BIOLEACHING

Bioleaching is the extraction of metals from their ores through the use of living organisms.
Bioleaching has been used to dissolve metals such as nickel, copper, zinc, cobalt, gold, lead,
arsenic and others. In modern mining industry several new processes are used which are easily
applicable and economical and environment friendly. Application of biotechnology in mining
industry to extract different metals is one of the above said new age techniques. The area of
discipline in which microbes are used to extract different metals from ores, is collectively termed
as bio-hydrometallurgy or microbial mining.

Bioleaching is a part of microbial mining. In microbial leaching or bioleaching metals can be

extracted from large quantities of low grade ores This process has been used for centuries to
recover copper form the drainage water of mines the role of microbes in the bioleaching came
into notice some 40 years ago.

MECHANISMS INVOLVED IN BIOLEACHING

Two processes are used for bioleaching, direct action and indirect action. In direct action bacteria
act on the ore directly to extract metal. In indirect action the bacteria produces some chemical
substance like acids or ions which lead to extraction of metal from the ore. In mining industry
both the direct and indirect method is used in combination to extract metal economically.

I) Direct Bioleaching

In direct bioleaching minerals which are susceptible to oxidation undergoes direct enzymatic
attack by the microorganisms. Certain bacteria such as Thiobacillus ferrooxidans oxidize ferrous
ions to ferric ions and in this process it transfers the electrons form iron to oxygen. Iron is
extracted from the iron ore pyrite (FeS2) in a more soluble form. Thibacillus thiooxidans extract
sulphur from iron ore pyrite in the form of sulphates which is also a more soluble form.
II) Indirect Bioleaching

In the indirect method of bioleaching of minerals bacteria produces strong oxidizing agents
which react with metals and extract them from the ores. Bacteria produce oxidizing agents such
as ferric ions and sulphuric acid on oxidation of soluble iron and soluble sulphur respectively.
Acidic environment is a necessary condition for indirect bioleaching to keep the ferric ion and
other metals in solution. The continuous oxidation of iron, sulphur, metal sulphides and
carbonate ions in solution help to keep the acidic environment.

MICROBES INVOLVED IN BIOLEACHING

The bacteria which are naturally involved in growing up among rocks are most suitable and
applicable ones for bioleaching. The most commonly used microorganisms for bioleaching are
Thiobacillus ferrooxidans and Thiobacillus thiooxidans. Thiobacillus ferrooxidans is a gram
negative, rod-shaped, motile, non-spore forming bacterium. It derives the energy of the growth
from the oxidation of iron or sulphur in ore. This microorganism oxidises ferrous ions (Fe2+) to
ferric ions (Fe3+) and converts sulphides, thiosulphate, sulphur to sulphate. Thiobacillus
thiooxidans has similar functions and it grows mostly on sulphur ores or rocks.

Sulfolobus acidocaldarius and Sulfolobus brierlevi are two other bacteria which are involved in
bioleaching of copper and molybdenum from ores like chalcopyrite and molybdenite. They are
thermophilic and acidophilic bacteria which mostly grow in acidic hot springs. Two other
bacteria like Leptospirillum ferrooxidans and Thiobacillus organoparpus work in a combination
to extract iron and copper from pyrite and chalcopyrite.

Some bacteria like Pseudomonas aeruginosa can also be used to extract high value metal like
uranium from low quality uranium ore containing around 0.02% uranium. This microorganism
extracts around 100mg uranium per one lire solution in less than 10 seconds. Rhizopus arrhizus
is another bacterium that can extract uranium from low grade uranium ores.
Fungi have not shown a broad area of examples in the field of bioleaching. Some of the fungi
associated with bioleaching are Aspergillus niger and Aspergillus oryzae. While A.niger extracts
copper and nickel, A.oryzae has been found to extract gold by bioleaching. The use of more
bacteria and fungi are still in the experimental stage.

COMMERCIAL PROCESS OF BIOLEACHING

The naturally occurring bioleaching process is a very slow process. For commercial extraction of
metals by bioleaching the process is optimized by controlling the temperature, pH, humidity, O2
& CO2 concentration etc. The appropriate microorganisms along with nutrients and acids are
pumped in to the ore bed and the extracted leach liquor is collected for metal recovery. The
different processes of commercial bioleaching are as follows.

I) Slope Leaching

In slope leaching the ore is finely ground and kept in large pile in a slope which is subjected to
continuous sprinkling of aqueous solution of microorganisms. The leach liquor collected at the
bottom of the ore is processed further for metal recovery.

II) In-situ Leaching

In in situ leaching ore is subjected to bioleaching in its natural occurrence. Aqueous solution of
microorganism is pumped through drilled passages within the ore. The leach liquor collected at
the bottom of the ore used for metal extraction.

III) Heap Leaching

In heap leaching the ore is arranged in heap and goes through the same treatment such as in slope
leaching. The aqueous solution containing microorganisms works on the heap of ore and
produces the leach liquor. The leach liquor is used for metal recovery.
In all these three processes of commercial bioleaching the aqueous solution of microorganisms
can be reused.

ADVANTAGES OF BIOLEACHING
1. Extraction of metals form low grade ores
2. Very economical process
3. Employed for collecting metals from wastes or drainages
4. Environment friendly process
5. Used to extract refined and expensive metals which is not possible by other chemical
processes

DISADVANTAGES OF BIOLEACHING
1. Very slow process
2. Dependency on several atmospheric conditions decreases the efficiency of the process.
 BIOFUEL

Biofuel is a fuel that is produced through contemporary biological processes, such as agriculture
and anaerobic digestion, rather than a fuel produced by geological processes such as those
involved in the formation of fossil fuels, such as coal and petroleum, from prehistoric biological
matter. Biofuels can be derived directly from plants, or indirectly from agricultural, commercial,
domestic, and/or industrial wastes. Renewable biofuels generally involve contemporary carbon
fixation, such as those that occur in plants or microalgae through the process of photosynthesis.
Other renewable biofuels are made through the use or conversion of biomass (referring to
recently living organisms, most often referring to plants or plant-derived materials). This
biomass can be converted to convenient energy-containing substances in three different ways:
thermal conversion, chemical conversion, and biochemical conversion. This biomass conversion
can result in fuel in solid, liquid, or gas form. This new biomass can also be used directly for
biofuels.

In recent years, as the need to develop alternative, non-petroleum-based transportation fuels has
become more pressing, there has been a growing interest in using advanced biotechnologies to
improve biofuel production. Specifically, numerous strategies have evolved by which
biotechnology is being used to create improved biofuels products or processes, involving the
creation of engineered or synthetic microorganisms for use in production of ethanol, biodiesel or
other fuels, or genetically engineered (transgenic) plants as improved fuel feedstocks. These
approaches can generally be summarized as follows.

POTENTIAL APPLICATIONS OF BIOTECHNOLOGY TO IMPROVE RENEWABLE

FUEL PRODUCTION.

Enhanced or engineered microorganisms for fermentation of ethanol, butanol, other fuels.

Engineered microorganisms or plants to manufacture enzymes used in fuel production.

Improved algal strains for biofuel production.

 Selected or engineered plant species with favorable traits for use as improved biofuel
feedstocks.

There are a number of ways in which microorganisms, algae or plants can be modified for
improved industrial performance. Some of the companies and technologies make use of
selected, often proprietary strains of production organisms, that have been derived from naturally
occurring organisms using traditional techniques of mutation and selection, or in the case of
plants by traditional crop breeding. These methods have been practiced in industry and in
agriculture for decades, and in many cases their use can lead to significant process
improvements, for example in the efficiency of ethanol fermentation.

However, in most cases, the technology strategies utilize genetic engineering methods based on
recombinant DNA. Recombinant DNA methods enable the insertion of genes from any source in
nature into a chosen host organism, thereby conferring on the host organism a genetic trait or a
biochemical capability not naturally found in that organism. Genes function in nature by
encoding the synthesis of specific protein molecules, most of which are enzymes whose role it is
to catalyze specific biochemical reactions inside living cells; and so by transplanting a gene into
a new host organism, under conditions in which the gene can actively direct the synthesis of its
corresponding enzyme, one can impart on the host organism new or improved biochemical
powers. In the years since recombinant DNA techniques were first developed in the mid-1970s,
techniques have been worked out for the genetic engineering of almost any species of organism
having medical, industrial or agricultural value, including most important plant species, almost
any microorganism, and many algal species. These techniques are now being used to improve
natural processes for synthesis of ethanol and other fuels.

There are also a number of companies and academic research laboratories using more advanced
technologies for improvement of microbial or plant performance. Many of these methodologies
utilize recombinant DNA, but in ways specifically designed to facilitate the creation of
organisms improved for a specific desired function. These newer techniques, along with more
traditional methods of organism improvement, are summarized as follows, with the
understanding that there can be some overlap in the way these terms are defined. Some of the
technologies, like directed evolution or DNA shuffling, use a combination of genetic tricks and
enhanced selective pressure to greatly enhance the activity of a targeted enzyme or pathway,
while other techniques like synthetic biology allow creation of novel or enhanced metabolic
pathways in organisms never before possessing such traits.

BIOTECHNOLOGIES APPLICABLE TO BIOFUELS.

Classical Mutation and Selection Or Plant Breeding: this

encompasses a variety of well-known, decades-old techniques for selectively breeding or
otherwise selecting naturally-occurring (or mutationally induced) variants of a starting
strain or plant variety.

Recombinant DNA: insertion and expression of heterologous genes into a desired

host organism (microorganism, algae or plant) to improve a desired trait or biochemical
function in the host organism.

Directed Evolution: this technique generally involves growing a desired microbial

strain under certain limiting conditions that impose selective pressure under which those
mutant overperforming strains that arise can eventually outcompete the starting strain.

DNA Shuffling: this is a form of directed evolution where the gene encoding the
enzyme targeted for improvement is mutated in millions of permutations using
recombinant techniques, followed by selection and isolation of superior performers, often
carried out in multiple iterations of selection.

Metabolic Engineering: this term refers to the use of recombinant DNA

technologies to create new metabolic or biosynthetic pathways in host organisms, or to
enhance existing pathways, through the engineered, coordinated expression of several
heterologous or enhanced enzymes in the desired pathway.

Synthetic Biology: in this technique biochemical pathways or even entire

microorganisms are created from scratch, to create pathways or organisms not
previously found in nature. It can be viewed as a more ambitious approach to metabolic
 engineering, as it often involves creation of biochemical pathways never before existing
in the host organism of choice.

BIOPLASTICS

Bioplastics are plastic materials that are either biodegradable or derived from renewable biomass
sources, such as vegetable fats and oils, corn starch, microbiota or both. Bioplastic can be made
from agricultural by-products and also from used plastic bottles and other containers using
microorganisms. Common plastics, such as fossil-fuel plastics (also called petrobased polymers),
are derived from petroleum or natural gas. Production of such plastics tends to require more
fossil fuels and to produce more greenhouse gases than the production of biobased polymers
(bioplastics). Some, but not all, bioplastics are designed to biodegrade. Biodegradable bioplastics
can break down in either anaerobic or aerobic environments, depending on how they are
manufactured. Bioplastics can be composed of starches, cellulose, biopolymers, and a variety of
other materials.

A reasonable amount of interest has been developed in the field of bioplastic as a result of
increased concern about non degradability of plastics and environmental hazards. Bioplastic have
been known to be produced by various means including vegetable fats, cornstarch etc

BIOTECHNOLOGICAL ROLE:

The role of biotechnology in the field of bioplastic has been accounted mainly to the presence of
the property of biodegradation by naturally occurring polyhydroxy- alkanoates(PHAs). These are
naturally occurring polyesters produced by bacterial fermentation. Polyhydroxybutyrate(PHB) is
a form of PHA. It is produced in the bacterial cell as a result of physiological stress in the form
of energy storing molecules. These PHAs are metabolized and used up by such bacteria when
none of the other forms of energy source is available under conditions of stress. The properties of
PHB include non toxic, water insoluble, high oxygen permeability and ultra violet resistance,
thermostable and biocompatible. Bacterial fermentation of Alcaligenes eutrophus produces
polyhydroxy alkanoates.

The organism is cultured under normal conditions to attain stable growth. The conditions of the
medium are then altered so that the bacterium induces production of PHA. This is usually
induced in deficiency conditions of macro elements, oxygen etc or due to the excess presence of
carbon sources in the medium. Acetyl-coA serves as a precursor in the production of
polyhydroxy alkanotaes. The microbial fermentation of Alcaligenes for the production of PHAs
is not favourable as the yield is very low. The cost involved in such mechanisms is substantially
higher.

Though plants do not produce PHB naturally, they can be programmed to do so. For the
production of PHBs by genetic engineering, the two genes involved in the production of
enzymes namely, aceto-acetyl-CoA reductase (phbB) and PHB synthase (phbC) are isolated.
These two enzymes are pivotal in the PHB synthesis from its precursor. The genes responsible
are transferred and expressed in Arabidopsis thaliana plant. These two genes are so targeted so
that they are expressed in plastids of plant. The PHB gets accumulated in leaves and can be
extracted easily.

Thus synthesis of polyhydroxyl butyrate is a classic example of application of biotechnology in

the production of goods of commercial value. Transgenic trees producing phbB and phbC are
also produced where the PHB are synthesised in the leaves which can be extracted. Studies
shows that plant produce PHB 20 -40% of their dry mass.
This method of production involves many advantages like;

ADVANTAGES
(i) Upstream production (the production of biomass from which the biochemical has to be
isolated) costs are much lower than microbial fermentation.
(ii) The post translation modification system of proteins in a plant cell is as advanced as in the
animal cell. As a result the PHB which is formed undergo appropriate modification so that when
extracted they do not require any further modification.

(iii) The plant seeds of transgenic plants can be stored and transported easily. These seeds could
be used to produce transgenic plants to synthesize bioplastic when required.

(iv) Since animal system is not used, it does not face any concerns regarding to ethical issues.

DISADVANTAGES:
(i) The production levels do not rise to the expected levels.
(ii) The increased level of trangenes in a plant genome results in accumulation of transgenic
product. This can affect the plant growth and stability.

RECENT DEVELOPMENTS:

Several studies involving production of PHB in corn and potato has been initiated. Transgenic
tobacco plants have also been extensively studied for PHB production. It is also estimated that
potatoes can be a good source of PHB production from which PHB can be easily obtained and
the resulting yield is also proposed to be higher is such plastic potatoes. If successful, it will be
the most cost effective method involved.

APPLICATION OF BIOPLASTICS

Increasing and sustainable use of bioplastics is possible in various industrial segments due to the
fact that the biodegradable property of the bioplastics doesnt depend upon the raw materials
used but on the molecular structure.

Bioplastics are currently playing important role in the fields of packaging,

agriculture/horticulture, gastronomy, consumer electronics/durables and automotive to name a
few. In the above mentioned fields, products used for short term or single use like, mulch films
or catering products (spoons, plates) utilize bioplastics. Even consumer durable products like
mobile phone covers or automobile interior parts are increasingly made out of bioplastic
materials.

The application of bioplastics is growing steadily. Fields like building and construction, clothing,
household items, toys are increasingly made of bioplastic materials. Due to the increasing
demand for environmentally friendly and sustainable material is leading to higher growth and
capacity addition of bioplastic materials. This is also leading to increase in the variety of the
bioplastics being offered in the market. The material properties of the bioplastics such as
durability, flexibility, heat resistance, printability, transparency, chemical resistance are being
improved to match that of current materials.

Bioplastics are improved and sustainable solution to the plastic industry. The bioplastic material
can be used in all the fields where plastics are currently being used and even extend to some of
the newer fields. Some products produced using bioplastics have an advantage at end of life as
they are biodegradable and can be easily disposed confirming to strict environmental laws in
some countries. These materials are also environmentally friendly, reducing fossil fuel utilization
and hence conserve precious non-renewable resources. These bioplastics also help in reducing
green house gas such as carbon dioxide emissions. Another major environmental benefit by
switching to bioplastics is the reduction in emissions like dioxins, which are harmful to
environment as well as health and are currently being emitted due to burning of the plastics. Use
of bioplastics can turn out to be a necessity in regions, such as European countries, which are
heavily dependent on imported fossil fuels.
 BIOFERTILIZERS

Biofertilizers are the substances which make use of microorganisms to fertile the soil. These
fertilizers are not harmful to crops or other plants like the chemical fertilizers. They are actually
taken from the animal wastes along with the microbial mixtures. Microorganisms are used to
increase the level of nutrients in the plants. They let the plants grow in a healthy environment.
They are also environment friendly and do not cause the pollution of any sort. Use of
biofertilizers in the soil, makes the plants healthy as well as protect them from getting any
diseases.

TYPES OF BIOFERTILIZERS:-

1. Nitrogen Biofertilizers:-

This type of biofertilizers helps the agriculturists to determine the nitrogen level in the soil.
Nitrogen is a necessary component which is used for the growth of the plant. Plants need a
limited amount of nitrogen for their growth. The type of the crops also determines the level of
nitrogen. Some crops need more nitrogen for their growth while some crops need fewer amounts.
The type of the soil also determines that which type of biofertilizers is needed for this crop. For
example, Azotobacteria is used for the non legume crops; Rhizobium is needed for the legume
crops. Similarly blue green algae are needed to grow rice while Acetobacter is used to grow
sugarcane. It means almost all the crops need different types of biofertilizers depending on their
needs.

2. Phosphorus biofertilizers:-

Phosphorus biofertilizers are used to determine the phosphorus level in the soil. The need of
phosphorus for the plant growth is also limited. Phosphorus biofertilizers make the soil get the
required amount of phosphorus. It is not necessary that a particular phosphorus biofertilizers is
used for a particular type of crop. They can be used for any types of the crops for example;
Acetobacter, Rhizobium and other biofertilizers can use phosphotika for any

3. Compost Biofertilizers:-

Compost biofertilizers are those which make use of the animal dung to enrich the soil with useful
microorganisms and nutrients. To convert the animals waste into a biofertilizers, the
microorganisms like bacteria undergo biological processes and help in breaking down the waste.
Cellulytic fungal culture and Azetobacter cultures can be used for the compost biofertilizers.

ADVANTAGES OF BIOFERTILIZERS
1) They help to get high yield of crops by making the soil rich with nutrients and useful
microorganisms necessary for the growth of the plants.

2) Biofertilizers have replaced the chemical fertilizers as chemical fertilizers are not beneficial
for the plants. They decrease the growth of the plants and make the environment polluted by
releasing harmful chemicals.

3) Plant growth can be increased if biofertilizers are used, because they contain natural
components which do not harm the plants but do the vice versa.

4) If the soil will be free of chemicals, it will retain its fertility which will be beneficial for the
plants as well as the environment, because plants will be protected from getting any diseases and
environment will be free of pollutants.
5) Biofertilizers destroy those harmful components from the soil which cause diseases in the
plants. Plants can also be protected against drought and other strict conditions by using
biofertilizers.

6) Biofertilizers are not costly and even poor farmers can make use of them.

7) They are environment friendly and protect the environment against pollutants.

APPLICATIONS OF BIOFERTILIZERS TO CROP:-

I. Seedling root dip:-

This method is applied to the rice crop. A bed of water is spread on the land where the crop has
to grow. The seedlings of rice are planted in the water and are kept there for eight to ten hours.

II. Seed treatment:-

In this method, the nitrogen and phosphorus fertilizers are mixed together in the water. Then
seeds are dipped in this mixture. After the applications of this paste to the seeds, seeds are dried.
After they dry out, they have to be sown as soon as possible before they get damaged by harmful
microorganisms.

III. Soil treatment:-

All the biofertilizers along with the compost fertilizers are mixed together. They are kept for one
night. Then the next day this mixture is spread on the soil where seeds have to be sown.