 700 million Indians have no access to a proper toilet, and 1,000 Indian children

die of diarrhoeal sickness every day. Nearly 500 million Chinese lack access to safe

drinking water (The Economist, 2008). 656,000 people die prematurely each year in China

because of air pollution (The New York Times, 2007). In India, air pollution is believed to

cause 527,700 fatalities a year. Studies have estimated that the number of people killed

annually in the US could be over 50,000 (National Geographic News, 2007).

Oil spills can cause skin irritations and rashes. Noise pollution induces hearing loss,

high blood pressure, stress, and sleep disturbance. Mercury has been linked to

developmental deficits in children and neurologic symptoms. Older people are majorly

exposed to diseases by air pollution. Those with heart or lung disorders are under

additional risk. Children and infants are also at serious risk. Lead and other heavy metals

have been shown to cause neurological problems. Chemical and radioactive substances can

cause cancer and as well as birth defects.



Figure 2.1: Overview of main health effects on humans from some types of pollution.

Source: http://www.immukare.com/image/data/logo/351px-Health_effects_of_pollution.png

The broad spectrum of pollutants included textile dyes, wall paints and heavy

metals (hexavalant chromium) as the three major pollutants and their health effects are

discussed below.

2.1.1 Health effects of dyes:

According to European Commission (Atkins, 2000), some of the reactive dyes are

recognized respiratory sensitizers. Breathing in respiratory sensitizers can cause

occupational asthma. Once a person is sensitized, re-exposure to even very small amounts

of the same dye may result in allergic symptoms such as a runny or stuffy nose, watery or

prickly eyes, wheezing, chest tightness and breathlessness. Some dyes can cause similar

allergic skin reactions. Certain reactive, vat and disperse dyes are recognized skin

sensitizers. A small number of dyes, based on the chemical benzidine, are thought to

possibly cause cancer. Substitutes exist for these dyes in textile use (Health and Safety

Executive information sheet). Wastewaters and lands from an industrial area in India have

been studied to assess the possible genotoxic health risk and environmental genotoxicity

due to textile industry effluents (Mathur et al., 2005). Allergic dermatoses and respiratory

diseases are known to be caused by reactive dyes (Estlander, 1988; Hatch and Maibach,

1995; Manzini et al., 1996). Contact dermatitis, asthma (Thoren et al., 1980) and

http://www.immukare.com/image/data/logo/351px-Health_effects_of_pollution.png



immunosensitivity (Park et al., 1991) have caused in textile industry workers exposed to

dyes. Previous studies have also suggested increased risks of colon and rectum cancers;

however, these cancers relate mostly to dyes for synthetic fibres (De Roos et al., 2005).

2.1.2 Health effects of paints:

The United States of America’s Environmental Protection Agency (US EPA)

classifies paint as one of its top five most hazardous substances. Prolonged or high

exposure to paint and paint fumes can cause headaches, trigger allergies and asthmatic

reactions, irritate skin, eyes and airways, and put increased stress on vital organs. The

WHO has reported a 20% to 40% increased risk of certain types of cancer (in particular

lung cancer) for those who come into regular contact with, or work with paint while

Danish researchers have pointed out to the added possibility of neurological damage.

2.1.3 Health effects of heavy metal [Cr(VI)]:

Hexavalent chromium is recognized as a human carcinogen via inhalation [IARC

(International Agency for Research on Cancer), 1999]. Workers in many different

occupations are exposed to hexavalent chromium. Problematic exposure is known to occur

among workers who handle chromate-containing products as well as those who perform

welding, grinding, or brazing on stainless steel (IARC, 2006). Within the European Union

(EU), the use of hexavalent chromium in electronic equipment is largely prohibited by the

Restriction of Hazardous Substances Directive. Hexavalent chromium compounds are

genotoxic carcinogens. Chronic inhalation of hexavalent chromium compounds increases

risk of lung cancer (lungs are especially vulnerable, followed by fine capillaries in kidneys

and intestine). Soluble compounds, like chromic acid, are much weaker carcinogens.

Chromate-dyed textiles or chromate-tanned leather shoes can cause or exacerbate contact

dermatitis (Salnikow and Zhitkovich 2008). Ingestion of Cr(VI) can also cause irritation or

ulcers in the stomach and intestines

(http://www.epa.gov/region7/pdf/national_beef_leathers prime_tanning_chromiumVI_Fact_Sheet.pdf).

Considering the impact of health effects of pollutants, the environmental pollution

of the three major pollutants viz. textile dyes, paints and hexavalent chromium has been

discussed in detail.

http://www.epa.gov/region7/pdf/national_beef_leathers prime_tanning_chromiumVI_Fact_Sheet.pdf



2.2 Textile Dyes Pollution

Approximately 10000 different dyes and pigments are used industrially and over

0.7 million tons of synthetic dyes are produced annually throughout the world (Gomare et

al., 2009). Due to the poor exhaustive properties of the dyes, unfixed dyes ultimately find

its way into the environment. A very small amount of dye in water (10 to 50 mg/L) affects

the aesthetic value, water transparency and gas solubility of water bodies (Banat et al.

1996). Textile wet processing (i.e. preparation, dyeing, printing and chemical finishing)

has always been considered one of the worst industrial sectors in terms of water

consumption and pollution. In treating 1 ton of cotton fabric the composite waste stream

may have 200 to 600 ppm BOD (biological oxygen demand), 1000 to 1600 ppm of total

solids and 30 to 50 ppm of suspended solids contained in a volume of 50 to 160 m3

(Hirschler, 1996). Nowadays, the increasing demand for non-fading colored textiles has led

to a continuous growth in the use of azo-dyes. Azo dyes account for the majority of all

textile dyestuffs produced and are the most commonly used synthetic dyes in the textile,

food, paper making, color paper printing, leather and cosmetic industries (Chang et al.,

2001). Sulfonated azo dyes are a class of synthetic organic colorants having great structural

differences with a great variety of colors. Structurally azo dyes are characterized by their

typical (N=N-) azo bonding. These dyes are extensively used for dyeing cotton fabrics and

are consequently discharged into the effluents posing a serious environmental threat. The

discharge of these sulfonated azo dyes has not only a negative aesthetic effect but also

some azo dyes and their degradation products, sulfonated and unsulfonated aromatic

amines are toxic or even carcinogenic (Myslak and Bolt, 1998).

2.3 Pollution of Carbendazim, Diuron and Octhilinone from a Paint Preservative

(Troysan S89)

Paint, a generic term used for a wide range of surface coating products which

include conventional solvent-borne formulations, varnishes, enamels, lacquers and water-

based systems. The function of paints and coatings is to provide an aesthetically pleasing

appearance, preservation, decoration as well as to help metal and other substrates to

withstand exposure to both environment and every day wear and tear. Broadly, paint is

composed of two general ingredients, the pigment and the fluid medium. The pigment



usually consists of a mineral oxide or precipitated vegetable dye that determines its color,

while the fluid medium is of oil, varnish or water in which the pigment is suspended. It

contains binders, turpentines, cellulosic thickeners and other minor ingredients such as

coalescing agent, defoamers and biocide preservatives. Several reports have shown that

paints available in the market have harmful substances and containing volatile organic

compounds (VOCs); which give off the characteristic smell of a fresh wet paint and are

mostly found in the solvents and binders of the paint. These compounds have long lasting

harmful effects and can give off damaging fumes, years after the paint has been applied.

These harmful products can lead to asthma, dizziness, and headaches and can exaggerate

allergies. As a result, paints are not environmentally friendly and can lead to the

development of several health problems. Research has shown that the air inside the homes

is more harmful than the air outside. The preservatives help to protect the paints from

microbial attack since waterborne paints are prone to contamination and spoilage by fungi,

algae and many types of bacteria where the conditions of temperature and humidity are

conducive for microbial growth. The microbial growth is very common in exterior

emulsion paints but fungal growth also occurs in interior paints where the humidity level is

generally high (Hueck-Vander, 1968; Barry, 1978; Bravery, 1988). To prevent this, the

most important chemical classes of biocides used as film preservatives are derivatives of

urea, isothiazoline-3-one, dithiocarbamates, benzimidazole, triazines, benzothiazole,

carbamates, thiophthalimide, sulfenic acids, sulfones, triazoles and pyridine-N-oxide (VdL

2000). However, a range of anti-fouling paints does not contain biocides and exhibit a

toxic effect (Karlsson and Eklund, 2004; Jungnickel et al., 2008).

The preservative, Troysan S89, widely used in paint, as an algaecide as well as a

fungicide, is a mixture of three different chemicals namely carbendazim, diuron and

ochthilinone. The percentage by mass, chemical structure and CAS number of these

compounds is presented in Table 2.1. Diuron is a substituted urea algaecide classified as

phenyl urea herbicide, used to control a wide variety of broadleaf and grassy weeds of

agricultural crops (Giacomazzi and Cochet, 2004). Diuron inhibits photosynthesis hence

used to control weeds on hard surfaces, such as, roads, railway tracks, and paths. It can be

used for both pre-emergent and knockdown weed control. It is a white, crystalline,

odorless solid and is stable towards oxidation and moisture under normal conditions and



decomposes at 180 to 190 oC. This chemical has a low acute toxicity on mammals but

juveniles are more susceptible than adults (Hayes, 1982). It is less toxic to birds but

moderately toxic to fish and readily absorbed through the gut and lungs. It can be irritant to

eyes and throat but less irritant to intact skin. Male rats showed changes in their spleen and

bone marrow, when subjected to extremely high doses of diuron over a two-week period.

Changes in blood chemistry, increased mortality, growth retardation, abnormal blood

pigment and anemia are a few of the other chronic effects exerted due to moderate to high

doses of diuron over time. (Beste, 1983; Worthing, 1983; U. S. Environmental Protection

Agency, 1983, 1984, 1985, 1987; Food and Drug Administration, 1986; Chemical

Information Systems, 1988; Kidd and James, 1991; National Library of Medicine, 1992).

Diuron is known to be carcinogenic in rats and reported to cause bladder, kidney and breast

cancers (Cox, 2003). It is also having mutagenic and teratogenic effects such as delayed

bone formation and reduced birth weight (Khera et al., 1979).

Table 2.1: Active ingredients of Troysan S89

Another compound, carbendazim, is a systemic benzimidazole fungicide (Ministry

of Agriculture, Fisheries and Food, 1992) plays an important role in plant disease control

of arable crops (cereals, oilseed rape), fruits, vegetables and ornamentals (Quian, 1996). It

Active ingredient Chemical Structure CAS No. Percentage

by mass

Diuron

(Phenyl urea) 330-54-1 19

Carbendazim

(Benzimidazole) 10605-21-7 9.9

Ochthilinone

(Isothiazolone) 26530-20-1 2.25



is also used in post-harvest food storage and as a seed pre-planting treatment. It works by

inhibiting the development of fungi probably by interfering with spindle formation at

mitosis. Carbendazim has extensive applications worldwide (WHO/FAO Joint Meeting,

1994). Carbendazim is less toxic to rodent and non-rodent species via the oral, dermal,

inhalational and intraperitoneal routes. In mice, it shows increased tumor formation

(Scientific committee on plants, 2001). It is believed to affect hormone functions (Friends

of the earth, 2001; Commission of Europian community, 1999). Mantovani et al. (1998)

showed the teratogenic toxic effects of carbendazim such as serious deformities including

absence of eyes and hydrocephalus. It is also reported to disrupt the development of

sperms and damage testicular development in adult rats. It is reported as a potent aneugen

on cultured lymphocytes (Du Pont, 1991; Mahmood and Parry, 2001). Carbendazim is a

stable compound with a long half life in the environment. It is decomposed in the

environment with half-lives of 6 to 12 months on bare soil, 3 to 6 months on turf, and half-

lives in water of 2 and 25 months under aerobic and anaerobic conditions, respectively

(WHO, 1993).

Octhilinone (OIT) is a mild weedicide, fungicide and bacteriocide, used as a

preservative in various household products including paints. OIT is an isothiazolone

chemical, which can react with cellular thiols, such as cysteine residues in proteins,

because of its structure and electron density (Du et al., 2002; King et al., 2009).

Methylisothiazolinone (MIT), a derivative of OIT, was found to be toxic to neurons in

vitro involving in glutathion depletion (Du et al., 2002). OIT and MIT were found to

increase significantly the amount of oxidized glutathion in the human liver cell line Hep

G2 (Arning et al., 2008). OIT is responsible and associated with development of

occupational asthma and several cases of occupational allergic contact dermatitis, mostly

among the paint manufacturers (Thormann, 1982; Mathias et al., 1983; Bourke et al.,

1997; Korte-Aalto et al., 2007; Balaguer et al., 2008).

2.4 Chromium Pollution

Man and environment are combating pollution by industrial wastes since a few

decades. Industries often produce infinite toxic substances, organic and inorganic as well,

which are many of the times discharged directly in to the sea, rivers, lagoons and lands.



Due to the strong corrosion resistance, hexavalent chromium [Cr (VI)] has been widely

applied in a wide range of industries, including electroplating, wood preservation, leather-

tanning and alloy production (Yu et al., 2008). Disposal of these industrial wastes

containing high levels of chromates results in anthropogenic contamination of pristine

environments, in spite the industrial effluent standards must not exceed 0.75 and 0.25

mg/L of Cr(III) and Cr(VI) in water discharge, respectively (Mongkhonsina et al., 2011).

Chromium can exist in a number of states in natural environment, amongst; Cr (VI) draws

serious public health and legislative concerns because of its extremely high toxicity,

mutagenicity and teratogenecity and listed as class A human carcinogens by the US EPA

(Desai et al., 2008). Chromium oxyanoins can readily permeate through biological

membranes and their intracellular reduction results in the dire consequences of the

chromate induced toxicity by generation of Cr(V), Cr(III) valence states and reactive

oxygen species (ROS) which damage cellular components including DNA, proteins and

lipids (Rodriguez et al., 2011).

2.5 Physical and Chemical Methods for Remediation of Environmental Pollutants

The numerous physico-chemical methods for subtraction of pollutants from

environment have been reported (Lear et al., 2007, Shen et al., 2007, Hu et al., 2011) and

are still underway to discover a sustainable remediation technology. Dozens of remediation

technologies developed internationally could be divided in two general categories

incineration and non-incineration. The methods were applied depending on the source of

contamination. The contamination of groundwater by toxicants is a matter of utmost

concern to the public health. Remediation of contaminated groundwater is of highest

priority since billions of people all over the world use it for drinking purposes. Hashim et

al. (2011) has reviewed 35 approaches for groundwater treatment including chemical and

physico-chemical treatment processes for removal of heavy metals. Remediation of metal-

contamination countenances a particular challenge, because unlike organic contaminants,

metals cannot be degraded in their native toxic form to simpler, non/less toxic components

hence must be removed.

Dye wastewater is usually treated by physico-chemical treatment processes which

include flocculation combined with flotation, electroflocculation, membrane filtration,



electrokinetic coagulation, electrochemical destruction, ion-exchange, irradiation,

precipitation, ozonation, and katox treatment methods. However, these technologies are

generally ineffective in color removal, expensive and less adaptable to a wide range of dye

wastewaters (Banat et al., 1996). Adsorption has been observed to be an effective process

for color removal from dye wastewater. Use of activated carbon has been found to be

effective, but it is too expensive. Many studies have been undertaken to investigate the use

of low-cost adsorbents for color removal (Ramakrishna and Viraraghavan, 1997; Crini,

2006; Gupta and Suhas, 2009). However, these low-cost adsorbents have generally low

adsorption capacities and require large amounts of adsorbents (Srinivasan and

Viraraghavan, 2010). Therefore, there is a need to find new, economical, easily available

and highly effective forward-looking technologies for these methods.

The electrokinetic methods have been put for soil remediation that demonstrated

effectiveness of the technology in removal of pollutants either singly (Shen et al., 2007) or

in multiples (Alcántara et al., 2012). Diuron has been previously shown to be degraded by

using electro-Fenton process (Edelahi et al., 2004). Degradation of carbendazim by

physicochemical method by using UV/H2O2 was reported earlier (Mazellier et al., 2003).

The coupling of two or more methods for soil remediation suggested efficacy of the system

together (Davezza et al., 2011). Nanoremediation is an advanced technology that uses

nanoparticles (NPs) for the environmental remediation. The recent developments on the

use of inorganic NPs for environmental remediation in polluted soil, water and gas have

been reviewed by Sánchez et al. (2011). So far, in attempting to preserve environment,

new and traditional methods of remediation using physical, chemical and biological

principles are being studied (Agarwal et al., 2006). However, the physico-chemical

treatments have numerous disadvantages, including high cost, low efficiency, and

inapplicability, to a wide variety of metals, as well as formation of huge quantities of toxic

by-products, further creating disposal problems of contaminated wastes (Wani et al.,

2007).

Cleaning up contaminated soil by the chemical and physico-chemical methods is a

costly enterprise-the overall cost to remediate affected sites in the EU is estimated to be

between €59 and €109 billion (EC, 2002) that cannot be affordable to developing countries

like India. Furthermore, current methods of soil remediation do not really solve the



problem. In Germany, for instance, only 30% of soils from contaminated sites are cleaned

up in soil remediation facilities (SRU, 2004); the remaining soil must be stored in waste

disposal facilities. This does not solve the problem; it merely transfers it to future

generations.

2.6 Microbial Remediation of Environmental Pollution:

Considering the hazards and disadvantages of physico-chemical remediation

processes, alternative approach is shifting towards the use of conventional biological

methods to treat wastes (Jadhav et al., 2010). These methods are gaining more importance

nowadays because of their lesser cost, effectiveness and eco-friendly nature. The

metabolites produced after biodegradation are mostly non toxic or comparatively less

toxic. Microbial remediation is the process whereby wastes are biologically degraded

under controlled conditions to an innocuous state, or to levels below concentration limits

established by regulatory authorities.

Several reports have been mentioned here for microbial bioremediation of

pesticides from Troysan S89. The white rot fungus Phanerochaete chrysosporium BKM-F-

1767 was shown to degrade diuron in nitrogen limited synthetic medium and on ash wood

chips, respectively. Co-immobilized bacterial cultures of Arthrobacter sp. and Delftia

acidovorans were found to be effective for diuron removal when compared to their free

cell cultures. Previous studies have showed that various microorganisms were able to

degrade diuron partially in to 3,4-dichloroaniline (3,4-DCA) which was found to be

accumulated in the medium (Tixier et al., 2000, 2001, 2002). Arthrobacter sp. N2 was

shown to degrade diuron completely (Widehem et al., 2002). Several researchers have

detected the degradation of herbicide carbendazim by using microbial sources either by

pure cultures or in consortia. A microbial consortium capable of degrading the herbicide

carbendazim was developed by enrichment of soil samples obtained from paddy fields and

the degradation has been studied in glass column reactors using carbendazim as a sole

source of carbon (Pattanasupong et al., 2004), while Zhang et al. (2005) had demonstrated

carbendazim degradation by the new bacterial species.

The use of biological agents (plants and microbes) for decolorization of hazardous

textile dyes is becoming a promising option. Microbial degradation of sulfonated azo dyes



with possible degradation pathways have been reported earlier (Lu and Hardin, 2006;

Kalme et al., 2007a; Dawkar et al., 2010). Recalcitrant nature of azo dyes led failure in

their degradation by typical biological (e.g. activated sludge) and physico-chemical (e.g.

flocculation, coagulation) treatments, which largely promoted the transfer of the azo-dyes

from the wastewater to the sludge, causing additional disposal problems. Furthermore, the

incomplete degradation of azo-dyes may lead to the production of toxic by-products, such

as aromatic amines (Heinfling et al., 1998; Kim and Shoda, 1999; Manu and Chaudhari,

2002) that can be transformed into highly reactive electrophiles, which have been

described as mutagenic and carcinogenic (Gottlieb et al., 2003; Yoo et al., 2001), thus

posing a significant health risk.

Figure 2.2: Bioremediation methods

Types of Biological remediation………….

•Microorganisms are used to breakdown

contaminants Into harmless products.

•Process requires high microbial

density & a continuous nutrient

input in case of bioreactors

•The use of plants (directly or indirectly)

to remediate contaminated

soil or water is known as phytoremediation.

• cost effective, noninvasive, eco-

friendly and publiclyAcceptable.

•The cultivation and harvest of animals to remediate nutrient and pathogenic

microorganism pollution in aquatic systems is the most

common form of zooremediation.

•Rarely considered for bioremediation owing to

ethical concerns or because many of the aquatic

organisms currently cultured or harvested commercially

are bound for human consumption

Microbial bioremediation for the removal of hazardous compounds has received

quite a lot of attention from researchers all over the world with the high potentiality of

prokaryotic systems to perform a variety of functions, but has some limitations. The use of



microbes might lead to infections to humans that is why the method could not readily be

used and required special restrictions. Obviously, there is an urgent need for alternative,

cheap and efficient methods to clean up heavily contaminated industrial areas. This could

be achieved by a relatively new technology known as phytoremediation, which uses plants

to remove pollutants from the environment. Plants also possess some inherent metabolic

pathways that can breakdown a wide range of toxicants but the fact was much less realized,

hence the use of phytoremediation processes for the removal of toxicants is comparatively

an unexplored methodology.

2.7 Phytoremediation: a Brief Overview- from a Concept to the Application

Phytoremediation is the use of plants and/or their associated microorganisms for

the environmental cleanup. This is an emerging biotechnological application which

operates on the principles of biogeochemical cycling (Raskin and Ensley, 2000; Raju et al.,

2008). Phyoremediation technology makes the use of the naturally occurring processes by

which plants and their associated rhizospheric microflora degrade and sequester organic

and inorganic pollutants (Pilon-Smits, 2005). Plants are autotrophic organisms capable of

using sunlight and carbon dioxide as sources of energy and carbon. However, plants rely

on the root system to take up water and other nutrients, such as nitrogen and minerals,

from soil and groundwater. As a side effect, plants also absorb a diversity of natural and

man-made toxic compounds for which they have developed diverse detoxification

mechanisms (Eapen et al., 2007). Pollutant-degrading enzymes in plants probably originate

from natural defense systems against the variety of allelochemicals released by competing

organisms, including microbes, insects and other plants (Singer, 2006). From this

viewpoint, plants can be seen as a natural, solar-powered pump-and-treat systems for

cleaning up contaminated environments, leading to the concept of phytoremediation

(Pilon-Smits, 2005).

The term phytoremediation, from the Greek phyto, meaning ‘‘plant’’, and the Latin

suffix remedium, ‘‘able to cure’’ or ‘‘restore’’, was coined by Ilya Raskin in 1994 and is

used to refer to plants which can remediate a contaminated medium. Phytoremediation

takes advantage of the plant’s ability to remove pollutants from the environment or to

make them harmless or less dangerous (Raskin, 1996). It can be applied to a wide range of



organic and inorganic contaminants. During the 1980s, the US Government initiated a

large program for the development of environmental cleanup technologies (The

Comprehensive Environmental Response, Compensation, and Liability Act or Superfund),

which has accelerated the growth of a new productive research field worldwide (Krammer,

2005). Though, this technology has been used for hundreds of years to treat human wastes,

reduce soil erosion and protect water quality, research focusing specially on the

phytoremediation of contaminated soils has only grown significantly in the last 25 years.

2.7.1 Mechanism of Phytoremediation

Understanding the basic physiology and biochemistry that underlie various

phytoremediation processes is very important to improve the applicability of this plant

based method. In the following section (Table 2.2 and Figure 2.3), basic processes for

phytoremediation are briefly summarized (Morikawa and Erkin, 2003).

2.7.1.1 Phytostabilization:

Certain heavy metals and organic contaminants in soils can be concentrated and

contained in the rhizosphere. This process is not to degrade but to reduce the mobility of

the contaminant and prevent migration to the deeper soil or groundwater. Rhizosphere

processes enhance the precipitation and conversion of soil pollutants to insoluble forms.

Table 2.2: Differing areas of phytoremediation

Technology DescriptionPhytostabilisation Reduction of mobility and bioavailability of pollutants in

environmentRhizodegradation Co-metabolic degradation of pollutants by soil rhizosphere

microorganisms Phytoextraction/ phytoaccumulation

Uptake of pollutants from environment and their concentration in harvestable plant biomass

Phytotransformation/phytodegradation

Chemical modification of pollutants as a result of plant metabolism, both in planta and ex planta, often resulting in their inactivation, degradation (phytodegradation) or immobilisation (phytostabilisation)

Phytovolatilisation Removal of pollutants from soil or water and their release into air, sometimes as a result of phytotransformation to more volatile and/or less polluting substances

Evapoptranspiration Combined effects of plants both to evaporate water on their leaf surfaces and to vaporize water at the stomata

Rhizofiltration Use of plant roots to absorb and adsorb pollutants or nutrients from water and wastewater (e.g. buffer strips)Table has been adapted from Vamerali et al. (2010).



Figure 2.3: Basic processes for phytoremediation

Bioconcentration

Translocation

Cr, Cd, As, Hg

Phytostimulation

Evapotranspiration

Source- modified figure from Aken (2008).

2.7.1.2 Rhizodegradation:

Plants are reported to excrete about 20% of the total photosynthesis products,

including sugars, organic acids and amino acids, to the rhizosphere (Campbell and

Greaves, 1990), and thereby stimulating the growth of microorganisms. In the rhizosphere

region (extending approximately 1 to 3 mm from the root surface), the proliferation of soil

microorganisms can be 3 or 4 orders of magnitude greater than in nonvegetated soils

(Shimp et al., 1993). Some soil microorganisms co-metabolically degrade or mineralize

organic contaminants such as PAHs and PCBs (Donnelly and Fletcher, 1994).

2.7.1.3 Phytoaccumulation / Phytoextraction:

Phytoextraction refers to the extraction of metals or organics by plant roots from

contaminated soil and water to translocate them to aboveground shoots. Metal

hyperaccumulators are those plants which accumulate more than 1.0% (Mn) or 0.1% (Co,

Cu, Pb, Ni, Zn), or 0.01% (Cd) of leaf dry matter (Baker et al., 2000). The standard

definition of a Cr hyperaccumulator plant has previously been described (Mongkhonsin et

al., 2011) that included three distinct criteria; first, the heavy metal concentration in the



plant shoots reaches the hyperaccumulating level (Cr > 50 mg/kg dry mass); second, the Cr

concentrations in the above-ground biomass are 10 to 500 times greater than that in non-

metallophytes (0.2 to 5 mg/kg Cr in dry matter) and third, the Cr concentrations in shoots

are invariably greater than those in roots (translocation factor > 1).

2.7.1.4 Phytodegradation:

Organics including PAHs, TPHs and PCBs, and inorganics including atmospheric

nitrogen oxides and sulfur oxides can be taken up by plants and transformed or degraded.

Whether or not organics are taken up into a plant cell is judged by the octanol–water

partition coefficient, logKow. Chemicals that can enter into the plant have roughly log Kow

values between 1 and 3.5 (Schnoor et al., 1995).

2.7.1.5 Phytovolatilization:

This is the volatilization through stomata of volatile chemicals taken up by plants

from the media. Phytovolatilization of trichloroethylene (TCE) by poplar (Chappell, 1998)

and methyl tertiarybutyl ether (MTBE) by eucalyptus (Newman et al., 1999), selenium by

Indian mustard (de Souza et al., 2000) and methyl mercury by tobacco (Heaton et al.,

1998) and by yellow poplar (Rugh et al., 1998) have been reported. Once volatilized, these

compounds may be degraded by hydroxyl radicals in the atmosphere or stay as an air

pollutant.

2.7.1.6 Evapotranspiration:

Evapotranspiration mechanism is attributed to the combined effects of plants both

to evaporate water on their leaf surfaces and to vaporize water at the stomata. This process

is used in hydraulic control of groundwater (Viessman et al., 1989). Mature phreatophyte

trees such as poplar, eucalyptus and river cedar, which are known to be deep-rooted,

typically can transpire (200 to 1100) L of water per day out of the ground. Hardwood trees

transpire about half the water of a phreatophyte. Two meters of water per year is reported

to be a practical maximum for transpiration in a system with complete canopy coverage (a

theoretical maximum is 4 m/year) (Schnoor et al., 1995). This process always comprises

together with other processes such as phytodegradation.

2.7.1.7 Rhizofiltration:

Rhizofiltration refers to the approach of using hydroponically cultivated plant roots

to remediate contaminated water through absorption, concentration, and precipitation of



pollutants. Khilji and Bareen (2008) investigated the efficient removal of excess toxic and

heavy metals from tannery sludge by growing a tolerant anchored hydrophyte, Hydrocotyle

umbellata in different concentrations of tannery sludge.

2.7.2 Recent reports on phytoremediation:

Phytoremediation has been implemented for environmental remediation since

1980s and its applicability is still underway of progress for sustainable remediation. A lot

of advancement has been progressed in the utilization of plants for cleaning up

environment. The Table 2.3 summarizes the recent research carried out worldwide. In the

present thesis, out of various pollutants mentioned so far, phytoremediation of textile dyes,

pesticides from Troysan S89 and heavy metal (chromium) has been discussed

comprehensively.

Plants are natural attenuators to stress in the environment usually possessing

properties to detoxify their surroundings, and may be suitable for use in phytoremediation.

Under normal circumstances, plants have to metabolize endogenous chemicals (e.g. natural

pesticides and growth hormones) and exogenous xenobiotics (e.g. herbicides, synthetic

pesticides and textile dyes) (Scott-Craig et al., 1998; McCutcheon and Schnoor, 2003;

Ghodake et al., 2009). Plants have also shown to possess metabolic pathways for

degradation of textile dyes (Kagalkar et al., 2009, Patil et al., 2009). Phytoremediation

dominates over microbial and other physico-chemical methods because of cost

effectiveness, safety, easiness to manage due to the autotrophic system of larger biomass

requiring little nutrient inputs (Cunningham and Berti, 2000). Depending on these facts,

some of the plants have been tested on field for phytoremediation studies (Cunningham

and Ow, 1996) and on constructed wetlands have been used to treat dye wastewater

domestic and industrial effluents (Carias et al., 2007; Nilratnisakorn et al., 2008). Salsola

vermiculata, a desert plant, has been proved to be a low cost option for the removal of

large organic molecules (Bestani et al., 2008).

Phytoextraction is an aspect of phytoremediation that involves the removal of

toxins, especially heavy metals and metalloids, by the roots of hyperaccumulator plants

with subsequent transport to aerial plant organs which are able to accumulate

concentrations up to 100-fold more than those normally found in non-accumulators species

(Brunetti et al., 2011). A number of plants have been studied for Cr uptake that included,



Prosopis sp., Typha angustifolia, and Convolvulus arvensis (Haque et al., 2009; Dong and

Wu, 2007; Gardea-Torresdey et al., 2004). In addition, Leersia hexandra Swartz and

Salsola kali have been reported as Cr hyperaccumulator (Zhang et al., 2005; De la Rosa et

al., 2007). Moreover, Prosopis and C. arvensis have been accounted to tolerate, uptake,

and reduce Cr(VI) to the less toxic Cr(III) (Aldrich et al., 2003; Montes-Holguin et al.,

2006).

Though, extensive research has been focused to develop effective and efficient

phytoremediation techniques for hyperaccumulation of metals (Padmavathiamma and

Loretta, 2007) and other organic molecules such as herbicides, pesticides (Kawahigashi,

2009; Benekos et al., 2010), phytoremediation is still at its initial stages of research and

development. Available data on phytoremediation of environmental pollutants is limited.

Many of the plants should be checked for their phytoremediation potential and the

knowledge should be explored for environmental welfare. Thus, in this concern, present

research is focused on phytoremediation capability of a tropical plant i.e. cactus (Nopalea

cochenillifera).



Table 2.3: Recent selected examples of phytoremediation

Pollutant Plant species Summary ReferenceOrganicTextile dyesRed HE7B Nopalea

cochenilliferaSalm. Dyck.

Cactaceae N. cochenillifera cell cultures and intact plants (cladodes) transformed various toxic textile dyes, including Red HE7B into less phytotoxic, non-hazardous metabolites.

Adki et al. (2011)

Malachite Green Blumea malcolmiiHook

Phytodegradation of triphenylmethane dye Malachite Green mediated by cell suspension cultures of B. malcolmii

Kagalkar et al. (2011)

Methyl orange Brassica juncea L. Biochemical characterization of laccase from hairy root culture of B. juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

Telke et al. (2011)

Brilliant Blue R Typhonium flagelliforme

In vitro cultures of T. flagelliforme decolorized a variety of dyes, along with Brilliant Blue R, to varying extents within 4 days.


Direct Red 5B Blumea malcolmii Tissue cultured shrub plants of B. malcolmii decolorized Malachite green, Red HE8B, Methyl orange, Reactive Red 2 but potently Direct Red 5B


Remazol Black B

Zinnia angustifolia Consortium ZE degraded efficient and faster RBB when compared to degradation by Z. angustifoila and E. aestuariiindividually

Khandare et al. (2011)

Navy Blue HE2R

Portulaca grandiflora Hook.

Wild and tissue cultured plants of P. grandiflora decolorized a sulfonated diazo dye Navy Blue HE2R up to 98% in 40 h.


Remazol Red Aster amellusLinn.

Potential of A. amellus to decolorize a sulfonated azo dye Remazol Red, a mixture of dyes and a textile effluent


Remazol Orange 3R, Green HE4B

Aster amellusLinn., Glandularia pulchella (Sweet) Tronc.

Plant consortium-AG of A. amellus and Glandularia pulchella (Sweet) Tronc. showed complete decolorization of a dye Remazol Orange 3R in 36 h, while individually A. amellus and G. pulchella took 72 and 96 h respectively.

Kabra et al. (2011)

Glandularia Phytoremediation ability of G. pulchella in degrading Green Kabra et al. (2011)



pulchella (Sweet) Tronc.

HE4B into non-toxic metabolites.

Green HE4B Sesuvium portulacastrum

Potential of Sesuvium for the efficient degradation of textile dyes and its efficacy on saline soils contaminated with toxic compounds.

Patil et al. (2011)

Reactive Red 198

Tagetes patula L. (Marigold)

Degradation analysis of Reactive Red 198 by hairy roots of T. patula

Patil et al. (2009)

Acid orange 7 Phragmites australis

The role of antioxidant and detoxification enzymes of P. australis(a sub-surface vertical flow constructed wetland), in the degradation of acid orange7

Carias et al. (2008)

The role of peroxidases extracted from the vertical flow constructed wetland P. australis leaves in the decolourization of AO7

Carias et al. (2007)

Integrated study of the role of P. australis in azo-dye treatment in a constructed wetland: From pilot to molecular scale.

Davies et al. (2009)

Phytoremediation of textile effluents containing azo dye by using Phragmites australis in a vertical flow intermittent feeding constructed wetland

Davies et al. (2005)

Textile wastewater

Typha angustifoliaLinn.

A constructed wetland model for synthetic reactive dye wastewater treatment by narrow-leaved cattails

Nilratnisakorn et al. (2009)

Synthetic reactive dye wastewater treatment by narrow-leaved cattails (T. angustifolia): Effects of dye, salinity and metals.

Nilratnisakorn et al. (2007)

polymeric dye R-478

Mentha pulegium Peroxidase activity and phenolic content in elite clonal lines of M. pulegium in response to R-478 and Agrobacterium rhizogenes.

Strycharz and Shetty(2001)

dye solutions of different colors

Helianthus annuus Phytoremediation of textile dyes used as a scientific experiment or demonstration in teaching laboratories of middle school, high school and college students.

Ibbini et al. (2009)

Herbicides and pesticidesAtrazine switchgrass Phytoremediation capacity for detoxifying atrazine was found in Murphy and Coats



(Panicum virgatum)

the leaf biomass of switchgrass. (2011)

Transgenic tobacco

Transgenic tobacco plants expressing atzA exhibit resistance and strong ability to degrade atrazine.

Wang et al. (2010)

Vetiver grass (Vetiveria zizanioides)

Vetiver grass studied for phytoremediation of heavy metals and organic wastes.

Danh et al. (2009)

Lolium multiflorum

Phytoremediation potential of the novel atrazine tolerant L. multiflorum and studies on the mechanisms involved.

Merini et al. (2009)

a mixture of prairie grasses

Fate of atrazine in a grassed phytoremediation system was found by using radiolabelled atrazin.

Henderson et al. (2007)

rice plants (Oryza sativa cv. Nipponbare)

The ability of transgenic rice plants (pIKBACH) to remove atrazine and metolachlor from soil was confirmed in large-scale experiments.

Kawahigashi et al. (2006)

three species of poplar tree

Phytoremediation of atrazine by poplar trees: toxicity, uptake, and transformation.

Chang et al. (2005)

Brassica juncea CG, Lindblom SD, Smits EA. Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea.

Flocco et al. (2004)

Metolachlor Prairie grasses Mass balance of metolachlor in a grassed phytoremediation system.

Henderson et al. (2007)

rice plants (Oryza sativa L. cv. Nipponbare)

The human cytochrome P450 gene CYP2B6 was introduced into rice plants, and the CYP2B6-expressing rice plants became more tolerant to various herbicides than nontransgenic Nipponbare rice plants.

Kawahigashi et al. (2005)

Arabidopsis thaliana

Gene expression and microscopic analysis of Arabidopsis exposed to chloroacetanilide herbicides and explosive compounds

Mezzari et al. (2005)

glyphosate Lemna minor Potential use of L. minor for the phytoremediation of isoproturon and glyphosate

Dosnon-Olette et al. (2011)

diphenyl ether transgenic tobacco Overexpression of a specific soybean GmGSTU4 isoenzyme Benekos et al. (2010)



and chloroacetanilide

plants improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants

glufosinate Zea mays L ssp mays, Brassica napus L var napus

Distribution and metabolism of D/L-, L- and D-glufosinate in transgenic, glufosinate-tolerant crops of maize and oilseed rape.

Ruhland et al. (2004)

Endosulfan Brassica campestris Linn.,Zea Maize

The phytoextraction potential of mustard and maize to remove a organochlorine pesticide endosulfan was investigated.

Mukherjee and Kumar(2011)

Nitroaromatics and explosives2,4,6-trinitrotoluene(TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)


Arabidopsis plants have engineered to degrade RDX, whilst

withstanding the phytotoxicity of TNTRylott et al. (2011)

the potential role of upregulated genes in Arabidopsis thalianaplant metabolism, phytoremediation, and phytosensing

Rao et al. (2009)

TNT Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants.

Beynon et al. (2009)

2,6-dinitrotoluene (2,6-DNT)

Phytotoxicity and phytoremediation of 2,6-dinitrotoluene using Arabidopsis plant

Yoon et al. (2007)

Phytotransformation of 2,4-dinitrotoluene in arabidopsis thaliana: toxicity, fate, and gene expression studies in vitro.

Yoon et al. (2006)

TNT Vetiver grass(Vetiveria zizanioides)

Vetiver grass is capable of removing TNT from soil in the presence of urea.

Das et al. (2010)



The efficiency of vetiver grass in removing 2,4,6-trinitrotoluene (TNT) from aqueous media was explored in the presence of a common agrochemical, urea, used as a chaotropic agent.

Makris et al. (2007)

tobacco (Nicotiana tabacum)

Enhanced transformation of TNT by tobacco plants expressing a bacterial nitroreductase.

Hannink et al. (2007)

The transgenic tobacco plants overexpressing a bacterial nitroreductase gene detoxify soil contaminated with the high explosive 2,4,6-trinitrotoluene (TNT), with a significantly increased microbial community biomass and metabolic activity in the rhizosphere of transgenic plants compared with wild type plants.

Travis et al. (2007)

Rumex crispus Phytoremediation of soil contaminated with cadmium and/or 2,4,6-trinitrotoluene.

Baek et al. (2006)

Polycyclic aromatic hydrocarbons (PAH)phenanthrene, pyrene, and benzo[a]pyrene

Rice Evaluating the spatial dissipation gradient of PAHs, including phenanthrene, pyrene, and benzo[a]pyrene in rice rhizosphere, with various bioavailability represented with sequential extraction.

Ma et al. (2012)

phenanthrene, pyrene, and benzo[a]pyrene

side oats grama (Bouteloua curtipendula)

Phytoremediation and removal mechanisms in B. curtipendulagrowing in sterile hydrocarbon spiked cultures

Reynoso-Cuevas et al. (2011)

Study of PAH dissipation and phytoremediation in soils: comparing freshly spiked with weathered soil from a former coking works.

Smith et al. (2011)

dibenzofuran bermuda grass (Cynodon

Evaluation of the phytoremediation potential of four plant species for dibenzofuran-contaminated soil.

Wang and Oyaizu (2009)



dactylon), bent grass (Agrostis palustris Huds.), lawn grass (Zoysia japonica), and, white clover (Trifolium repensL.)

Polychlorinated biphenyls

perennial weed species

Phytoextraction and uptake patterns of weathered polychlorinated biphenyl-contaminated soils using three perennial weed species.

Ficko et al. (2011)

pumpkin and weed species

Effect of pumpkin root exudates on ex situ polychlorinated biphenyl (PCB) phytoextraction by pumpkin and weed species.

Ficko et al. (2011)

Salix alaxensis(felt-leaf willow) and Picea glauca(white spruce)

Assessing the potential for rhizoremediation of PCB contaminated soils in northern regions using native tree species.

Slater et al. (2011)

alfalfa Influence of arbuscular mycorrhiza and Rhizobium on phytoremediation by alfalfa of an agricultural soil contaminated with weathered PCBs: a field study.

Teng et al. (2010)

alfalfa, ryegrass, tall fescue and rice

Beta-cyclodextrin enhanced phytoremediation of aged PCBs-contaminated soil from e-waste recycling area.

Chen et al. (2010)

Chenopodium album, Vicia cracca, Cirsium vulgare, Solidago canadensis

Potential for phytoextraction of PCBs from contaminated soils using weeds.

Ficko et al. (2010)

pumpkin(Cucurbita pepo)

Effect of plant age on PCB accumulation by pumpkin Low et al. (2009)



halogenated hydrocarbons

2,4-Dichlorophenol (2,4-DCP)

tobacco (Nicotianatabacum cv. Wisconsin)

Tobacco hairy roots efficiently transformed high concentrations of 2,4-DCP in the medium to products with the lignin-type nature, which compartmentalized in hairy root cell walls.

Talano et al. (2010)

Inorganic (metals and metalloids)Chromium

Crambe abyssinica Identifying genes and gene networks involved in chromium metabolism and detoxification in Crambe abyssinica.

Zulfiqar et al. (2011)

Spirodela polyrrhiza

Phytoremediation of Cr(VI) by Spirodela polyrrhiza (L.) Schleiden employing reducing and chelating agents.

Bala and Thukral et al. (2011)

rice (Oryza sativaL.), paragrass (Brachiaria mutica), and an aquatic weed (Eichhornia crassipes)

Bio-concentration of chromium-an in situ phytoremediation study at South Kaliapani chromite mining area of Orissa, India.

Mohanty et al. (2012)

water spinach (Ipomonea aquatica)

Phytoremediation of Cr(III) by I. aquatica from water in the presence of EDTA and chloride.

Chen et al. (2010)

hybrid willows Effect of temperature on phytoextraction of hexavalent and trivalent chromium by hybrid willows

Yu et al. (2010)

ArsenicHydrilla verticillata (L.f.)

The accumulation of As in the shoot and immobilization of As below ground in roots proved H. verticillata as a potential As

Xue and Yan (2011)



Royle phytofiltrator for bioremediation.maize (Zea maysL.)

Identification of QTLs for arsenic accumulation in maize using a RIL population.

Ding et al. (2011)

Pityrogramma calomelanos and Pteris vittata L.

Phytoremediation potential of P. calomelanos var. austroamericana and P. vittata L. grown at a highly variable arsenic contaminated site.

Niazi et al. (2011)

hyacinth Batch and continuous removal of arsenic using hyacinth roots. Govindaswamy et al. (2011)

cottonwood Enhanced arsenic tolerance of transgenic eastern cottonwood plants expressing gamma-glutamylcysteine synthetase.

LeBlanc et al. (2011)

CadmiumAlyssum species. Cadmium phytoextraction potential of different Alyssum species. Barzanti et al. (2011)Ricinus communis The phytoremediation potential of bioenergy crop R. communis

for DDTs and cadmium co-contaminated soilHuang et al. (2011)

Solanum nigrumL.

In-situ cadmium phytoremediation using S. nigrum L.: the bio-accumulation characteristics trail.

Ji et al. (2011)

S. nigrum effective in phytoextracting Cd and enhancing the biodegradation of PAHs in the co-contaminated soils with assistant chemicals (EDTA, cysteine, salicylic acid, and Tween 80).

Yang et al. (2011)


Heterologous expression of a N. nucifera phytochelatin synthase gene enhances cadmium tolerance in A. thaliana.

Liu et al. (2011)

Expression of the bacterial heavy metal transporter MerC fused with a plant SNARE, SYP121, in A. thaliana increases cadmium accumulation and tolerance.

Kiyono et al. (2011)

Solanum nigrum Chemical-assisted phytoremediation of CD-PAHs contaminated soils using S. nigrum L.

Yang et al. (2011)

SeleniumStanleya pinnata The salt/B tolerant S. pinnata genotypes selected represent a

promising new tools for the successful phytoremediation of Se Freeman and Bañuelos (2011)



from salt/B and Se-laden agricultural drainage sedimentscanola, mustard, broccoli, spearmint, sugarcane, guar, wheat, and poplar

Developing a sustainable phytomanagement strategy for excessive selenium in western United States and India.

Bañuelosm and Dhillon (2011)

MercuryImpatiens walleriana

Mercury uptake and translocation in I. walleriana plants grown in the contaminated soil from Oak Ridge.

Pant et al. (2011)

----------------- Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

Ruiz et al. (2011)

Atriplex codonocarpa, Austrodanthonia caespitosa and Vetiveria zizanioides

Chelate-assisted phytoextraction of mercury in biosolids. Lomonte et al. (2011)

Chlamydomonas reinhardtii

Removal of mercury from sediment by ultrasound combined with biomass (transgenic C. reinhardtii).

He et al. (2011)

CesiumCalendula alata, Chenopodium album, Amaranthus chlorostachys

Phytoremediation of stable Cs from solutions by C. alata, A. chlorostachys and C. album.

Moogouei et al. (2011)

Sorghum hybrid and Trifolium pratense

Using elevated CO2 to increase the biomass of a S. vulgare x S. vulgare var. sudanense hybrid and T. pratense L. and to trigger hyperaccumulation of cesium.

Wu et al. (2009)

Chromolaena odorata

Potential of C. odorata for phytoremediation of (137)Cs from solution and low level nuclear waste.

Singh et al. (2009)



Coppersunflower Copper phytoextraction in tandem with oilseed production using

commercial cultivars and mutant lines of sunflower.Kolbas et al. (2011)

marigold The effect of the symbiosis between Tagetes erecta L. (marigold) and Glomus intraradices in the uptake of Copper(II) and its implications for phytoremediation.

Castillo et al. (2011)

perennial peanut Potential phytoextraction and phytostabilization of perennial peanut on copper-contaminated vineyard soils and copper mining waste.

Andreazza et al. (2011)

ZincPhysalis alkekengi Zinc accumulation and synthesis of ZnO nanoparticles using P.

alkekengi L.Qu et al. (2011)

Leadbuttonwood Phytoremediation of lead in urban polluted soils in the north of

IranHashemi (2011)

willow varieties The pot experiment suggested that Salix varieties have the potential to take up and translocate significant amounts of Pb into above-ground tissues using EDTA.

Zhivotovsky et al. (2011)

Nickelrape shoots (Brassica napus L.)

Nickel accumulation in rape shoots (B. rassica napus L.) increased by putrescine.

Shevyakova et al. (2011)

MiscellaneousFe, Cu, Zn, Ni, Al, Cr, Pb, Si, and As

Pteris vittata L. P. vittata is confirmed to be a heavy metals accumulator and a highly suitable candidate for phytoremediation of metal contaminated wastelands.

Kumari et al. (2011)

Cd, Cr, Cu, Mn, Fe, Ni, Pb and Zn

Phragmites cummunis, Typha angustifolia, Cyperus

Phytoremediation of Cd, Cr, Cu, Mn, Fe, Ni, Pb and Zn from aqueous solution using Phragmites, Typha and Cyperus.

Chandra and Yadav (2011)



esculentusPb, Cr and Mn Typha latifolia L.

and Scirpus americanus pers

Lead, chromium and manganese removal by in vitro root cultures of two aquatic macrophytes species.

Santos-Díaz Mdel and Barrón-Cruz Mdel (2011)

Cu, Ni and Cr rapeseeds, sunflowers, tomatoes, and soapworts

Combined mild soil washing and compost-assisted phytoremediation in treatment of silt loams contaminated with copper, nickel, and chromium.

Sung et al. (2011)

Cr and Cd Prosopis laevigata Prosopis laevigata a potential chromium (VI) and cadmium (II) hyperaccumulator desert plant.

Buendía-González et al. (2010)

Pb, Zn, Fe and Cr

Eichhornia crassipes, Hydrilla verticillata

Evaluation of uptake rate of heavy metals by two macrophytes Eichhornia (free-floating) and Hydrilla (submerged).

Dixit and Dhote (2010)

Zn, Pb, and Cd Inula viscosa, Euphorbia dendroides, and Poa annua, A. donax, Cistus salvifolius and Helichrysum italicus

Uptake of heavy metals by native species growing in a mining area in Sardinia, Italy: discovering native flora for phytoremediation

Barbafieri et al. (2011)

Te Indian mustard Distinct uptake of tellurate from selenate in a selenium accumulator, Indian mustard (Brassica juncea).

Ogra et al. (2011)

Cs, Pb Calendula alata Potential of C. alata for phytoremediation of stable cesium and lead from solutions.

Borghei et al. (2011)

Vetiveria zizanoides

Phytoremediation of 137cesium and 90strontium from solutions and low-level nuclear waste by V. zizanoides.

Singh et al. (2008)



2.7.3 Applications of plant biotechnology in phytoremediation studies:

Several plant-based experimental systems can be employed for the

phytoremediation research viz. cell extracts, differentiated organ cultures such as roots and

shoots, dedifferentiated plant cell cultures such as callus and cell suspensions, explants

such as leaf disks and excised roots, plants in hydroponic culture or in potted soil under

greenhouse cultivation or in the field. As shown in Figure 2.4, plant tissue cultures share

several common features with intact plants grown either hydroponically or in the field;

however, these different culture systems also possess important unique properties.

Figure 2.4: Phytoremediation technology

Plant tissue

cultures

Plants in hydroponic

culture

Plants in the field

Intrinsic genetic and metabolic capacity of

cells

Enhanced bioavailability of

medium components.

Ease of medium manipulation

Mature plants.Reduced bioavailability of

components in soil.Exposure to aged and weathered pollutants.

Subject to site and weather conditions.

Source: Doran, 2009



Table 2 .4 . Examples of plant tissue cultures applied in metabolic studies of organic pollutants (Table has been adapted from Doran, 2009)

Organic pollutantForm of planttissue culture Plant species Summary References

Bisphenol A(2,2-bis(4-hydroxyphenol)propane)

Cell suspension Eucalyptus perriniana Cells capable of regioselective hydroxylation andglycosylation; 3 products identified

Hamada et al. (2002)

Polychlorinated biphenyls (PCBs) Callus, hairy root,shooty teratoma

Solanum aviculare,Solanum nigrum

Greater metabolic capacity in organized tissuesthan callus; tetrachlorinated and pentachlorinatedPCBs mostly did not produce metabolites

Kucerova et al. (1999),Rezek et al. (2007)

Phenol Hairy root Brassica napus,Lycopersicon esculentum

Brassica juncea, Beta vulgaris,Raphanus sativus,Azadirachta indica

B. napus roots reused in 3–4 treatment cycles;overexpression of peroxidase in tomato increasedphenol removal by ca. 20%

Extent of phenol removal varied between species;peroxidase activity and H2O2 levels increasedafter phenol exposure; phenol removal by B. junceadid not require H2O2 addition

Coniglio et al. (2008), Wevar Oller et al. (2005)

Singh et al. (2006)

Phenol and chloroderivatives Hairy root Daucus carota Metabolism accompanied by increasein peroxidase activity

2,4-Dichlorophenol Cell suspension Nicotiana tabacum Complex glycoside conjugates identifiedas main metabolites

Hairy root Brassica napus Peroxidase isoenzyme profiles affected by repeated treatment cycles

de Araujo et al. (2002)

Laurent et al. (2007)

Agostini et al. (2003)

Pentachlorophenol Cell suspension Triticum aestivum,Glycine max

Metabolism mainly to polar metabolites; G. max cellsreleased large amounts of metabolites; T. aestivummetabolites mainly cell-associated; glucoside production higher during the lag phase

Harms (1992), Harms andLangebartels (1986), Langebartels andHarms (1986)

Phenoxyacetic acid Cell suspension Glycine max Metabolism to soluble metabolites and insolubleresidues; bound residues associated mainly with hemicelluloses and lignin

Laurent and Scalla (1999)

2,4-Dichlorophenoxyacetic acid (2,4-D)

Cell suspension Glycine max Metabolism to at least 5 components; detoxificationby ring-hydroxylation followed by O-glucosylation

Lewer and Owen (1989)

4-Chloroaniline Cell suspension Triticum aestivum, Glycine max Metabolism to polar compounds; G. max cellsreleased large amounts of metabolites; high levelsof bound residues complexed with pectin and lignin in T. aestivum

3,4-Dichloroaniline Cell suspension Glycine max, Daucus carota G. max produced mainly N-malonyl conjugatesreleased into the medium; glucoside levelsdecreased in D. carota cultures as malonyl conjugates accumulated

Root, cell suspension Arabidopsis thaliana Metabolism mainly to N-glucosyl conjugatesreleased into the medium

Harms (1992), Harms andLangebartels (1986), Langebartels andHarms (1986)

Gareis et al. (1992),Schmidt et al. (1994)

Brazier-Hicks et al. (2007),Lao et al. (2003), Loutre et al. (2003)



(Continued)4-Nitrophenol Cell suspension Glycine max, Triticum

aestivum, Daucuscarota, Daturastramonium

Metabolism to glucosylated and malonylatedderivatives; metabolism in 3-L bioreactor notsignificantly different from that in shakeflasks; conjugate turnover during late growthand stationary phases of D. stramoniumcultures

Knops et al. (1995), Schmidt et al. (1993,1994, 1997)

4-Nonylphenol Cell suspension Lycopersicon esculentum Complete conversion to polar metabolites Harms (1992)Pentachloronitrobenzene Cell suspension Arachis hypogaea Primary pathway involved initial conjugation

with glutathione; 7 major metabolites detectedTNT (2,4,6-trinitrotoluene) Hairy root Catharanthus roseus Transformed to products including aminated

nitrotoluenes found in the medium and biomassCell suspension Datura innoxia Metabolised to aminodinitrotoluenes (ADNTs)

accumulated transiently in cells and medium;virtually all TNT and ADNTs cleared within 12 h

Cell suspension Nicotiana tabacum Five metabolites purified and characterized; almostcomplete transformation tohydroxylamino-4,6-dinitrotoluene

Cell suspension Saponaria officinalis Plant oxidoreductase isolated for reductionof nitro groups

Lamoureux et al. (1981)

Hughes et al. (1997)

Lucero et al. (1999)

Vila et al. (2005)

Podlipna et al. (2007)

Pyrene Cell suspension Glycine max, Triticum aestivum,Datura stramonium,Digitalispurpurea

Benzo[a]pyrene Cell suspension Glycine max, Triticum aestivum,Atriplex hortensis

No soluble metabolites found in G. max andD. stramonium; hydroxypyrene and carbohydrateconjugates identified in T. aestivum;hydroxypyrene methyl ester identified in D. purpurea

Metabolized to mainly polar compounds; conversionhigher in G. max than T. aestivum

Huckelhoven et al. (1997)

Harms (1992),Harms and Langebartels(1986)

Phenanthrene Cell suspension Lycopersicon esculentum Compound taken up almost completely butonly 7% conversion to polar metabolites

Dibenz[a,h]anthracene Cell suspension Atriplex hortensis Poor absorption by cells and little evidenceof metabolism

Harms (1992)

Harms (1992)

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine)

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Hairy root Catharanthus roseus RDX removed from medium and accumulated inbiomass as intact RDX and unidentified bound forms;HMX more recalcitrant with minimal removal

Green cell aggregate Populusdeltoides P. nigra Kinetic models developed and tested against experimental data;models represented uptake, transformation, mineralization,formation of bound residues, and exchange of intermediateswith medium

Partial reduction in the dark;further transformation required exposureto light; significant mineralization to CO2; methanol and formaldehyde also produced

Bhadra et al. (2001)

Mezzari et al. (2004)

Van Aken et al. (2004b)



Table 2.5. Examples of plant tissue cultures applied in studies of metal uptake, toxicity, and tolerance (Table has been adapted from Doran, 2009)

Metal pollutant Form of plant tissue culture Plant species Summary References

Aluminium Cell suspension Nicotiana tabacum Al uptake by actively growing but not stationary-phase cells;pre-chelation of Al to citrate reduced uptake

Cadmium Cell suspension Nicotiana tabacum Rapid H2O2 generation by Cd-treated cells; starting pointfor oxidative burst located in the plasma membrane;oxidative burst may be mediated by calmodulin and/orcalmodulin- dependent proteins

Cell suspension Nicotiana tabacum Three waves of reactive oxygen species contribute toCd-induced cell death

Vitorello and Haug (1996)

Olmos et al. (2003)

Garnier et al. (2006)

Hairy root Adenophora lobophylla,A. potaninii

Thlaspi caerulescens(Cd hyperaccumulator),Nicotiana tabacum

Cobalt Cell suspension Crotalaria cobalticola (Co hyperaccumulator), Rauwolfiaserpentina, Silene cucubalus

Copper Hairy root Hyptis capitata, Polycarpaealongiflora, Euphorbia hirta

A. lobophylla (endangered) accumulated higher levels of Cd thanA. potaninii (not endangered); reduced glutathione (GSH) andcysteine levels higher in A. lobophylla than A. potaninii

Higher antioxidative enzyme and glutathione levels in T.caerulescens

than in N. tabacum; strong catalase induction in Cd-treatedT. caerulescens; low levels of Cd complexation with organic acids;most Cd localized in cell walls in both species

Co did not induce phytochelatin synthesis in C. cobalticola; cysteinelevels increased after Co treatment in all species; Co inC. cobalticola extracts coeluted with cysteine

Lower Cu uptake by E. hirta than other species; growthof H. capitata tolerant to Cu added together with EDTA

Wu et al. (2001)

Boominathan andDoran (2003a,b)

Oven et al. (2002)

Nedelkoska and Doran(2000a)

Mercury, methyl Cell suspension Daucus carota, Lactucasativa Toxicity dependent on plant growth regulators and light Czuba (1987)Nickel Cell suspension Nicotiana tabacum Ni tolerance not dependent on phytochelatin synthesis;histidine,

oxalate, citrate, 2-oxoglutarate, and glutamate levels higher inNi-tolerant cells than controls; Ni localized in vacuoles

Nakazawa et al. (2004),Saito et al. (2005)

Hairy root Alyssum bertolonii(Ni hyperaccumulator),Nicotiana tabacum

Alyssum bertolonii(Ni hyperaccumulator)

Superoxide dismutase and catalase activities higher inA. bertolonii than N. tabacum; Ni treatment increasedH2O2 levels in both species; Ni complexed withorganic acids in A. bertolonii

Ni-rich bio-ore containing up to 82% Ni generated byfurnace treatment of dried biomass

Boominathan and Doran(2002, 2003b)

Boominathan et al. (2004)

Selenium Cell suspension Coffea arabica Lipid peroxidation and alterations in antioxidative enzyme activities

Strontium Shoot Solanum laciniatum Sr accumulated at levels up to 0.13% of dry biomass;morphological changes to the shoot vascular systemafter exposure to Sr

Gomes-Junior et al. (2007)

Kartosentono et al. (2001)

Uranium Hairy root Brassica juncea,Chenopodium amaranticolor

B. juncea better than C. amaranticolor for U removal;growth unaffected by up to 500 mM U

Eapen et al. (2003)



Plant tissue culture is a convenient laboratory tool for phytoremediation studies

(Figure 2.5). As indicated in Tables 2.4 and 2.5, in vitro plant cell and organ cultures have

been applied in numerous studies intended to identify the capacity of plant cells’ tolerance

to, assimilation, detoxification, metabolism, and storage of a wide variety of organic and

heavy metal pollutants. The complex network of interactions of various factors in stress

tolerance could be studied at the initial stages of plant development under controlled

conditions (Kumar et al., 2008). Plant tissue culture involves the growth of plant systems

in vitro in an axenic environment. Dedifferentiated plant cells are cultured in the form of a

callus or cell suspensions; differentiated organs such as roots and shoots can also be

propagated in vitro. Because they grow, relatively quickly and do not require exogenous

hormones in the medium, genetically transformed hairy roots and shooty teratomas are

often used in tissue culture instead of untransformed roots and shoots. The forms of tissue

culture most frequently employed are cell suspensions and hairy roots (Doran, 2009).

Plant biotechnology using tissue culture, somaclonal variation and in vitro

selection, offers the opportunity to develop new germplasm, better adapted to the changing

demands (Skirvin et al., 1993; Alibert et al., 1994). Somaclonal variation and in vitro

selection seem to be an appropriate technology for the development of new plant variants

with enhanced metal accumulation and extraction properties (Herzig et al., 1997;

Guadagnini et al., 1999). Further, improvement of a plant variety with better

phytoremediation capacity by transgenic technology primarily requires plant tissue

cultures.

2.7.3.1 Differentiated organ cultures for phytoremediation:

Differentiated organ cultures include multiple shoots and root cultures that have

been established by using suitable growth regulators, either auxins, or cytokinins or

combinations of both. Once established, these in vitro cultures can be propagated

indefinitely and are available on demand. For the plants with slower growth rate (like

cacti) micropropagation could enhance their rapidity and availability. Therefore, the time

required to carry out experimental investigations may be substantially reduced using plant

tissue cultures rather than whole plants. In this regards, Smykalova et al. (2010) has

reported large scale and rapid screening of heavy metal tolerance in flax/linseed (Linum

usitatissimum L.) tested in vitro. Because in vitro plant cultures are grown and maintained



free from microbial contamination, they can be used to distinguish the responses and

metabolic capabilities of plants from those of microorganisms normally present in the

rhizosphere or in plant tissues (Chaudhry et al., 2005; Lebeau et al., 2008). Plant cultures

can help to carry out studies under more but easily controlled conditions than with soil-

growing plants, particularly with regard to medium composition, nutritional parameters,

growth regulator levels, and medium additives. Although effecter substances can be added

to soil, they may be rendered unavailable to plants due to the adsorption or binding with

soil components. Extracellular complexation of substrates is minimized in tissue cultures,

thus facilitating substance transport and uptake directly by the cells.

2.7.3.2 Callus and cell suspension cultures for phytoremediation:

Main advantage in use of callus and suspended plant cells is lacking many of the

barriers used by ex vitro plants to regulate penetration of chemicals from the environment,

such as leaf wax, bark, cuticles, epidermis, and endodermis, and do not depend on

translocation processes for tissue-specific metabolic activity. Hence, better and more

uniform uptake of external components is generally expected in plant cell cultures (Camper

and McDonald, 1989; Lucero et al., 1999). The ability to feed in vitro cultures relatively

large amounts of contaminants that would be unavailable from the soil at similar levels

(Lucero et al., 1999) allows the recovery of metabolites and intermediates in quantities

suitable for analysis (Laurent et al., 2007), providing a significant advantage for

biochemical and metabolic research. This benefit is amplified when plant cells are

cultivated in bioreactors in vitro in multiliter volumes (Knops et al., 1995). Because of the

reduced amounts of starch, chlorophyll, and other pigments in cultured plant cells,

compared with whole plants, isolation of reaction products from plant tissue cultures may

be easier, require fewer purification steps, and yield samples of higher purity than from

intact plants (Lamoureux et al., 1981; Mumma and Davidonis, 1983; Schmidt, 2001).

The new plant variants with enhanced phytoremediation properties could be

developed with somaclonal variation and in vitro selection technology. The isolation of

highly metal-tolerant cell lines from the callus or suspension cultures of non-metallophytes

like Datura innoxia L. has been reported by Jackson et al. (1984), suggesting that non-

metallophytes should possess the ability for high level metal tolerance. Huang et al. (1987)

have reported that tomato callus cell lines, selected on 5 mM Cd, showed a higher



tolerance of Cd, a slightly higher tolerance of Cu, but not improved Hg, Zn, Pb or Ag

tolerance. Nickel-tolerant callus lines of Setaria italica L. were developed by Rout et al.

(1998). Callus cultures of Ginkgo biloba L. have been selected on a medium containing 10

or 100 μM Cd (Nehnevajova et al. 2002). Aluminium tolerance has been induced by in

vitro selection in rice, maize and wheat (Jan et al., 1997; Ramgareeb et al., 1999; Sibov et

al., 1999).

2.7.3.3 Protoplasts culture and Somatic hybridization in phytoremediation research:

Plant breeding could help bringing together the characteristics of two or more

plants with counter-properties of phytoremediation to get a hybrid, ideal phytoremediation

plant. But owing to the drawbacks such as time consumption, more manpower

requirement, the plant breeding could not be used. Though, the possibility of using

molecular techniques to engineer a larger plant for phytoremediation is attractive;

unfortunately, the number or types of genes responsible for tolerance to and removal of

pollutant have not been elucidated. Ingrouille and Smirnoff (1986) observed that tolerance

and hyperaccumulation were genetically independent in Thlaspi caerulescens, and most

genetic studies of metal tolerance in other species have concluded that tolerance is a

polygenic trait (Antonovics et al., 1971). As an alternative to the isolation and

characterization of all genes involved in metal tolerance, Brewer et al. (1999) has

attempted to produce somatic hybrids between the zinc hyperaccumulator hybridize T.

caerulescens with a related species of higher biomass Brassica napus by electrofusion of

protoplasts isolated from each species and the growth of the plant in tissue cultures on

high-zinc media as well as on high-Zn soil conclusively demonstrated the utility of somatic

hybridization as a technique for the production of metal tolerant plants with greater

phytoremediation capacity.

There have been numerous successful attempts at interspecific and intergeneric

hybridization, both sexual and somatic, within the Brassicaceae (Prakash and Hinata ,1980;

Glimelius et al., 1991). Interspecific sexual hybridization may require the use of

techniques to overcome initial sexual incompatibility, such as in vitro fertilization or

embryo rescue. Somatic hybridization, which is accomplished by hybrid regeneration from

tissue culture after protoplast fusion, encompasses species with complete sexual

incompatibilities. Somatic hybrids from parents of a widely divergent genetic background



may have low fertility and viability. But repeated backcrossing to one parent, while

screening for a desired trait, may provide an opportunity to transfer genes from one

genome to another via non-homologous recombinations (Chevre et al., 1994).

2.7.3.4 Hairy Roots:

Hairy roots are produced by genetic transformation using Agrobacterium

rhizogenes. An overview of hairy root biology and applications, including detailed

procedures for inducing hairy root cultures, is provided by Hamill and Lidgett (1997) and

Bapat and Ganapati (2005). Suza et al. (2008) has highlighted in their review the advances

in the use of hairy roots to assess plants for their potential in removing important water and

soil pollutants such as metals, explosives, radionuclides, insecticides, and antibiotics. Hairy

roots offer the important advantages of greater genotypic and phenotypic stability

compared with dedifferentiated plant cells (Doran, 2009), thus providing a more reliable

and reproducible experimental system over time. They also have simpler culture

requirements as exogenous plant growth regulators are not needed. Because hairy roots are

themselves the products of genetic transformation of plant cells with bacterial DNA,

further genetic modification to introduce genes for improved phytoremediation traits via

the Ri (root-inducing) plasmid of A. rhizogenes is relatively straightforward. Alternatively,

transgenic hairy roots can be initiated from already transformed plant material. Transgenic

hairy root cultures are a useful tool in metabolic studies and for screening genetic

transformants prior to regeneration of whole plants with enhanced phytoremediation

potential.

2.7.3.5 Transgenic plants:

Typically, transgenic plants exhibiting new or improved phenotypes are engineered

by the over expression and/or introduction of genes from other organisms, ranging from

bacteria to mammals. Historically, transgenic plants for phytoremediation were first

developed in an effort to improve heavy metal tolerance; for example, tobacco plants

(Nicotiana tabacum) expressing a yeast metallothionein gene for higher tolerance to

cadmium, or Arabidopsis thaliana over expressing a mercuric ion reductase gene for

higher tolerance to mercury (Misra and Gedamu, 1989; Rugh et al., 1996). Transgenic

narrow-leaved cattails (Typha angustifolia Linn.) created by transforming a

hyperaccumulation gene into this plant, grown in contaminated sites have been reported to



remediate heavy metals (Lincoln and Eduardo, 2002). The first attempts to transform

plants for phytoremediation of organic compounds targeted explosives and halogenated

organic compounds in tobacco plants (French et al., 1999; Doty et al., 2000). These initial

efforts in developing transgenic plants for phytoremediation lead to their applications to

remediate contaminated sites. Transgenic poplars with improved phytoremediation abilities

plants have been applied in the field to remove metal contaminants (Peuke and

Rennenberg, 2005).

Although the motivation to exploit plant cultures in phytoremediation research are

convincing, this approach also has its limitations and drawbacks (Doran, 2009). Plant

tissue cultures cannot represent or simulate many aspects of whole plant cultivation, hence,

the applicability of in vitro cultures depends in many ways on the original purpose of the

research and is neither a practical nor a commercially feasible technology for direct

application in large-scale phytoremediation operations; require sterile culture conditions

throughout the process. However, a rapid progress in plant biotechnology is very

promising to remediate hazardous chemicals using tissue culture technology.

2.7.4 Phytoremediation applicability

Phytoremediation technologies could be subdivided into two broad fields

(Schröder, 2003): the first dealing with the removal of compounds from the environment

by phytoextraction, phytodegradation, phytoaccumulation, and pump-and-treat. The

second field deals with the stabilization of compounds within the site of interest (e.g.

phytostabilization and hydraulic control). Many phytotechnologies are at the

demonstration level, but relatively a few have been applied in practice on large sites. Those

options that may prove successful at a higher scale are (a) phytoextraction of metals, As

and Se from marginally contaminated agricultural soils, (b) phytoexclusion and

phytostabilisation of metal- and As-contaminated soils, (c) rhizodegradation of organic

pollutants and (d) rhizofiltration/rhizodegradation and phytodegradation of organics in

constructed wetlands. Each incidence of pollution in an environmental compartment is

different and successful sustainable management requires the careful integration of all

relevant factors, within the limits set by policy, social acceptance and available finances.

Many plant stress factors that are not evident in the short-term laboratory experiments can

limit the effective deployment of phytotechnologies at field level. The current lack of



knowledge on physicochemical and biological mechanisms that underpin

phytoremediation, the transfer of contaminants to bioavailable fractions within the

matrices, the long-term sustainability and decision support mechanisms should be

understood to identify future R&D priorities that will enable potential end-users to identify

particular technologies to meet both statutory and financial requirements.

There are many remediation techniques available, but due to cost, time, and

logistical concerns, relatively a few are applicable to contaminated soils and waters.

Whichever may be the technique, in general, remediation technologies are concerned with

the two facts: they either remove the contaminants from the substratum (“site

decontamination or clean-up techniques”) or reduce the risk posed by the contaminants by

reducing exposure (“site stabilization techniques”). Numerous plants have been attempted

for phytoremediation of a variety of pollutants on the field, a few of them have been

summarized in the Table 2.3. More than 400 plant species have been identified to have

potential for soil and water remediation. Among them, Thlaspi, Brassica, Sedum, and

Arabidopsis species have been mostly studied for metal remediation (Lone et al., 2008). A

significant uptake and transport of chromium in all the three tree species viz. Azadirachta

indica A. Juss. (Neem), Melia azedarach Linn. (Wild Neem) and Leucaena leucocephala

(Lam) de Wit (Subabool) raised over the tannery sludge suggesting that these plants could

be employed in phytoremediation of soils contaminated with heavy metals (Sakthivel and

Vivekanandan, 2009).

Direct in situ appliances whether to contaminated sites or to industrial effluent

treatment plants could result in the considerable removal of the pollutants, but the positive

modifications in the technology might increase the potentiality of the phytoremediation

process. A few of the advancements in in situ phytoremediation applications include

hydroponics and constructed wetlands for phytoremediation.

2.7.4.1 Hydroponic phytoremediation:

The cultivation of plants and their phytoremediation applicability by hydroponic

cultures has been reported for many of the pollutants (January et al., 2008; James and

Strand 2009; Liu and Shnoor, 2008). Hydroponic solutions are enriched with various

macro and micro nutrients, providing a nutrient status which is closer to that of the soil in

which the plant usually grows and can be used for the cultivation and/or maintenance of



plants for phytoremediation. As a phytoremediation application, the use of hydroponics

provides a cost effective method. Aubert and Schwitzguébel (2004) carried out the

screening of four different plant species (Rheum rabarbarum, Rumex acetosa, Rumex

hydrolapatum and Apium graveolens), in hydroponic solutions for the removal of

sulfonated anthraquinones. Many plant species have the capacity to absorb large quantities

of water from hydroponic solutions. The water absorption capacity of a plant is a factor

that should be considered while performing studies in hydroponic solutions because it

reflects the overall health of the plant. Lower water absorption capacity for the plant R.

acetosa in hydroponic solution indicated that the plant might not be in optimum health

under hydroponic conditions and thus the metabolism and transpiration could probably be

reduced as compared to native growth conditions. Owing to the difficulties in using adult

terrestrial plants such as Rhubarb and common sorrel, plants could be cultured under

hydroponic conditions (Aubert and Schwitzgue´bel, 2004). But, the cultivation and

experimentation with plants in hydroponic systems showed a few major disadvantages that

included the leaves of the same age and same stage of growth and development could not

be collected in case of plants grown in hydroponics (Page and Schwitzgue´bel, 2009), the

level of enzymes like cytochrome P450 and peroxidases changed with the growth of the

plants; which made difficulty in exactly confirming the role of these enzymes in the

detoxification of dyes (Page and Schwitzgue´bel, 2009). To overcome these problems with

wild plants grown in hydroponics or in wetlands for phytoremediation, the importance of

tissue culture based technologies has been stressed.

2.7.4.2 Constructed wetlands:

CW are basically treatment systems that mimic the functions of natural wetlands by

the use of processes involving wetland vegetation, soils and their associated microbial

populations to improve water quality, with the benefit that the specific design of CW

allows higher treatment efficiencies (Davies et al., 2005). The main role of wetland

vegetation is attributed to the modification of soil texture, hydraulic conductivity and soil

chemistry by the growth of plant roots and rhizomes. Phytoremediation with CW uses the

storage of inorganics and the degradation of organics to clean contaminated waters. A

broad range of effluents can be managed, e.g. municipal, domestic and industrial

wastewaters, landfill leachates or products of sludge dewatering, containing organic



pollutants, trace elements or radionuclides (Schröder et al., 2007). Different models of CW

could be engineered to achieve conditions closer to those prevalent at the contaminated

sites or in order of achieving sustainable remediation management. A vertical flow

constructed wetland was designed so as to work in intermittent feeding mode (8 feeding

cycles per day) which enhanced the characteristics like constant hydrolic permeability and

maximized the oxygen transfer rate and was tested for the removal efficiency of the dye

Acid Orange 7 (Davies et al., 2005). CW with a continuous re-circulating system has been

employed for phytoremediation of chromium by two tropical plants Penisetum purpureum

and Brachiaria decumbens individually from tannery waste waters (Mant et al., 2006).

Environmental parameters such as BOD, COD, TOC content, hardness and alkalinity etc

of the industrial effluents were evaluated to assess the applicability of CW as a

phytoremediation technology.

2.7.5 Phytoremediation: pros and cons

Most scientific and commercial interest in phytoremediation now focuses on

phytoextraction and phytodegradation that use selected plant species grown on

contaminated sites. The plants are then harvested to remove the pollutants that have been

accumulated in their tissues and also to get biomass together. Depending on the type of

contamination, the plants can either be disposed off or used in alternative processes, such

as burning for energy production. In essence, phytoextraction removes pollutants from the

contaminated soils, concentrates them in biomass and further concentrates the pollutants

by combustion. And such mixed-benefit strategies could be considered ‘para-

phytoremediation’. This etymological construction recognizes concurrent or post-

remediation uses for phytoremediation plants in addition to the environmental

detoxification (Rugh, 2004).

Some of the metals can be recovered from plant tissue (phytomining) [e.g. humans

have restored potassium (potash) for centuries] that has economic importance (Meagher,

2000). In addition to accumulating toxic minerals in their tissues, plants are also able to

take up a range of harmful organic compounds, including some of the most abundant

environmental pollutants such as polychlorinated biphenyl (PCB), halogenated

hydrocarbons (trichloroethylene, TCE) and ammunition wastes (nitroaromatics such as

trinitrotoluene (TNT) and glycerol trinitrate (GTN)). Subsequent metabolism in plant



tissues then mineralizes or degrades such pollutants to non- or less-toxic (Peuke and

Rennenberg, 2005) Compared with conventional methods of soil remediation, the use of

plants provides several striking advantages. It is cheap: after planting, only marginal costs

apply for harvesting and field management, such as weed control. It is a carbon-dioxide

neutral technology: if the harvested biomass is burned, no additional carbon dioxide is

released into the atmosphere beyond what was originally assimilated by the plants during

growth. Phytoremediation is also a potentially profitable technology as the resulting

biomass can be used for heat and energy production in specialized facilities.

Table 2.4: Phytoremediation- a comparative analysis

Advantages Disadvantages

It is cheap Time consuming, relatively slow technology

It is a carbon-dioxide neutral technology Site availability for phytoremediation

Para-phytoremediation provided better avenues

Toxicity of pollutants to plants

A potentially profitable technology Limited plants can be used

Plants supply nutrients to rhizospheric bacteria

Chance of posing risk to consumers of plants

Production of very less waste (no secondary pollution and wastes)

Mostly depending on root system of plant

Solar-driven Unfamiliar to many regulators

Transfer is faster than natural attenuation

Limited to shallow soils, streams, and groundwater

High public acceptance Mass transfer limitations

Soils remain in place and are usable for following treatment

Only effective for moderately hydrophobic contaminants

Fewer air and water emissionsContaminants may be mobilized into the groundwater

A major disadvantage of phytoremediation is its relatively slow pace, because it

requires several years to remove metal contamination in soil considerably (McGrath and

Zhao, 2003). Furthermore, during the process of phytoremediation, a contaminated site is

not available for sale or rent, which can cause problems for economic development (SRU,

2004). The challenge for plant scientists is therefore to improve the plants’ performance in

removing toxicants from the soil in a short time, which will require more basic research

and knowledge on the natural detoxification mechanisms of plants.



2.8 A Brief Review on Opuntia and Other Cacti: Applications and Biotechnological

Insights

2.8.1 General introduction of Opuntia and other cacti

Cacti are the most conspicuous and characteristic plants of warm arid regions. Cacti

are remarkable for their diversity of growth forms and their ability to not only grow, but to

thrive under environments recognized as stressful for most plant speciess. Cacti can be

used to prevent soil erosion and act as an effective living fence for land reclamation while

functioning as a commercial crop with unique attributes. Cacti do not need much water and

accordingly they exhibit unusual physiological and morphological features (Scheinvar,

1995; Le Houerou, 2000). Cacti are known to contain several useful chemical compounds

having nutritional and medicinal desirable properties (Agurell, 1969; Knishinsky, 1971;

Rosemberg, 1973; Bruhn and Agurell, 1974; Bruhn and Lindgren, 1976; Ferrigni, 1984;

Wang, 1988; Frati, 1990; Fernandez, 1992; Kazemini, 1994; Sahelian, 2001; Fernández-

López, 2002; Alarcon-Aguilar, 2003; Galati, 2003; Oliveira, 2003; Gentile, 2004;

Sirivardhana, 2004; Tesoriere, 2004; Zou, 2005; Saleem, 2006). Cacti have been exploited

as a cheap, alternate source of food suitable for humans and feed for animals and are

cultivated as ornamental crops (Estrada-Luna, 2008). A review has been published that

outlined the significance of cacti highlighting opportunities for utilizing this group of

plants in numerous applications.

The Cactaceae family includes approximately 130 genera and 2000 species, which

were originally native to the New World. Different growth forms or habits of cacti are

depicted in Table 2.5. Opuntia and Nopalea are the most important genera due to their

many applications (Hollis, 1978; Hollis and Mejorada, 1992a,b; Flores-Valdez and Osorio,

1996). Many species of cactus are found growing either as wild plants in arid and semiarid

regions of India. Gurbachan Singh (2003) has reviewed the introduction of cacti into India.

Generally, these species are used as live fences to protect agricultural fields from human

and animal encroachments or as an ornamental plant in urban homes and gardens. With

few exceptions, there has, so far, been no attempt to cultivate this plant as a horticultural or

fodder crop in India. While, in the countries such as Mexico, USA, Spain, Italy, and in

northern Africa, where the crop is commonly known, it already forms an integral part of

the people’s dietary requirement. As exotic plants, a few of the species from Cactaceae



family found in Kolhapur, India are Cereus perucianus, Epiphyllum oxypetatum, Nopalea

cochenillifera, Opuntia elatior and Pereskia aculeata (Yadav and Sardesai, 2002).

Table 2.5: Types of Cacti

Type of Cactus Photograph Example

Columnar cactus Carnegiea (Saguaro), Lophocereus (Senita), Packycereus (Cardon), Stenocereus(Organpipe Cactus)

Barrel cactus Ferocactus (Fishhook Barrel) Echinocactus(Many-headed Barrel)

Hedgehog cactus Echinocereus (Hedgehog Cactus)

Pincushion cactus Mammillaria (Fishhook Pincushion Cactus)

Cholla cactus Opuntia (Chainfruit, Christmas, Staghorn, and Teddybear Cholla)

Prickly pear cactus Opuntia (Prickly Pear)

(Source: http://wc.pima.edu/~bfiero/tucsonecology/plants/cactus_types.htm)

http://wc.pima.edu/~bfiero/tucsonecology/plants/cactuses_sagu.htm



As a part of an Indo-US collaborative research program on Opuntia in India

initiated by Dr. Peter Felker, Texas, USA, 33 Opuntia clones were introduced at the

Nimbkar Agricultural Research Institute at Phalton, India, in 1987. All these clones grew

well under the semiarid agroclimate of western Maharashtra and it is reported that some

clones also produced fruits. In 1991, Central Soil Salinity Research Institute, Karnal

obtained five fruit, forage, and vegetable clones from Dr. Peter Felker’s collection in

Texas. The author of this paper worked with Dr. Felker in Texas for four months as a FAO

fellow. As a followup of this programme, this germplasm exchange occurred. Again, in

January 1997, 51 additional Opuntia clones were introduced from Texas A&M University-

Kingsville at the National Research Centre for Arid Horticulture in Bikaner.

Cacti have evolved to grow in to water scarce environments. Cacti stems are

swollen with water-storage tissues and are sometimes termed stem succulents. Cacti stems

have a thick water proof epidermis covered with a waxy cuticle and many are grooved with

ribs so that the stem can expand and contract without damage to the surface tissues in the

process of storing and utilizing water. These traits evolved to allow the stems to function

as the main photosynthetic system. In fact, most cacti do not produce leaves at all. The

main exceptions are the species of Opuntia (prickly pears and chollas) that have

rudimentary leaves in their juvenile stages. However, even these juvenile leaves are

cylindrical and fleshy, adapted to withstand water loss and drought conditions. Cacti

generally have extensive, shallow root systems, spreading just below the soil surface, so

that they can absorb water, even the relatively small amounts that moisten the soil surface

during light rain showers. Plants in the genus Opuntia and Nopalea prefer dry, hot areas

populated with perennial shrubs, trees, and creeping plants.

Cacti can grow 3 to 19 m in height and can spread over an area of up to 40 m in

diameter. Among cacti, Pachycereus pringlei is recorded as reaching a height of 19.2 m

and Carnegiea gigantea to a height of 15 m with a girth of 3.1 m. Stems of cacti are

branched, green, and broadly oblong to ovate to narrowly elliptic. Areoles, depending on

the species, give rise to spines and/or small, detachable barb-tipped bristles, known as

glochids which may be origin of flowers (Anderson, 2001). Leaves are cylindrical in

shape, and the plant is covered with glochids that are unique to Opuntia. Glochids are

small and are present along the adaxial margin of the areoles and under an inconspicuous



tuft of yellowish hair. This hair on aging turns brown and is composed of 100% crystalline

cellulose (Pritchard and Hall, 1976). Spines, 1 to 6 per areole, may be absent or very

highly reduced, or on the margins of nearly all areoles. Spines are erect to spreading,

whitish, tan or brown, setaceous only, or setaceous and subulate, straight to slightly

curved, basally angular-flattened, or appear as small bristle-like deflexed spines to 5 mm.

Spines are constituted of 96% polysaccharides consisting of 49.7% cellulose and 50.3%

arabinan. The cellulose microfibrils, 0.4 mm length and (6 to 10) mm in diameter, are

loosely imbedded in an arabinan matrix. The latter is partly present as a solid gel, partly

woven tightly with the cellulose fibers (Malainine et al., 2003). Functions of the spines

include mechanical protection from herbivores, reflection of light, and shading of the stem

to reduce water loss and for condensing fog (Anderson, 2001).

Cacti flowers, petals and sepals are numerous in quantity and color. The inner

petals are generally yellow to orange throughout, filaments and anthers are yellow; the

style is bright red, and the stigma lobes yellow. Fruits are oval, pear-shaped, yellow to

orange to purple, fleshy (pulpy), juicy, sweet, glabrous, usually spineless, but they may be

covered with spines or bristles. Areoles on fruit are 45-60 in number and evenly

distributed. The seeds within the pulp are disk-shaped, sub circular and have numerous

colors but are generally pale tan. (Chevallier, 1996; Hocking, 1997; Le Houerou 2000; Van

Sittert, 2002; Defelice, 2004; Synman, 2006; Saleem et al., 2006). O. ficus-indica seeds, as

in many other Opuntia species, have low germination capacity mainly due to their hard

lignified integuments, the most inward of which is the funiculus that envelops the embryo

and obstructs radicle protrusion. Certain physical and chemical treatments can overcome

low seed germination (Altare et al. 2006, Ochoa-Alfaro et al. 2008).

2.8.2 Applications of Opuntia and other cacti

2.8.2.1 Health Benefits of Cacti:

Cacti (Opuntia) have been used for centuries as common vegetables and medicines

by Native Americans to treat a variety of ailments and disorders (Knishinsky, 1971; Kay,

1996; Cornett, 2000; Tesoriere et al., 2004). The nutritional properties of the fresh stems

(cladodes) have long been known (Frati, 1990; Fernandez, 1992). Prickly pear fruits are a

rich source of flavanoids, including kaempferol and its methyl ether, quercetin and its

methyl ether, narcissin, dihydrokaempferol (aromadendrin, 6), dihydroquercetin, and



eriodictyol (Table 2.6). Flavanoids, as those in prickly pear fruit, are known for their

affirmative health-benefits (Knishinsky, 1971; Iwashina et al., 1984, Wang, 1988;

Goycoolea and Cardenas, 2003; Tesoriere et al., 2004). Cacti are reported to produce

alkaloids, which is a characteristic feature of Cactacea family (Agurell, 1969; Rosemberg

and Paul, 1973; Bruhn and Agurell, 1974; Bruhn and Lindgren, 1976; Gabermann, 1978;

Ferrigni et al., 1984).

Table 2.6: Active components of cacti

Name of the component

Structure Occurrence Reference

A. Flavanoids Kaempferol

O. ficus-indicaO. lindheimeriO. streptacanthaO. ficus-indica var. Saboten

Kuti, 2004;Lee et al., 2003

.Quercetin

O. ficus-indica Kuti and Galloway, 1994

Narcissin Opuntia dillenii Qiu et al., 2007

B. Antioxidants Pectin

Opuntia ficus-indica var Ia O. ficus-indica var IIa O. spp. (Blanca I)O. spp (Blanca II)O. amylaceaa O. megacanthaa O. steptracanthaa O. robustaa

Goycoolea and Cardenas, 2003



Carotenes (α and β)

Opuntia ficus-indica L Ramadan and Morsel, 2003

Betalains Opuntia ficus-inadicaL, Opuntia stricta, Opuntia undulata

Gentile et al., 2004; Fernández-Lopez et al., 2002

Ascorbic acid

O. ficus-indicaO. lindheimeriO. streptacantha

Kuti, 2004

Prickly pear cacti have shown promise for diabetes treatment. Clinical trials

indicate that cacti help to stabilize blood sugar levels, and are effective for the treatment of

type-II (adult onset) diabetes (Alarcon-Aguilar, 2003). Research has shown that cacti can

help to reduce the effects of excessive alcohol consumption if used prior to drinking

alcoholic beverages (Wiese et al., 2004).

In Chinese medicine, cactus fruits are considered weak poisons and are used for

treatment of inflammation, pain and as detoxification agents for snake bites (Wang, 1988).

Aqueous extracts of prickly pear cacti were found to inhibit in vitro cell growth effectively

in several different immortalized and cancer cell cultures including ovarian, cervix, and

bladder cancer cells and suppressed tumor growth in a nude mouse of ovarian cancer

models. The mechanism of anticancer effect of cactus pear extracts is not understood (Zou

et al., 2005). Prickly pear cacti are well recognized for their wound healing properties and

anticancer effects (Zou et al., 2005, Galati et al., 2003).

2.8.2.2 Food industry applications of cactus pear:

The use of natural sweeteners other than sucrose is a priority area for the food

industry. Saenz et al. (1998) demonstrated that cactus fruit can be used as a raw material in

the creation of natural sweeteners. Sawaya et al. (1983) attempted to use prickly pear fruits

in the manufacturing of jam. Cactus pear juice has been used as a health drink. Orange-



yellow cactus pear pulp has been used to produce a dehydrated cactus pulp sheet and

pasteurized cactus pear juices (El-Samahy et al., 2007). Sáenz and Sepúlveda (2001)

reported technological characteristics and main difficulties in producing high-quality

cactus pear juice. Red cactus pear juice contains betalain and green cactus pear juice

contains chrolorphyll. Heat treatment of the juices affects cactus pear juice color, but the

purple juice is more stable. Blends with other fruit juices (e.g. pineapple) could enhance

the quality of the juices. Cactus pear juice has also been used as a good feedstock for

fermentation using Saccharomyces cerevisiae as the fermentation microorganism (Turker

et al. 2001). Turker and co-workers (2001) reported that the fermentation process did not

affect the thermo stability of the betalain pigments obtained from the cactus pear. Pulp of

the red cactus pear (O. ficus–indica) has been used for ice cream production (El–Samahy et

al., 2009).

Sáenz (1997) reported the potential use of cactus cladodes as a new source for fiber

in human diet. Based on cactus pear (O. boldinghii) stems (cladodes) having high fiber

content and potential health benefits, bakery products have been produced and evaluated

from composite flours of wheat and cactus pear stems (Moreno–Álvarez et al., 2009).

The quality of nopalitos (O. ficus-indica) during cold storage suffers weight loss

and chilling injury. These disorders have been reduced in several fruits and vegetables,

including cladodes or nopalinas, by covering the product with edible coatings. Rodríguez-

Félix et al. (2007) reported the effect of edible coatings on nopalitos of Opuntia sp. during

cold storage. The effect of application of two edible coatings (Semperfresh® and another

formulation based on carboxymethylcellulose, CMC, named Wax 1) on the quality of

nopalitos of two cultivars stored at 5 °C and 10 °C for 30 d was evaluated.

Cerezal and Duarte (2004) studied the effect of a combination of common food

additives as sodium bisulphite (0, 50 and 100 ppm), phosphoric acid (50% v/v), citric acid

(50% v/v), ascorbic acid (500 ppm), calcium chloride (120 ppm) and potassium sorbate

(1000 ppm), on the sensory quality and storage of nopalitos canned in glass jars.

Apart from food applications of cacti for human consumption, cactus pear has been

used as a supplemental feed for animals including small goats (Fuentes-Rodrίguez, 1997)

and for finishing lambs Aranda-Osorio (2008).



2.8.2.3 Cacti for phytoremediation:

In vitro cultures of cacti were used to test the effect of degradation and/or

biotransformation of dye precursors by plants (Golan-Goldhirsh et al., 2004). Cacti were

shown to produce protective compounds, such as alkaloids, from a family of aromatic

hydrocarbons (Oliveira and Da Machado, 2003). Thus, it was hypothesized that the

metabolic pathways and enzymes related to biotransformation of xenobiotic aromatics

would be more likely to be found in these plants (Golan-Goldhirsh et al., 2004).

Synthesis of metal-binding peptides, known as phytochelatins, is one defense

mechanism used by higher plants in exposure to non-essential metal/metalloids or toxic

concentrations of essential elements. Phytochelatins synthesis has been evaluated in

cladodes of O. ficus in relation to plant and soil levels of Cd, Pb, Cu and Ag (Figueroa et

al., 2007). The feasibility of genetic engineering to assure higher rates of phytochelatin

induction has been explored for phytoremediation purposes (Wojas et al., 2008; Guo et al.,

2008; Couselo et al., 2010).

Ancient seas once covered the west side of California's San Joaquin Valley (SJV),

and those seas left behind marine sediments, shale formations and deposits of selenium and

other minerals in the soil. Crops grown there need to be irrigated, but the resulting runoff,

when it contains high levels of selenium, can be toxic to fish, migratory birds, and other

wildlife that drink from waterways and drainage ditches. Selenium runoff is subject to

monitoring by regional water quality officials. Gary Bañuelos from U.S. Department of

Agriculture (USDA) has discovered that a drought-tolerant cactus may be an effective tool

for cleaning up soils and waterways in parts of the Valley (O'Brien, 2012).

For this water-scarce region, Opuntia ficus-indica (prickly pear cactus), a plant

species that can be found across the world was grown with minimal water. Opuntia’s low

requirement of water could be especially advantageous for introducing this crop to semi-

arid agricultural regions where water supplies are limited. Bañuelos and Cantonese has

previously evaluated the tolerance of prickly pear cactus to poor quality drainage water

under greenhouse conditions (unpublished observation, 2007). Based upon their

preliminary greenhouse observation with cactus and Se-contaminated drainage water,

Bañuelos hypothesized that cactus might be a potential and unique Se-biofortified crop

species for growing in the Se-laden and arid environment in the westside of the SJV.



Further, the ability of five prickly pear cactus clones [Opuntia ficus-indica (L.) Mill., no.

243, 248, 250, 252, and 255] to grow, accumulate and volatilize Se from drainage sediment

high in sulphate salinity, soluble B and Se concentrations has been evaluated (Bañuelos

and Lin 2010) and concluded that the successful growth of prickly pear cactus and its

accumulation and volatilization of Se under adverse soil conditions may provide growers

with an alternative Se-biofortified crop in the westside of the SJV in central California.

While Opuntia has been studied for its tolerance to high salinity, aridity and high

temperatures, the fundamental mechanisms determining the tolerance responses of this

species to salinity, B, and Se are not well understood (Amzallag et al., 1990; Murillo-

Amador et al., 2001).

2.8.2.4 Use of cacti for degraded lands:

Land erosion and degradation coupled with levels of water insufficient for most

agriculture systems are serious problems posing threat to agricultural sustainability. The

arid and semi-arid areas of North Africa are becoming deserts (Alary et al., 2007). Most

research and development projects in these areas aim at developing alternative

technologies to reduce land degradation and favor sustainable economic activities. The

‘spineless cactus-alley cropping system’ is an interesting approach for the low rainfall

areas of North Africa since it limits land degradation by the use of a perennial crop for the

production of cheap and drought resistant sources of feed and biomass in the inter row

spaces between food crops. A bio-economic model has been developed to identify the

conditions for applying the ‘spineless cactus-alley cropping system’ in an agro-pastoral

community of Central Tunisia. Findings suggested that extension services will play a

crucial role in creating awareness among farmers of the impact of this technology on yields

and income diversification (Alary et al., 2007).

2.8.2.5 Additional potential benefits of cacti:

Several bioassays by Han et al. (2001) indicated inhibitory activity of monoamine

oxidase from methanolic extracts of cacti fruits. Four compounds isolated were tested for

in vitro monoamine oxidase inhibitory activities and the authors concluded that industrial

applications for these molecules may be found through further investigations (Han et al.

2001). Cactus pear cladodes have been used in many building construction applications in

Mexico, since ancient times. Hernández-Zaragoza et al. (2008) found that mortar made



with cladodes as an additive had a greater compressive strength than mortars made without

the additive and suggested that cactus pear acts as a retardant of slowing of the setting time

in mortar manufacturing process. An electrochemical cell was fabricated by Deshpande

and Joshi (1994) from cactus cladodes acting as an electrolyte. The discharge

characteristics revealed that, at a current drain of 100 φA, the cell gave an optimum energy

density of 175 mWh/kg. The power generated by these cells was sufficient to run a

piezoelectric buzzer and a LCD calculator for a few hours. This work opens up a new

interdisciplinary area for physicists, botanists and electro-chemists.

2.8.3 Plant biotechnology research on cacti

Methods for propagation of succulent plants are by seeds, cuttings, or grafting

(Krulik, 1980). Cacti can be propagated from seeds, but seed propagation has problems of

genetic segregation, slow growth, and a long juvenile stage compared to asexually

propagated material (Mohamed-Yasseen et al., 1995). Tissue culture can be an excellent

tool for cacti micropropagation and has been used on species in the genera Cereus,

Equinocereus, Ferocactus, Mammillaria, and Opuntia (Escobar et al., 1986; Machado and

Prioli, 1996; Pérez-Molphe et al., 1998; Juárez and Passera, 2002) (Table 2.7). Malda et

al. (1999) demonstrated in vitro culture as a potential method for the conservation of

endangered plants, including cacti, that possess crassulacean acid metabolism. Massive in

vitro production of new propagules resulted in regenerated plants for two endangered cacti,

Obregonia denegrii Fric. and Coryphantha minima Baird.

Khalafalla et al. (2007) demonstrated the micropropagation of cactus (O. ficus-

indica) to combat desertification in arid and semi arid regions. These workers studied the

effects of benzyladenine (BA) and kinetin (Kin) alone or in combination with 1-

naphtalenacetic acid (NAA) on morphogenetic responses of O. ficus-indica on Murashige

and Skoog (MS) medium (Murashige and Skoog, 1962). This work demonstrated that BA

at the highest concentration (5.0 mg/L) gave the highest number of shoot per explant

(26.5±1.74) after three months of culture. The combinations of NAA and the cytokinins

BA and Kin did not increase the number of shoots per explant. The effect of two different

media on in vitro growth of cactus (O. ficus-indica) explants was studied by Aliyu and

Mustapha (2007). Medium A contained MS basal salt and vitamins supplemented with 5%

sucrose and 1% BA; medium B contained vitamins and MS salt supplemented with 3%



sucrose, 1.25 mg/L BA, and 0.25 mg/L IAA. There were no significant differences in shoot

height or days to shoot emergence between the two media over a period of 30 d.

Estrada-Luna et al. (2008) established conditions for micropropagation of the

ornamental prickly pear cactus O. lanigera Salm–Dyck through axillary shoot

development from isolated areoles and studied the effects of sprayed gibberelic acid (GA3)

after transplantation to ex vitro conditions. Escobar et al. (1986) studied the effect of BA

on axillary bud development in O. amyclaea and reported the best response at 10 µM.

Llamoca- Zárate et al. (1999a) proposed 2.2 µM BA while Mohamed-Yasseen et al.

(1995) reported 8.8 µM BA as optimal for O. ficus-indica micropropagation.

Many cacti produce an excess of auxin under in vitro culture conditions, and this

stimulates callus production (Clayton et al. 1990). Garcίa-Saucedo et al. (2005) developed

a micropropagation system for three Opuntia lines used as vegetables in Mexico. Balch et

al. (1998) developed a micropropagation system for 21 species of Mexican cacti

(Astrophytum, Cephalocereus, Coryphantha, Echinocactus, Echinocereus,

Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, Stenocactus) using explants

from seedlings germinated in vitro or shoot segments of juvenile 2-3 year old greenhouse

plants. Several workers have achieved success in micropropagation of species including

Astrophytum myriostigma, Mammillaria carmenae, M. prolifera, and Trichocereus

spachianus (Vyskot and Jara, 1984), Echinocereus engelmanii, E. pectinatus, Ferocactus

covillei and F. wislizenii (Ault and Blackmon, 1985), Ferocactus acanthodes (Ault and

Blackmon, 1987), Mammillaria san-angelensis (Martínez-Vázquez and Rubluo, 1989),

Mediocactus cocciaeus (Infante, 1992) and Cereus peruvianus (Machado and Prioli, 1996),

Turbinicarpus laui (Rosas et al., 2001). The effect of different growth regulators on the

morphogenesis of O. polycantha, a non-edible Opuntia, has been determined by Mauseth

and Halperin (1975) and Mauseth (1976).

De Medeiros et al. (2006) worked to establish a protocol for in vitro culture and

plant regeneration of Notocactus magnificus, an ornamental species called the blue cactus

that is native to Brazil. These workers observed callus formation in Notocactus magnificus

when the explants were cultured on MS medium supplemented with sucrose at 2% (w/v),

2,4-dichlorophenoxyacetic acid 0.5 µM, BA 4.4 µM, thiamine HCl 0.4 mg/L and i-inositol

100 mg/L. The highest number of shoots was obtained when MS medium was



supplemented with benzylaminopurine 22.2 µM, sucrose 3% (w/v) and agar 0.6% (w/v). In

vitro spontaneous rooting of shoots was observed after eight months on MS medium. In

vitro rooted shoots developed into normal plants under glasshouse culture conditions.

Table 2.7: Plant biotechnology research on cacti

Cactus species PurposePlant growth

regulatorsReference

Nopalea cochenillifera micropropagationBA, IAA, NAA

Brasil et. al. (2005),Houllou-Kido et. al. (2009)

O. ficus-indicamicropropagation BA, Kin

Khalafalla et al. (2007)Mohamed-Yasseen et al. (1995)

callus and cell suspension culture

FAP, 2,4-D, ATCPA

Llamoca-Zárate et al. (1999b)

O. lanigera micropropagation Estrada-Luna et al. (2008)

Opuntia (three lines) micropropagationGarcίa-Saucedo et al. (2005)

Astrophytum,Cephalocereus, Coryphantha, Echinocactus, Echinocereus, Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, Stenocactus

micropropagation Balch et al. (1998)

O. polycantha morphogenesisMauseth and Halperin (1975) and Mauseth (1976)

Notocactus magnificusin vitro culture and plant regeneration

2,4-D and BA

De Medeiros et al. (2006)

Cereus peruvianus Callus inductionOliviera and Da Machado, 2003

Advantages of the propagation systems outlined above include (1) the time required

for production of cacti shoots by microprogagation is quite short compared to the time

required for germination of seedlings (Ault and Blackmon, 1985) and (2) micropropagated

cacti from axillary buds are genetically stable compared to other methods of in vivo

propagation (Vyskot and Jara, 1984; Machado and Prioli, 1996).



Relatively little work has been reported on the establishment of callus and cell

suspension cultures of cacti. Llamoca-Zárate et al. (1999b) developed a method for

establishment of callus and cell suspension culture of O. ficus-indica. Friable callus

cultures were initiated when cotyledons and hypocotyls of O. ficus-indica were used as

explants. Explants from cotyledons produced significantly more callus than those from

hypocotyls. Optimum callus growth was observed on MS medium supplemented with 0.9

μM 6-furfurylaminopurine (FAP), 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 μM

4-amino 3,5,6-trichloropicolinic acid, 400 mg/L casein hydrolysate and 3% sucrose. The

same medium without agar was used for establishing cell suspensions.

Callus tissue of Cereus peruvianus was established and used for alkaloid

production (Oliveira and Da Machado, 2003). Morphologically undifferentiated callus

cells of Cereus peruvianus cultured in the original medium and in medium supplemented

with tyrosine were used as the alkaloid source. Culture medium supplemented with

tyrosine showed higher levels of alkaloids than the original medium. Alkaloid levels were

almost twice as high in callus tissues as in shoots of C. peruvianus plants. The ratio of

alkaloid concentration between mature plant shoots and morphologically undifferentiated

cells of callus tissue was 1:1.7. Since increased alkaloid production can be induced by

factors such as tyrosine, the levels of tyrosine or other conditions of the culture medium

may be responsible for the higher levels of alkaloid production by the callus tissues.

2.8.4 Plant biotechnology research on Nopalea cochenillifera

Brasil et al. (2005) established a protocol for the mass propagation of N.

cochenillifera (Table 2.7). Areoles were isolated from young cladodes of field-grown

plants and cultivated in Murashige and Skoog (MS) medium supplemented with 6-

benzyladenine (BA) at 7.5 μM and indolacetic acid (IAA) at 1.0 μM. Axillary bud

proliferation was achieved by transferring explants containing up to 8 areoles to MS

medium supplemented with BA at 7.5 μM and IAA at 0.2 μM. Rooting was achieved by

transferring in vitro grown shoots to hormone-free MS medium. The plantlets successfully

survived acclimatization ex vitro. By using the protocol by Brasil et al. (2005), it was

possible to reach a propagation rate of up to 20 individuals for each cycle of multiplication.

Houllou-Kido and his coworkers’ (2009) study aimed to evaluate the apical

meristem absence effect on in vitro development of fodder cactus pear plants (N.



cochenillifera, IPA Sertânia clone) in the growth, micropropagation and shoot tip rooting.

Explants were cultivated in induction medium [Murashige and Skoog salts, supplemented

with 6-benzylaminopurine (BAP) and naphthalene acetic acid (NAA)]. The treatments

used were: (1) explants with or without apical dominance breakdown; (2) horizontally or

vertically inoculated in the medium. Apical dominance breakdown induced a significant

increase of the number of new adventitious shoots tip formed from in vitro explants of N.

cochenillifera. The same medium was used for in vitro shoot tips rooting. There was no

influence of apical meristem absence on root development. All micropropagated plants

were transferred to greenhouse and all of them survived acclimatization process.

2.8.5 Genetic engineering and molecular biology studies on cacti

Molecular marker characterization of plants in the germplasm banks reduced the

time for selection of elite genotypes. Souto Alves et al. (2009) attempted to evaluate the

level of genetic diversity in cactus clones by molecular markers to gather information for

future breeding programs. Wang et al. (1998) analyzed eight Opuntia species consisting

of five fruit varieties from Mexico and Chile, two ornamental Texas accessions, and one

vegetable accession from Mexico. Differences among fruit cultivars were smaller and the

results demonstrated the potential for use of RAPD markers to distinguish cacti from one

another.

DNA polymorphisms in callus tissues of Cereus peruvianus Millwere detected by

using RAPD markers (Mangolin et al., 2002). There was a higher level of genetic

variability in callus tissue maintained with the highest kinetin (8.0 mg/l) versus 2, 4-D

(4.0 mg/l) concentration. RAPD analysis was also used to estimate the genetic

relationship among five Hylocereus and nine Selenicereus species (Tel-Zur et al., 2004).

Twenty-two cactus pear varieties were characterized with RAPD and ISSR markers using

DNA from seeds of Opuntia spp. (Luna-Paez et al., 2007). The RAPD and ISSR marker

profiles revealed differences between the varieties and species under study.

Expression of ripening-related genes in fruit of prickly pear (Opuntia sp.) was

studied by Collazo-Siques et al. (2003) and concluded with the agreement that an active

metabolic role of ethylene during non climacteric prickly pear fruit ripening.

A system for genetic transformation of an elite prickly pear cactus (O. ficus-indica

L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed by Silos-Espino et



al. (2006) and the method described may be useful for routine transformation and

introduction of agronomically important genes in prickly pear cactus.

2.9 Characteristics of Nopalea cochenillfera Salm. Dyck.

Erect shrubs, trunk thick, branches ascending with obovate-oblong joints, spineless;

glochids numerous; flowers 4-5cm long, sepals and petals scarlet, petals longer than

sepals, stamens pinkish, exerted. Native of Mexico, grown in gardens and also found

escape near human habitation

Location: Ichalkaranji, Kolhapur (Yadav and Sardesai, 2002)



2.10 Research Objectives

The main aspect of the present dissertation is the evaluation of the

phytoremediation capabilities of cactus plant (N. cochenillifera) for removal of various

environmental pollutants viz. textile dyes, pesticides (from paint preservative Troysan

S89) and hexavalent chromium [Cr(VI)]. Many of the plants have been reported for

phytoremediation of numerous pollutants but the need of searching for an ideal candidate

plant that can remove maximum number and higher concentrations of pollutants with ease

effectively and economically is enduring. Hence, the present research work is to meet

phytoremediation potential of cactus with the criteria of the ideal phytoremediation plant.

The outline of the present thesis is as follows:

Checking phytoremediation ability of N. cochenillifera plant for removal of pollutants viz.

textile dyes, Troysan S89 and Cr(VI)

Establishment of micropropagation protocol and acclamation to soil for rapid

multiplication of N. chenillifera

Establishment and growth characterization of callus and cell suspension cultures of N.

cochenillifera and Blumea malcolmii

Decolorization and optimization for removal of textile dyes by in vitro cultures of N.

cochenillifera

Phytotoxicity analysis of Red HE7B and its degradation metabolites

Determination of the activities of enzymes such as lignin peroxidase, tyrosinase, laccase,

DCIP reductase, azoreductase, MG reductase etc. in these plants that could play active

roles in the degradation of dyes

Spectroscopic (UV-Vis, FTIR and GC-MS) and chromatographic (HPLC, HP-TLC and

GC-MS) analysis of metabolites obtained after degradation of textile dye (Red HE7B) by

N. cochenillifera cell cultures

Detoxification of Troysan S89 by cell suspensions of B. malcolmii and N. cochenillifera

Spectroscopic (UV-Vis and FTIR) and toxicity analysis (phytotoxicity, carcinogenicity and

cytogenotoxicity) of Troysan S89 and its detoxified metabolites

Phytoextraction of Cr(VI) by in vitro shoots of N. cochenillifera

Spectroscopic analysis (AAS) of Cr accumulation and checking hyperaccumulation

capacity of N. cochenillifera by using bioconcentration factor and translocation factor

review of literature -...

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