Biofertilizer For Bioremediation: January 2015

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Biofertilizer for Bioremediation
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13
Biofertilizer for Bioremediation
Subash Chandra Santra, Anusaya Mallick and Alok Chandra Samal
Department of Environmental Science, University of Kalyani, West Bengal, India
E-mail: scsantra@yahoo.com
ABSTRACT
Industrial revolution is the mother of environmental pollution. This affects the quality of life and
ecosystem. To reduce the environmental pollution, various attempts are being taken in different
ways. Among these attempts bioremediation is one of the good remedial techniques. In this technique
microorganisms are used to reduce the toxicity of harmful substances from contaminate environments.
Bioremediation solves the problem in the field of solid and liquid wastes by the process of detoxification.
It is an attractive clean-up technology due to its cost effectiveness and environmental-friendly nature.
Biofertilizer is a large population of a specific or a group of beneficial microorganisms incorporated
aseptically into sterile carrier materials. Microorganisms perform several important processes such as
cycling of nutrients and degradation of various compounds. In sustainable agriculture, algae, bacteria
and fungi are being utilized as biofertilizers. Plant growth promoting bacteria (PGPB) plays important
role in soil fertility and can be used as biofertilizers that stop deteriorations of soil caused by excessive
application of chemical fertilizers. Different microbes were utilized in degradation of different
environmental contaminants like hydrocarbons, heavy metals, and toxic substances. In recent years
researchers are carried out to study the effect of bioremediation by using biofertilizer in reducing the
environmental hazards.
Keywords: Microorganisms, Biofertilizer, Bioremediation, Biodegradation.
13.1 INTRODUCTION
Biofertilizer use is the best modern tools for sustainable agriculture. Biofertilizer is an
important component of integrated nutrient management in agricultural system. It is a cost
effective and renewable source of plant nutrients to supplement the chemical fertilizers.
The commercial history of biofertilizers began by Nobbe and Hiltner, a laboratory culture
of Rhizobia in 1895, followed by the discovery of Azotobacter and then the blue green
algae and a host of other microorganisms. In India the first study on legume Rhizobium
symbiosis was conducted by N.V. Joshi and the first commercial production started in
middle of 1950s.
206 Recent Trends in Biofertilizers
Biofertilizers are the most advanced biotechnology tool to support developing organic
agriculture, sustainable agriculture, green agriculture, and non-polluting agriculture.
This bioorganic fertilizer can increase the output, improve the quality in agriculture. In
recent years the biofertilizer has been widely used with excellent results in agriculture all
over the world. It is well known that continuous and excess use of chemical fertilizers and
pesticides have caused a detrimental effect to our environment and health. The alternate
sources of environment-friendly plant nutrients are the immediate need for the recent
times. Biofertilizers has become a hope for the future world as far as economical and
environmental viewpoints concerned. Especially in developing countries like India it can
solve the problem of high cost of chemical fertilizers and help in saving the economy of
the country [1, 2]. The disposal of bio-digested slurry after biogas production is a major
concern for the environment [3-8]. It contains considerable amount of plant nutrients and
helps to improve crop production, also preventing adverse environmental impacts of
waste disposal [9, 10]. The application of biodigested slurry in agricultural field increases
the soil fertility by fixing atmospheric nitrogen, solubilize soil phosphates and produces
plant growth substances in the soil. The role and importance of biofertilizers in sustainable
crop production has been reviewed by several authors [11-13]. The degradation of
organophosphorus insecticides, chlorpyrifos, chlorpyrifos – methyl, cyanophos and
malathion in mineral salts media were studied by several researchers [14-18]. The effect
of additional biofertilizers, individually or combined with organic amendments, on
chlorpyrifos and cyanophos degrading activity in soil were investigated. Paenibacillus
polymyxa (Bacillus polymyxa) and Azospirillum lipoferum (Beijerinck) were found to degrade
the organophosphorus insecticides, chlorpyrifos, chlorpyrifos — methyl, cyanophos and
malathion in mineral salts media as a carbon and phosphorus source. Bacillus polymyxa
appeared to be more effective than Azospirillum lipoferum in degrading all the tested
organophosphate pesticides in mineral salts media.
There is a continuous interest in algal based waste stabilization pond systems that
are inexpensive and are known for their ability to achieve good removal of pathogens
and organic pollutants all over the world [19]. Cyanobacteria and microalgae plays
an important role by supplying molecular oxygen to heterotrophic partners and thus
support the initial steps of degradation [20]. Nutrient removal with aid of algae compares
favourably with other conventional technologies [21-23]. It also found that some
cyanobacteria and algae might remove xenobiotics from the environment by sorption,
transformation and degradation [24]. Several attempts have been made to explore the
efficiency of microalgae for metal removal [25-27]. Increasing the light intensity can lead
to a higher microalgal activity and an increased removal of nutrients from wastewater
[28].
Phycoremediation is the process of employing algae for improving water quality
which can fix carbon dioxide by photosynthesis and remove excess nutrients effectively
at minimal cost [29-31]. The use of algae in purification of wastewater and to eliminate the
nutrients was studied widely [32, 33]. Additionally, these photosynthetic microorganisms
are also useful in bioremediation applications and as nitrogen-fixing biofertilizers [34-36].
Anabaena is capable of evolving hydrogen through the process of indirect biophotolysis
of water, using nitrogenase as a catalyst. Hydrogen stands a promising alternative to
Subash Chandra Santra, Anusaya Mallick and Alok Chandra Samal 207
fossil fuels, being renewable, eco-friendly and efficiently [37]. Photobiological hydrogen
production by cyanobacteria is an ideal process, owing to their simple nutritional
requirements from air, water, mineral salts, and light as the main energy source [38,
39]. There are several reports concluding that the metal-binding capacities of several
biomasses including marine algae, fungi, and yeast are very high [40-44].
13.2 MICROBES IN BIOFERTILIZER PRODUCTION

Biofertilizers are products containing living cells of different types of microorganisms
which, applied to seed, plant surface or soil, colonize the rhizosphere or the interior of
the plant and promotes growth by converting nutritionally important elements (nitrogen,
phosphorus) from unavailable to available form through biological process such as
nitrogen fixation and solubilization of rock minerals [45]. Beneficial microorganisms in
biofertilizers accelerate and improve plant growth and protect plants from pests and
diseases [46]. There is important role of soil microorganisms in sustainable development
of agriculture [47-48]. Organisms that are commonly used as biofertilizers component are
nitrogen (N) fixers, potassium (K) solubilizer and phosphorus (P) solubilizer, or with the
combination of molds or fungi. Most of the bacteria included in biofertilizer have close
relationship with plant roots. Rhizobium has symbiotic interaction with legume roots,
and Rhizobacteria inhabit on root surface or in rhizosphere soil.
13.2.1 Nitrogen-Fixing Microorganism

Nitrogen is one of the major important nutrients essential for plant growth. Nitrogen
fixation in soil by different microorganism is a complex biochemical process by which
atmospheric elemental nitrogen (unavailable to plants) is transformed into organic
nitrogenous compound, which is available to plants. Different species of bacteria, some
actinomycities and blue-green algae are agriculturally important Nitrogen fixers. The
value of nitrogen-fixing legumes in improving and higher yield of legumes and other
crops can be achieved by the application of biofertilizers [49]. Nitrogen-fixing bacteria
(NFB) that function transform inert atmospheric N2 to organic compounds usable for
plants [50-51]. Nitrogen fixer organism are used in biofertilizer as a living fertilizer
composed of microbial inoculants or groups of microorganisms which are able to fix
atmospheric nitrogen. They are grouped into free-living bacteria (Azotobacter and
Azospirillium) and the blue-green algae and symbionts such as Rhizobium, Frankia
and Azolla [52]. The N2-fixing bacteria associated with non-legumes includes species
of Achromobacter, Alcaligenes, Arthrobacter, Acetobacter, Azomonas, Beijerinckia, Bacillus,
Clostridium, Enterobacter, Erwinia, Derxia, Desulfovibrio, Corynebacterium, campylobacter,
Herbaspirillum, Klebsiella, Lignobacter, Mycobacterium, Rhodospirillum, Rhodo-pseudomonas,
Xanthobacter, Mycobacterium and Methylosinus [53].
13.2.1.1 Symbiotic nitrogen-fixing microorganism for leguminous plants
Symbiotic bacteria infecting plant roots, numerous taxa of less intimately associated
N2-fixing bacteria can be considered for improvement of crop yield. Examples of such
bacteria include Acetobacter diazotrophicus and Herbaspirillum spp. associated with sugar
cane, sorghum, and maize [54-56], Azoarcus spp. associated with kallar grass (Leptochloa
fusca) [57], and Alcaligenes, Azospirillum, Bacillus, Enterobacter, Herbaspirillum, Klebsiella,
Pseudomonas, and Rhizobium associated with rice and maize [58]. The genus Azospirillum
colonizes a great variety of annual and perennial plants, many of which have never been
reported to be colonized by N2-fixing bacteria. Accordingly, Azospirillum possesses a
great potential as a general root colonizer, whose use is not limited by host specificity
[59]. Several studies indicate that Azospirillum can increase the growth of various crops,
include sunflower, carrot, oak, sugar beet, tomato, eggplant, pepper, and cotton in
addition to wheat and rice [59, 60].
Some species of Rhizobium bacteria forms nodule by colonizing in the roots of
different leguminous plants. They derive carbohydrates (energy) and water from the root
tissues of the host plants, which enables them to directly fix atmospheric nitrogen for the
production of amino acids and protein to make cell materials. In return, the host plant
received the mentioned nitrogenous compounds synthesized by the bacteria for their
own growth and development.
13.2.1.2 Free-living or non-symbiotic nitrogen-fixing microorganism
Many free-living bacteria also fix atmospheric N2 like Azotobacter, Beijerinckia, and
Clostridium. In favourable condition nodulating bacterial symbionts (e.g., Frankia) of plant
roots can also fix N2 without symbiotic association (free-living) with their plant host [61].
More interestingly, it has been found that Frankia can occur and possibly fixes atmospheric
N2 in the rhizosphere of non host plants. Frankia has been recorded in the rhizosphere of
Betula pendula and in soil where actinorhizal plants were not present [61, 62]. Certain free-
living microorganisms exist in soil and water is able to fix atmospheric nitrogen without
symbiosis. These organisms are mainly bacteria (i.e., Azotobacter, Clostridium, Verxia,
Azospirillum, etc.). Among the free-living nitrogen-fixing bacteria, Azotobacter is most
important. They colonize in rhizosphere and derived their energy by oxidation of soil
organic matter and fix atmospheric nitrogen into the soil and subsequently increase the
soil fertility.
13.2.1.3 Blue green algae and azolla
N-fixing blue-green algae (BGA) or cyanobacteria and Azolla (free floating water-borne
fern), have been shown to be the most important in maintaining and improving the
productivity of rice fields [63]. It has been demonstrated that the N fertility of soil is
sustained better under flooded conditions than under dryland conditions [64]. Favourable
conditions for biological N2 fixation by such BGA is considered to be one of the reasons for
the relatively stable yield of rice under flooded condition. Unlike chemical N fertilizers,
BGA and Azolla neither contaminate the environment nor consume the photosynthates of
rice plants [65]. The importance of N2- fixing BGA was first recognized by De [66, 67], who
attributed the self-maintenance of the N status of tropical rice-field soils to the growth of
N2-fixing BGA. Similarly, the fertilizing value of Azolla in rice fields is well-known and
has been utilized over year old in China and Vietnam [68, 69]. The plant-available N of rice
soils is increased considerably by the growth of N2-fixing BGA and Azolla [70-80]. BGA
liberate extracellular organic compounds and photosynthetic O2 during their growth,
while Azolla prevent a rise in the pH, reduce water temperature, curb NH3 volatilization
and suppress weeds; and both of them contribute biomass.
The symbiotic association of Anabaena and Azolla produces 40-60 tonnes organic matter
and at the same time they are capable of fixing 100 to 150 kg of atmospheric nitrogen per
hectare per year. In India, their use as biofertilizer is yet to be popularized and their
location specific and thermo-sensitive cultures are not easily available in the market.
Another difficulty in mass production of these biofertilizer is costly.
13.2.2 Phosphate Solubilizing Microorganisms

Phosphorus is one of the least mobile elements in soil. In most soils (especially in acid
soil), soluble phosphatic fertilizers locked in the soil by reacting with calcium, ferrous
and aluminium and converted into insoluble phosphate. Phosphorus is the second most
limiting plant nutrient after nitrogen [81]. Total P content in soil is usually high, but most
of this soil P pool is not in forms available for plant uptake. Bacteria that can mobilize P
from unavailable soil pools and increase P availability to plants are of great importance.
Most predominant phosphorus-solubilizing bacteria (PSB) belong to the genera Bacillus
and Pseudomonas [82]. The fixed phosphorus in the soil can be solubilized by phosphate
solubilizing bacteria, which have the capacity to convert inorganic unavailable phosphorus
to available for the plants. Bacteria are more effective in phosphorus solubilization than
fungi [83]. Among the whole microbial population in soil, phosphate solubilizing bacteria
(PSB) constitute 1 to 50%, while phosphorus solubilizing fungi (PSF) are only 0.1 to 0.5%
in P solubilization potential [84].
Microorganisms involved in phosphorus acquisition include mycorrhizal fungi and
phosphate solubilizing microorganism (PSMs) [85]. Among the soil bacterial communities,
ectorhizospheric strains from Pseudomonas and Bacilli, and endosymbiotic rhizobia have
been described as effective phosphate solubilizers [86]. Strains from bacterial genera
Pseudomonas, Bacillus, Rhizobium and Enterobacter along with Penicillium and Aspergillus
fungi are the most powerful P solubilizers [87]. Bacillus megaterium, B. circulans, B. subtilis,
B. polymyxa, B. sircalmous, Pseudomonas striata, and Enterobacter could be referred as the
most important strains for phosphate solubilizer [88, 89]. A nemato fungus Arthrobotrys
oligospora also has the ability to solubilize the phosphate rocks [90].
13.2.3 Potassium Accumulating Organism

Potassium is one of the important nutrients for plants. There are a number of
microorganisms which can also accumulate potassium and thus help in enhancing
soil fertility. Some microorganisms in the soil are able to solubilize unavailable forms
of K-bearing minerals, such as micas, iolite and orthoclase, by excreting organic acids
which either directly dissolves rock K or chelating silicon ions to bring the K into solution
[91,92]. These microorganisms are commonly known as potassium solubilizing bacteria
(KSB) or potassium dissolving bacteria or silicate dissolving bacteria. Some research
has been made about the use of potassium dissolving bacteria, known as “biological
potassium biofertilizer (BPF)”, particularly in China and South Korea to investigate the
bio-activation of soil K-reserves so as to alleviate the shortage of K-fertilizer. It was shown
that KSB increased K availability in soils and increased mineral uptake by plant [93, 94].
Therefore, application of KSB holds a promising approach for increasing K availability
in soils.
13.2.4 Sulphur Solubilizing Organism

Sulphur is generally regarded as trace element in majority of crop plants. But this is one of
the major elements in oilseed crops, some important vegetables (onion, oat, cauliflower,
etc.) and in some spices (ginger, garlic, etc.). Sulphur is essential for biochemical synthesis
of some important glycosides, pungent compound and disease resistant properties.
Deficiency of sulphur in agricultural soil could be corrected using Azotobacter pasturianam
as boifertilizer.
It is essential for all plants and is indispensable for the oxidation of pyrite can be coupled
to solubilization growth and metabolism. Khandkar et al., observed that the nodule in
blackgram was increased due to sulphur application [95]. Sulphur is involved in the
formation of nitrogenase enzyme and is known to promote nitrogen fixation in legumes
[96]. Sulphur is involved in the formation of chlorophyll [97]. Hanesklaus and Schnug
reported that sulphur is associated with production of crops of superior nutritional and
market quality [98]. The sulphur application increased the total chlorophyll content of
greengram [99]. Sulphur plays a dominant role in improving the quality of pulses [100].
13.2.5 Mycorrhiza
Mycorrhizal fungi form mutualistic symbiosis with a vast majority of land plants [101].
Possibly more than 80% of all land plants form mycorrhizal symbiosis. The extent of
mycorrhizal symbioses emphasizes the ancient evolutionary history and potential
importance of fungal symbioses for plant production and physiology. The association
between plants and their root-colonizing mycorrhizal fungi is a functional symbiosis
in which the mycorrhizal fungus is obligately or facultatively dependent on host
photosynthates and energy. Mycorrhizal fungi may also improve soil quality by having a
direct influence on soil aggregation and therefore aeration and water dynamics [102]. An
interesting potential application for mycorrhizal fungi is their ability to allow plant access
to nutrient sources generally unavailable to the host plants. For example, crop plants may
be able to use insoluble sources of P when inoculated with mycorrhizal fungi but not in
the absence of inoculation [101].
13.2.6 Cellulose Decomposing Inoculants

Cellulases are especially common in soil and plant inhabiting fungi. Many fungi in the
Ascomycotina and Basidiomycotina are able to digest cellulose. The necessary enzymes
are less common in members of the Zygomycotina. Presence of the cellulase enzymes in
moist soil may indicate release of glucose dimers available for absorption by all microbes
in the near vicinity which indicating a continuing and community-based process of
cellulose degradation by microbes. Cellulose is the most abundant constituent of plant
residues. It has been reported that cellulose decay would be enhanced by nitrogen addition
due to the increased cellobiases [103,104]. Serpula lacrimans, a cellulose degrading fungus,
produced more mycelium and faster decay of cellulose under nitrogen fertilization [105].
Excess doses of manure or sludge fertilizers can harm mesofauna because of toxicity
(e.g., anhydrous ammonia) and high osmotic pressure create due to salt accumulation
[106], or heavy metal accumulation [107]. The repellent nature of ammonium can affect
soil invertebrates adversely [108]. The potential of toxic effects can be decreased by
applying composted manure or sludge [109]. The time period for material is composted
prior to incorporating it into soil must be considered. For example, the plant-pathogenic
fungus Rhizoctonia solani may cause damping-off disease in soil when fresh or immature
compost material of high content of cellulose is added. However, in aged compost,
cellulose is degraded and the biocontrol fungus Trichoderma spp. can grow and parasitise
the pathogen effectively. Many soil-borne fungal species like Aspergillus, Penicillium,
Tricoderma, Chaetomium, etc. acts as activator in the decomposition process of plant bodies
containing cellulose or lignin.
13.3 CONSTRAINTS AND SUCCESS OF BIOFERTILIZER

TECHNOLOGY
Though the biofertilizer technology is a low cost, eco-friendly technology, several
constraints limit the application or implementation of the technology. The constraints
may be environmental, technological, infrastructural, financial, human resources,
unawareness, quality, marketing, etc.
13.3.1 Technological Constraints

• Use of improper, less efficient strains for production.
• Lack of qualified technical personnel in production units.
• Unavailability of good quality carrier material or use of different carrier materials
by different producers without knowing the quality of the materials.
• Production of poor quality inoculants without understanding the basic
microbiological techniques.
• Short shelf life of inoculants.
13.3.2 Infrastructural Constraints

• Non-availability of suitable facilities for production.
• Lack of essential equipments, power supply, etc.
• Space availability for laboratory, production, storage, etc.
• Lack of facility for cold storage of inoculant packets.
• Financial constraints.
• Non-availability of sufficient funds and problems in getting bank loans.
• Less return by sale of products in smaller production units.
13.3.3 Environmental Constraints

• Seasonal demand for biofertilizers.
• Simultaneous cropping operations and short span of sowing/planting in a
particular locality.
• Soil characteristics like salinity, acidity, drought, waterlogging, etc.
13.3.4 Human Resources and Quality Constraints

• Lack of technically qualified staff in the production units.
• Lack of suitable training on the production techniques.
• Ignorance on the quality of the product by the manufacturer.
• Non-availability of quality specifications and quick quality control methods.
• No regulation or act on the quality of the products.
• Awareness on the technology.
• Unawareness on the benefits of the technology.
• Problem in the adoption of the technology by the farmers due to different methods
of inoculation.
• No visual difference in the crop growth immediately as that of inorganic fertilizers.
13.3.5 Awareness on the Technology

• Unawareness on the benefits of the technology.
• Problem in the adoption of the technology by the farmers due to different methods
of inoculation.
• No visual difference in the crop growth immediately as that of inorganic fertilizers.
• Unawareness on the damages caused on the ecosystem by continuous application
of inorganic fertilizer.
13.3.6 Marketing Constraints

• Non-availability of right inoculant at the right place in right time.
• Lack of retain outlets or the market network for the producers.
13.4 BIOREMEDIATION PROCESS: AN OVERVIEW

Bioremediation is defined as a process whereby hazardous wastes are biologically
degraded under controlled conditions to an innocuous state, or to levels below
concentration limits established by regulatory authorities [110]. This uses living
organisms, especially plants and microorganisms, to reduce, eliminate, transform,
and detoxify the unwanted products present in soils, sediments, water, and air.
Phytoremediation technology, one of its many approaches, uses plants as filters for
accumulating, immobilizing, and transforming the contaminants to less harmful form
[111]. More specifically, it is the utilization of vascular plants, algae, and fungi to
control, breakdown, remove wastes, or to encourage degradation of contaminants in the
rhizosphere [112]. Phytoremediation has recently become a tangible alternative to the
traditional methodology in restoring the polluted sites [113].
Bioremediation technologies currently used in many countries are mostly directed to
the remediation of oil spillages on land and include in-situ biotreatment of contamination,
for example, the addition of bacterial fertilizers, mineral, and organic nutrients to the
oil-contaminated soil [114-116]. However, these technologies are not acceptable for the
treatment of oil wastes, as the high concentration of toxic contaminants and anaerobic
conditions in the pit content prevent the development of an active oil-oxidizing microbial
consortium.
Rhodococcus biosurfactants have been used for the bioremediation of oil-contaminated
agricultural soils after an accidental oil spill [117, 118]. The application of composting
systems enhanced by nutrient addition, bulking with straw and inoculation of
Rhodococcus-biosurfactant complexes provided a 57% decrease in oil contamination
during a 3-month treatment. An ex situ biotechnology developed to employing a
Rhodococcus biosurfactant-based biofertilizer for the decontamination of heavily oil-
polluted soil [119].
13.5 BIOFERTILIZER TECHNOLOGY IN BIOREMEDIATION

Success or failure of bioremediation depends on several factors such as the competitive
ability of the bioremedial agents and biotic factors such as soil moisture, pH, and
temperature [120, 121. Inoculated biofertilizers (Phosphoren, Microbien, Cerealin and
Azospirillum) may act as potential agent for soil inoculation to bioremediate pesticides
contaminated soil [122]. Successful removal of pesticides by the addition of bacteria
(bioaugmentation) has been reported for many compounds including, coumaphos
(ethoprophos, dicofol and malathion) [123-126].
13.5.1 Use of Microorganisms in Bioremediation

Plant growth-promoting bacteria (PGPB) are mainly used to improve agricultural
yields along with solving environmental problems [127]. A number of microorganisms
(bacteria and fungi) have been used in soil inoculations intended to improve the supply
of nutrients to crop plants, to stimulate plant growth, to control or inhibit the activity of
plant pathogens and to improve soil structure (Table 13.1). Other more recent, objectives
for the introduction of microorganisms into soil are the mineralization of organic
pollutants [121]. Phosphoren, micrebien, cerealin and azospirillum are the biofertilizers
(derived from the living biomass of different microbial species) produced by general
organization belongs to the Egyptian Ministry of Agriculture as a mixed inoculums are
beneficially applied to many fields and horticultural crops [128].
Table 13.1 Plant growth promoting bacteria (PGPB) applied in bioremediation strategies
Characteristics of PGPB Microorganism Plants associated References
Nitrogen fixation
Freely associated Azotobacter chroococcum Brassica juncea Wu et al., (2006) [129]
bacteria
Symbiotic bacteria Sinorhizobium meliloti Medicago truncatula Bianco and Defez
(2009) [130]
Enhanced the nodule Bradyrhizobium sp. Greengram (Vigna Wain et al., (2007) [131]
numbers, seed yield, (vigna) radiate)
grain protein, root and
shoot
Phosphate
mobilization
Inorganic P source Pseudomonas aeruginosa Vigna mungo Ganesan (2008) [132]
Organic P source Bacillus Zea mays Idriss et al., (2002)
amyloliquefaciens [133]
Phosphate solublizing Pseudomonas putida, Artichoke (Cynara Jahanian et al., (2012)
bacteria Azospirilium, scolymus) [134]
Azotobacter
Siderophore release
Hydroxamates Streptomyces Cicer arietinum Dimkpa et al., (2008)
acidiscabies [135]
Phenol catecholates Rhizobium sp. Sesbania procumbens Sridevi et al., (2008)
[136]
Carboxylates Pseudomonas fluorescens Arachis hypogaea Dey et al., (2004) [137]
Salicylic acid Arthrobacter oxidans Pinus sp. Barriuso et al., (2008)
[138]
Stimulated plant Pseudomonas aeruginosa Indian mustard and Sinha and Mukherjee
growth, reduced Cd pumpkin (2008) [139]
uptake
Auxin production
Indole acetic acid Enterobacter chloacae Oryza sativa Mehnaz et. al., (2001)
[140]
Gibberellin Bacillus pumilus Alnus glutinosa Gutierrez-Man˜ero
et al., (2001) [141]
Influence on metal
toxicity
Increased Ni Bacillus subtilis Brassica juncea Zaidi et al., (2006) [142]
accumulation
Increased Cd Xanthomonas sp. Brassica napus Sheng and Xia (2006)
accumulation [143]
Reduction of Cr(VI) to Ochrobactrum Helianthus annuus Faisal and Hasnain
Cr(III) intermedium (2005) [144]
Stimulated plant Bacillus edaphicus Indian mustard Sheng et al., (2008)
growth, facilitated (Brassica juncea) [145]
soil Pb mobilization,
enhanced Pb
accumulation
13.5.1.1 Bioremediation by microalgae
Algae are universally known as playing a very important role in natural water purification
process [24, 146]. Thus, the use of microalgae for removal of nutrients from different
wastes has been described by a number of authors [21, 147-156].
Industrialization has led to increased emission of pollutants into ecosystems. Metal
pollutants can easily enter the food chain if heavy metal-contaminated soils are used
for production of food crops. Farm productivity has decreased in toxic metal polluted
areas [157]. Accumulation of toxic metals, e.g., Hg, Cu, Cd, Cr and Zn in humans has
several consequences such as growth and developmental abnormalities, carcinogenesis,
neuromuscular control defects, mental retardation, renal malfunction and wide range
of other illnesses. Elevated levels of such metal ions are generally toxic and cause
major damage to cell [158]. Conventional technologies, such as ion exchange or lime
precipitation, are often ineffective and/or expensive, particularly for the removal of heavy
metal ions at low concentrations (below 50 mg/L). Furthermore, Most of these techniques
are based on physical displacement or chemical replacement, generating yet another
problem in the form of toxic sludge, the disposal of which adds a further burden on the
techno-economic feasibility of the treatment process. In view of this, the development
of new techniques is necessary to meet the environmental standards at affordable costs.
They can provide solutions to the twin challenges of energy security and environmental
pollution. They have great potential for the removal of excess nitrogen and phosphorus
from wastewater including the farm runoff. They can capture carbon dioxide in the flue
gas from coal fired power plants thereby reducing greenhouse gas and also producing
algal biomass, which can be converted into biofuel Chlorella, Scenedesmus and Spirulina
are the most widely used algae for nutrient removal.
The ability of algae to absorb metals has been recognized for many years [159]. In
natural environments, algae play a major role in controlling metal concentration in lakes
and oceans [160,161]. Algae possesses the ability to take up toxic heavy metals from the
environment, resulting in higher concentrations than those in the surrounding water
[140, 162].
13.5.1.2 Bioremediation by N2 - fixing bacteria
N2 fixation is an energy-consuming process and has been widely observed in symbiotic,
associative and free living bacteria. This bacterial physiological feature plays a tremendous
role in ecological sustenance, world agriculture and global nitrogen cycle. Nitrogen
availability can limit microbial growth and affect ecosystem activity [163]. Although
N2 fixation is widely distributed among Bacteria and Archaea [164], this significant
physiological feature has not been revealed in reductively dechlorinating bacteria.
Nitrogen is essential to microorganism metabolism and it is necessary to biosynthesis
of amino acids, protein and nucleic acids [165-167]. The soil rich in nitrogen has a
good metabolic activity and good microbial biomass. Therefore, its presence in the
soil for a good bioremediation process is necessary. However, some authors claim that
biodegradation in negligible amounts of nitrogen is possible, but the efficiency is lower
[168-170].The literature quotes different bacterial genera, which have the capacity to
degrade hydrocarbons in poor concentration of nitrogen [169]. The genera Pseudomonas
[168, 171], Agrobacterium, Alcaligenes [172], Arthrobacter [173], Azotobacter [174] have the
capacity to fix nitrogen in soil with deficiency of nitrogen. The effects of bio-stimulation
on petroleum hydrocarbon degradation have been investigated in different conditions
[175, 176]. However, the effects of nitrogen affect the biodegradation of hydrocarbon
classes, i.e. aliphatic, aromatic and polar hydrocarbons have not been completely studied
in the Patagonian soil, which has a poor concentration of nitrogen and it is an important
oil production area.
Nitrogen and phosphorus are the nutrients that most frequently limit bioremediation
[177]. The presence of petroleum products in the soil can widen the C:N ratio, therefore
limiting available nitrogen for degradation processes [178]. In addition, microbes able to
metabolize hydrocarbons will quickly immobilize the mineral nitrogen that is available,
leaving unfavourable conditions for other microorganisms and growing plants [179].
The absence of sufficient nitrogen in the soil will, in turn, slow the degradation process
resulting from microbial metabolism [177]. Therefore, adding nutrients in the form of
either organic or inorganic fertilizers can stimulate contaminant degradation [180]. Free-
living nitrogen-fixing bacteria can fix atmospheric nitrogen into a more usable form such
as ammonia. Thus, the bioaugmentation of hydrocarbon polluted soil with free-living
nitrogen-fixing bacteria may indirectly contribute to soil nitrogen by releasing nitrogenous
biomass [181]. This study was undertaken to isolate hydrocarbon-degrading free-living,
nitrogen-fixing bacteria from the soil and to determine their potential in bioremediation
of hydrocarbon polluted soil.
13.5.2 Role of Azolla in Phytoremediation of Heavy Metals

Among various water pollutants, heavy metals are of major concern because of their
persistent and bioaccumulative nature [182-185]. Water is an indispensable part for the
sustenance of mankind and the increasing awareness about the environment; especially
aquatic ecosystems have attracted the attention of researchers worldwide. A definite need
exists to develop a low cost and eco-friendly technology to remove pollutants particularly
heavy metals, thereby improving water quality. Phytoremediation offers an attractive
alternative. Among these, Azolla, a free-floating, fast growing, and nitrogen-fixing
pteridophyte seems to be an excellent candidate for removal, disposal, and recovery of
heavy metals from the polluted aquatic ecosystems [186, 187].
Both living and dead biomass of Azolla have been exploited for the removal of heavy
metals from industrial effluents and sewage water [188-191]. Bioaccumulation potential
of different species of Azolla for various heavy metals. Azolla has great possibility of use
in bioremediation of wastewaters and soils. There are three main aspects of use of Azolla
in bioremediation. These areas are:
(a) For treatment of wastewaters rich in heavy metal pollutants.
(b) Treatment of domestic sewage effluents which are rich in N and P wastes.
(c) Bioremediation of saline soils.
A study in China stated tolerance of four Azolla species to Cu, Mn, Fe, Zn, Mo, Co,
Cd, etc. under laboratory conditions and found that concentration capacity of Azolla for
metals affected its growth only slightly without any detrimental effect or not at all [192].
13.6 CASE STUDIES ON BIOREMEDIATION WITH

BIOFERTILIZER
13.6.1 Bioremediation of Sewage Wastewater Using Selective
Algae for Manure Production
Phycoremediation is the process of employing algae for removing excess nutrient
load from wastewater and subsequently diminish the pollution load. It is an alternative
technology of treating sewage wastewater compare to conventional treatment process
in economical and sustainable way. IARI gives the effort to phycoremediate sewage
wastewater with different microalgae, viz., Chlorella minutissima, Scendesmus spp. & BGA
(Nostoc) and their consortium. Algae were very effective in reduction of BOD5, COD, NO3–
, NH4, PO4–3 and TDS in sewage wastewater. Further, it has been observed that Chlorella
was having best phycoremediation potential as well as manure production among all three
microalgae and even better than consortium. Among the potential uses of algal biomass
from such systems is its use as a slow release fertilizer. After 20 days microalgae were
harvested using muslin cloth and fresh and dry weigh were determined. The maximum
biomass was observed in Scendesmus spp. and Chlorella minutissima while percentage of
nitrogen and phosphorus was highest in Chlorella minutissima. So Chlorella minutissima
has the best manurial potential. The algae in nutrient-rich sewage wastewater offers a
new option of applying algae to manage the nutrient load and after phycoremediation
the biomass itself can be utilized for manure application in agriculture, serving the dual
roles of nutrient reduction and valuable manure feedstock production [193].
13.6.2 Bioremediation of Olive Oil Mill Wastewater: Chemical

Alterations Induced by
An environmentally friendly bioremediation system of olive oil mill wastewater (OMWW)
is studied with respect to its physicochemical characteristics and degradation efficiency
on major characteristic constituents. The method exploits the biochemical versatility of
the dinitrogen fixing bacterium Azotobacter vinelandii (strain A) to grow in OMWW at
the expense of its constituents and to transform it into an organic liquid fertilizer. The
system eliminates the phytotoxic principles from OMWW and concomitantly enriches it
with an agriculturally beneficial microbial consortium along with useful metabolites of
the latter. The end product, branded “biofertilizer”, is used as soil conditioner and liquid
organic fertilizer. Growth of A. vinelandii in OMWW results in the decline of content
of most of the compounds associated with phytotoxicity, and this is confirmed by the
assessment of degradation yields. In parallel, during the process several other compounds
noncommittally undergo degradation and biotransformation. More specifically, the
biofertilization system is capable of achieving removal yields as high as 90 and 96% after
3 and 7 days of treatment, respectively [194].
13.6.3 Biofertilizers for Bioremediation of Pesticide
Contaminated Soil
Biofertilizers by the General Organization of Agriculture Fund, Ministry of Agriculture,
Egypt (Phosphoren, Microbien, Cerealin and Azospirillum) to degrade five selected pesticides
representing different classes including organophosphate, carbamate and chlorinated
organic compounds. There were differences in rates of biotransformation, suggesting
the selective induction of certain metabolic enzymes. Inoculation of soil incorporated
with malathion, fenamiphos, carbaryl, aldicarb and dieldrin, resulted in 80-90% removal
of malathion and fenamiphos within 8 days, carbaryl and aldicarb within 11-15 days
respectively. Dieldrin removal occurred slowly within 2 months. These data suggest
that biofertilizers may act as potential candidates for soil inoculation to bioremediate
pesticide contaminated soil. The production of CO2 (soil respiration) was stimulated by
some pesticides. In samples with microbien, about 2 times higher CO2 production was
measured [195].
13.6.4 Bioremediation of Petroleum Pollutants

Bioremediation tries to raise the rates of degradation found naturally to significantly
higher rates. The two general approaches that have been tested for the bioremediation of
marine oil spills are the application of fertilizer to enhance the abilities of the indigenous
hydrocarbon-utilizing bacteria and the addition of naturally occurring adapted microbial
hydrocarbon-degraders by seeding. Bioremediation, accomplished by the application of
fertilizer to enhance the abilities of the indigenous hydrocarbon-utilizing bacteria, was
successfully applied for the treatment of the Alaskan oil spill in Prince William Sound,
Alaska [196].
13.6.5 Cyanobacterial Consortia for Bioremediation Purposes

Many studies have reported the ability of cyanobacteria to oxidize oil components and
other complex organic compounds such as surfactants and herbicides [22, 197-199].
Among these cyanobacteria were the nonaxenic cultures of Microcoleus chtonoplastes
and Phormidium corium, degrading n-alkane [200], Oscillatoria sp. and Agmenellum
quadruplicatum oxidizing naphthalene to 1-naphthol [201, 202], Oscillatoria sp. strain JCM
that oxidized biphenyl to 4-hydroxybiphenyl [203] and Agmenellum quadruplicatum that
metabolized phenanthrene into trans-9,10-dihydroxy-9,10-dihydrophenanthrene and
1-methoxy-phenenthrene [204]. Cyanobacteria those are responsible for the degradation
of these compounds but the associated aerobic organotrophic bacteria [205-207]. Presence
of cyanobacteria alongside with the aerobic organotrophs facilitated the degradation
process and both groups constituted idea consortia for degradation of petroleum and
other complex organic compounds [206].
13.6.6 Potential Use of Cyanobacteria Species in

Phycoremediation of Municipal Wastewater
The treatment of municipal wastewater becomes effective by using microalgae which
are superior as the wide range of toxic and other wastes can be reduced with them.
Locally available blue-green algae such as Oscillatoria limosa and Nostoc commune have
ability to remove of various nutrients to prevent further deterioration of water quality.
The phycoremediation experiments were conducted at Department of Environmental
Sciences, University of Pune, India using randomized complete block design with three
replications of each treatment. The results of present investigation clearly indicated that
both the algal species, viz., Oscillatoria limosa and Nostoc commune are highly efficient
for removal of NO3– 2, PO4–2, SO4–2, Cl– and for reducing EC values. The average reduction
was between 84 to 98%. The pollutant removal efficiency was increased with decreasing
concentration of wastewater. Amongst the selected algae Oscillatoria limosa was the
best candidate as compare to Nostoc commune. It was concluded that the cyanophycea
members would be the best options for phycoremediation [208].
13.6.7 Bioremediation of Wastewater by Using Microalgae

Autotrophs play an important role in remediation of wastewater particularly domestic
waste through it photosynthetic ability. Dominant and pollution tolerant algae such as
C. vulgaris and S. quadricauda cultures in BBM isolated form sewage wastewater treatment
plant Bopodi from Pune city and used for the treatment of the wastewater. To study
the role of microalgae in wastewater, the following protocols were used, (i) Wastewater
treated with culture of C. vulgaris and S. quadricauda; and (ii) Wastewater treated without
culture of C. vulgaris and S. quadricauda (Control). Samples were periodically (every 5th
day) analyzed for physico-chemical parameters such as pH, phosphate, nitrate, BOD and
COD using standard methods. C. vulgaris showed the best removal capacity of nitrate
and COD while S. quadricauda shows BOD and phosphate reduction [209].
13.6.8 Microalgae for Bioremediation of Distillery Effluent

Distillery effluents, also referred to as spent wash/stillage/slop/vinasses, are one of the
most environmentally aggressive industrial effluents. With the development of economies
and resultant growth of distillery industries, large volume of spent wash is produced
which is likely to cause extensive soil and water pollution due to the presence of high
amount of organic matter and dark brown coloured recalcitrant compounds. There have
been many isolated studies for treatment of distillery effluents and related compounds
using microalgae.
For treatment of wastewaters native microalgal strains are a favourable alternative to
the traditional wastewaters treatment systems. A consortium of Oscillatoria, Lyngbya and
Synechocystis decolorized melanoidin by 98% by absorption followed subsequently by
degradation of the organic compounds. The microalgal strains like Anabaena cylindrica,
Phormidium foveolarum, P. valderianum, Synechococcus, Ankistrodesmus braunii and
Scenedesmus quadricauda have been reported instrumental in degradation of phenol and
its derivatives, whereas the performances of Phormidium ambiguum, Chroococcus minutus,
Oscillatoria, and Anabaena azollae were found satisfactory for degradation of lignin.
Phormidium ambiguum and Chroococcus minutus were found to reduce lignin by over
73.0% from the pulp and paper mill wastes in 5 days; whereas Phormidium, Oscillatoria,
and Anabaena azollae were able to degrade lignin by 89% and hemicellulose by 92% from
coir waste [210].
The role of cyanobacteria in distilleries effluent was studied in Kanchipuram,
Tamilnadu, India. Totally 12 species of cyanobacteria belonging to 6 genera were identified.
Nostoc muscorum was found to be the most dominated genus in this effluent (heterocyst
organism). The inoculation of cyanobacteria Nostoc muscarum resulted in removal of
various chemicals as well as nutrients such as nitrogen, ammonia and phosphorus from
the effluent and could be potentially employed for the treatment of distilleries effluent
[211].
13.7 FUTURE SCOPE FOR BIOREMEDIATION BY

BIOFERTILIZER PRODUCING ORGANISMS
Bioremediation using biofertilizer producing microorganisms is though a very new
concept, still it is really advancing field of research. Different types of interactions
like biotransformation, biosorption and binding of heavy metals with extracellular or
intracellular organelles are effectively used and scientifically manipulated to perform
with higher effectiveness. Designing of different types of bioreactors, within which
microbes can accumulate or adsorb metals or radionuclides under controlled systems,
is also being modernized. Several strains having the capacity of promoting plant growth
and reducing environmental toxicants. However, more studies are needed to be done
on application fields, so that we can better understand the effect of natural factors and
of co-toxicants present in concerned environment. Apart from choosing and developing
such strains there remains a considerable amount of work required to be done on
effective and easy desorbtion of toxic contaminants from environment and regeneration
of the biomass for further use. To attract more usage of biosorbent technology using plant
growth promoting microorganism (PGPM), certain strategies have to be formulated and
promoted in agricultural and environmental sectors. The education, awareness and
biotechnological research in this area increased our crop production and decrease the
environmental contaminants. With so much to be done in this rapidly growing field of
biotechnology, hope that future promises us a better and healthier environment.
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Biofertilizer for Bioremediation

Chapter · January 2015

Subhas Chandra Santra Alok Chandra Samal

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13.2 MICROBES IN BIOFERTILIZER PRODUCTION

13.2.1 Nitrogen-Fixing Microorganism

13.2.2 Phosphate Solubilizing Microorganisms

13.2.3 Potassium Accumulating Organism

13.2.4 Sulphur Solubilizing Organism

13.2.6 Cellulose Decomposing Inoculants

13.3 CONSTRAINTS AND SUCCESS OF BIOFERTILIZER

13.3.1 Technological Constraints

13.3.2 Infrastructural Constraints

13.3.3 Environmental Constraints

13.3.4 Human Resources and Quality Constraints

13.3.5 Awareness on the Technology

13.3.6 Marketing Constraints

13.4 BIOREMEDIATION PROCESS: AN OVERVIEW

13.5 BIOFERTILIZER TECHNOLOGY IN BIOREMEDIATION

13.5.1 Use of Microorganisms in Bioremediation

13.5.2 Role of Azolla in Phytoremediation of Heavy Metals

13.6 CASE STUDIES ON BIOREMEDIATION WITH

13.6.2 Bioremediation of Olive Oil Mill Wastewater: Chemical

13.6.4 Bioremediation of Petroleum Pollutants

13.6.5 Cyanobacterial Consortia for Bioremediation Purposes

13.6.6 Potential Use of Cyanobacteria Species in

13.6.7 Bioremediation of Wastewater by Using Microalgae

13.6.8 Microalgae for Bioremediation of Distillery Effluent

13.7 FUTURE SCOPE FOR BIOREMEDIATION BY

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