Soil Microbial Diversity; Methods Used & Future Consideration

Contents

• Soil
• Soil microbial diversity
• Molecular methods:
 Gene probes
 Southern& Nouthernhybridization
 Microarray
 Phyloarray
 T-RFLP
 Metagenomics
 Sequence analysis
 Comparative genome

• Future consideration


Soil
Weathered end product of the action of climate and living organisms on soil parent material.

Soil microbial diversity
Diverse population of microorganisms inhabiting various horizons of soil.
Studied under following heads:
• Microorganisms in surface soil
• Microorganisms in Subsurface soil
• Microorganisms in Shallow subsurface soil
• Microorganisms in Deep subsurface soil

1. Microorganisms in surface soil

Surface soil is inhabited by Indigenous population of:
• Archaea
• Bacteria including actinomycetes
• Fungi
• Algae
• Protozoa
• Viruses.
In general, as Size of organism increases from bacteria to protozoa, the no. decreases.

(i) Bacteria – are almost always abundant in surface soil.
• Culturable bacteria- 107 to 108 / gm of soil
• Total population exceed 1010 / gm of soil.
• In Unsaturated soil aerobes outnumber anaerobes by 2 or 3 order.
• Anaerobic population increases with soil depth but rarely predominate unless soils are saturated and/ or clogged.

Based on DNA sequencing in combination with statistical approaches, diversity indicated by operational taxonomic units (OTU) where each OTU represents a different bacterial population in community. These provides estimates of diversity range upto 6300 OTU/ gm of soil.

Dominant culturable bacteria-

Mainly belong to the genus Arthrobacter, Streptomyces, Pseudomonas And Bacillus. All of them play role in nutrient cycling and biodegradation.




Examples of Important autotrophic soil bacteria

Organism Characteristics Function
Nitrosomonas Gram -, aerobe Nitrification
NH4+ to NO2-
Nitrobacter Gram -, aerobe NO2- to NO3-
Acidothiobacillus Gram -, aerobe S to SO4 2-
A. Denitrificans Gram -, fac. Anaerobe S to SO42-
A. ferrooxidans Gram -, aerobe Fe2+ to Fe3+

Important heterotrophic soil bacteria

Organism Characteristics Function
Bacillus Gram +, aerobe, spore forming C cycle, antibiotic production
Clostridium Gram +, anaerobe spore forming C cycle, toxin production
Methanotrophs Gram -, aerobe Cometabolize TCE
Ralstonia eutrophus Gram -, aerobe 2,4-D degradation
Via pJP4
Rhizobium Gram -, aerobe N2 fixation
Agrobacterium Gram -, aerobe Plant pathogen


(ii) Actinomycetes -
Characteristics-
• Structure – procaryotic
• Size- 1-2 micrometer in diameter
• Morphology – filamentous length of Cocci
• Gram stain- positive
• Abundance in soil- 106 to 108/ gm
Functions :
• Antibiotic source e.g. streptomycin from Streptomyces
• Produce goesmin, which gives soil, a characteristic earthy odour.
• Degrading complex molecules like chitin, cellulose. hemicellulose
• N2 fixation with non legume Frankia

(iii) Fungi
• Except yeast most are aerobic
• Their no. usually range from 105 to 106/gm of soil

Common genera of fungi found in soil are-

Organism Function
Penicillium & Aspergillus Nutrient cycling
White rot fungus Degrade lignin, DDT, TNT
Fusarium, Pythium, Rhizoctonia Plant pathogen
Coccidiodes immitis Human pathogen
Mycorrhiza Protection,P uptake

(iv) Algae – are typically phototrophic so found in areas where sunlight can penetrate. But actually algae are found at a depth of 1m because some algae can grow heterotrophically as well as photoautotrophically. Algal population are highest in surface 10 cm of soil.

Group of algae Examples
Chlorophyta Chlamydomonas. Found in acidic soil
Chrsophycophyta Navicula. Found in neutral & alkaline soil
Xanthophyta Botrydiopsis
Rhodophyta Porphyridium
Cyanobacteria Blue green algae

Functions:
• Carbon input by photosynthesis
• Weathered mineral particles so play role in soil formation.
• Produce extracellular polysaccharides which causes aggregation of soil particles.
• Cyanobacteria fix nitrogen, a nutrient that is usually limiting in a barren environment.

(v) Protozoa
• Unicellular, eukaryotic. The common genera found in soil include-
Heteromitra cucullus, Oikomonas & cercomonas (flagellates)
Colpoda cucullus & C. sitinii (cilliates)
Naegleria gruberi, Acanthoamoeba spp., Hartmanella hyalina ( amoebae)

Testaceous & rhizopodes restrict to acid soil.
Most of the protozoans are heterotrophic and survive by consuming other microflora

(vi)Viruses
• Bacterial viruses, as well as, plant & animal viruses find their way into soil through addition of wastes.
• Soil microflora may themselves harbor viruses.

2. Microorganisms in Subsurface soil

• In subsurface environment, the same patch like distribution of microbes exists that is found in surface soil.
• Culturable count 0 to 107/ gm of soil.
• Direct count 105 to 107/ gm of soil.
• Thus the difference between culturable and direct count is large in subsurface soil than in surface soils. This is mostly due to-
VBNC- viable but non culturable
VBDC- viable but difficult to culture
• Estimate that 99% soil microbes may be VBNC or VBDC

3. Microorganisms in Shallow subsurface soil

The study of subsurface microflora is new, beginning in 1980s only.
Complicated the study of subsurface life are the facts that sterile sampling is problematic and many subsurface microbes are difficult to culture. Some of the initial studies evaluating subsurface populations were invalidated by contamination with surface microbes. Because subsurface microbiology is still a developing field, so the information is limited. Yet Information is there about zone specifically rapid water discharge have a higher no. of microorganisms. Direct count remain fairly constant, ranging from 105 to 107/ gm of soil throughout the profile of shallow subsurface systems. This is low as compared to surface soil having 109 to 1010 cells/ gm Culturable count 0 to nearly equal to direct count
This is due to-
• Nutrients are limiting in subsurface, a greater proportion of population may be in the non culturable state.
• The Physiological & nutritional requirements of subsurface organisms are not understood

4. Microorganisms in Deep subsurface soil

Earlier it was thought that the deep subsurface environment contain few microbes because of oligotrophic conditions found there. But research has shown that microbes can be found at depth of > 3 km below earth’s surface.
First evidence regarding this was given by Edward bastin, a geologist at the university of Chicago, upon examine water form deep within oil fields
He found significant high levels of H2S and HCO3-. The presence of these materials could not be explained on a chemical basis alone. So suggested occurrence of sulfate reducing bacteria.

Subsequently, Frank Greer, a microbiologist at the University of Chicago, was able to culture sulfate reducing bacteria from water extracted from oil deposit 100s m below earth’s surface.
Types of organism mainly include Aerobe and facultative anaerobe chemoheterotrophs, denitrifiers, methanogens, S reducers & S oxidizers.



MOLECULAR METHODS TO STUDY SOIL MICROFLORA

Extraction of Nucleic acid from environmental samples-

The first step in molecular analysis is the extraction of nucleic acid from environmental samples. The most common approach to extraction of community DNA from soil is to lyse the bacterial cells in siyu (direct lysis)

Community DNA- DNA concurrently extracted from populations within a sample, generating a mixture of DNA referred to as Community DNA.

1. Gene probes and probing

Gene probes consisting of single stranded DNA can be used to identify the presence of a particular nucleic and sequence within an environmental sample. Typically, probes are short sequences of DNA, known as oligonucleotides, that are complementary to the target sequence of interest. These probes are labeled in some way that facilities their detection.

In order to design a gene probes, the DNA sequence of the gene interest must be known. This gene may be unique to a particular microbial species, in which case the gene probe would allow screening of an environment sample for the presence of that MOS.

Functional gene probes-
the target gene may code for production of an enzyme unique to a metabolic pathway. In this case, the gene probe results indicate that the environment sample contain the genetic potential for that particular activity referred to as gene probe.
e.g. gene probe complementary to genes coding for enzymes involved in N2 fixation.

Phylogenetic probe-
Probes designed against specific rRNA sequences known as Phylogenetic probe. These can be specific for groups of bacteria e.g. proteobacteria or its subgroups

Universal probes-
Probes designed to detect an entire domain ( bacteria, archaea or eucarya referred to as universal probes).

Size of probe-
Range from 18 bp to as many as several 100 bp.

Labeling of probes-
1. Earlier labeling option was radioactivity, done by labeling the sequence with a radioactive material such as P32 incorporated into DNA. After hybridization probe is detected by autoradiography.

2. non radioactive alternatives include probes labeled with Digoxigenin (DIG), biotin or fluorescein which can be incorporated by chemical synthesis. Different labels are detected by binding the respective Antibiotic or streptavidin- alkaline phosphate conjugate which when reacted with appropriate substrate will give a signal.

2. Southern and Northern Hybridization

Gene probes can also be used to detect target DNA or RNA on gels following electrophoreses in a process known as Southern hybridization (DNA) or Northern hybridization (RNA).
e.g. to know whether a gene is plasmid or chromosomally borne. This can be done using
Southern hybridization analysis. In this, all plasmids within microbe being studied are extracted and separated by gel electrophoresis. The plasmid DNA is then transferred on to a nylon membrane by blotting and membrane is subsequently probed, only the DNA molecules that contain target sequence hybridize with probe, thus allowing detection of these plasmids containing the target sequence.




Northern blotting is the analogous process used to analyse RNA. In this, total RNA has been extracted from environmental sample, run on gel and transferred to membrane and specific RNA is detected using appropriate probe.



Although extraction and stability of RNA are problematic, this technology can be used in gene expression studies to show induction of a specific gene.
Detection of DNA sequences give information about the presence of a gene in a population, whereas detection of RNA provides information about the expression of gene is given population.

4. Microarrays

A high throughput screening tool that is used to study gene expression. It is basically a collection of oligonucleotides or gene probes that have been “arrayed” on to a glass chip or slide. This microarray or gene array or gene chip is then hybridized with mRNA from a sample to determine which genes are being expressed.

Microarray is of two types:
• Printed or spotted-
 printed cDNA
 oligonucleotide
• Synthesized or in sillico


Printed cDNA arrays-
cDNA probes are made from mRNA transcripts of gene on interest using RT-PCR.

Printed oligonucleotide array
DNA sequences of gene of interest are used to design unique 35 – to 70- nucleotide probes which are then synthesized commercially and deposited on to the micro array slide and subsequently chemically linked to the slide.

In sillico synthesis
Some manufactures synthesize their oligonucleotides directly on the microarray chip called in sillico synthesis.
The upper range for printed microarrays is 1,60,000 probes/ array.
Agilent technologies’ high density microarrays format include 2,40,000 probes/ array Affymetrix microarrays contain over 1 million pobes/ array.

Problems
Only targets with high enough similarity or homology to the probes will bind, this can make specificity an issue.
e.g. when soil community DNA are analyzed, a reduced detection limit mat result, presumably due to target and probe sequences being diverse and consequently not highly homologous with designed probes. Inhibitors, cross reactive cDNAs and RNAses may also result in decreased sensitivity.

5.Phyloarrays

Phylochips allow to follow to follow population dynamics and community profile changes across a wide variety of species on the same array, based on 16s rRNA hybridizations.

Gary Andersen at Lawrence Berkeley national lab. Developed the idea of a phylochip
For the department of homeland securiby with DNA signatures for 9000 known species in phyla of bacteria and archaea.

A variety of less dense phyloarrays have also been developed to target a variety of microbial populations such as Enterococcus spp. Etc.

6. T-RFLP Analysis



Reffered to as terminal restriction fragment length polymorphism. In this method, DNA is extracted from microflora and digested with particular restriction endonuclease enzyme. For a T-RFLP analysis DNA is extracted from a microbial community and then amplified with a primer pair for a specific gene of interest, where one of the primers is flouresently labeled. The amplicon is subsequently digested with one or more restriction enzymes. Fragments are then separated on an automated DNA analyzer. And only fragments containing the fluorescently labeled primers are detected. Primer binding sites are located at the ends (or termini) of the amplicon, and fragments are differentiated based on sequence differences in regions extending from that binding site of the labeled primer, thus the name T-RFLP.
Microbial diversity in a community can be estimated based on the number and peaks heights of the terminal restriction fragment (T-RF) patterns. Which are easily visvualized on electropherograms. A variety of software programs are available for T-RFLP data analysis, facilitating highly sensitive and reproducible results.





7. Metagenomics

Refers to the genetic analysis of an entire microbial community. Term was coined by handelsman et.al. (1998).


This involve the cloning of large fragments of DNA extracted from the environment, allowing the analysis of multiple genes encoded on a continuous pieces of DNA as well as allowing screening of large environment fragments for functional activities.
Specialized vectors are required for the creation o large insert libraries.
1. Bacterial artificial chromosomes (BACs) can handle fragments of 300 kb.
2. Yeast artificial chromosomes (YACs) can incorporate upto 2MB inserts. But their transformation efficiency is 100 times less than bacs.
3. Most recent development in metagenomic sequencing is 454 sequencer.

In this technology, DNA is fractionated into small fragments that are fixed on to small beads. The small fragments then undergo a pyrosequencing step or sequencing by synthesis in which each DNA fragment is amplified by PCR to determine its sequqnce.

Its advantage is that it can be used to sequence large amount of DNA at low cost compared to BAC sequencing.

Its disadvantage that the length of 454 sequences is quite short (300 bp). Thus it becomes difficult to put these short sequences together to provide a picture of a microbial community.

Two main approaches exist for the analysis of metagenomic data-
1. Sequence based
2. Functional metagenomis

Sequence based-
Analysis can be directed e.g. clone libraries can be sequenced after PCR or probe screening for phylogenetic markers such as 16S rRNA genes present.

Functional metagenomics-
Refers to the use of a functional screen to identify clones for subsequent sequencing.

Metagenomic analysis are extremely data intensive. IMG/ M (available at http ://img.jgi.doe.gov/) is a metagenome data management and analysis system that provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative manner.
The potential of metegenomics is to allow insight into how microbial communities function and also to help unlock the vast genetic potential held with in the diverse population.

8. Sequence analysis

Advance is automated DNA sequencing have made the task of sequencing very routine and economical. DNA samples are generally sent o commercial sequencing lab or core facilities. A variety of sequence database have been compiled to catalog information and make it accessible to entire scientific community.
Several software programs allow researchers to utilize these database to identify sequences, translate a DNA sequence into protein, and look for homology or relatedness based on sequence.

The most common use of databases in studying soil microflora are in identification of isolates by their 16S rRNA gene sequence. The national centre for biotechnology information (NCBI) is an excellent resource for current freeware bioinformatics tools including the basic local alignment sequence tool (BLAST). This program allows researchers to submit a query sequence that is subsequently compared to all other sequences in database and scored for similarity and identity. Other tools such as Clustalw used to perform sequence comparisons where homologous sequences or unique sequence can be identified. Some time this information is used in phylogenetic analyses to identify genetic relationships and ancestory.
e.g. 16S rRNA gene sequence from an unknown oil isolate can be compared with other sequences in databases to identify the organism.


9. Comparative genomics

Advents in recombinant DNA and Sequencing tech. have resulted in the availability of large amount of sequence information. Sequencing is no longer limited to specific gene targets or relatively short DNA fragments, but can be applied to whole genomes. The first organism whose genome was completely sequenced was Haemophilus influenzae in 1995
According to Genome online database (GOLD) as of Jan 2007 >600 genome have been sequenced & 2200 ongoing projects. The availability of vast amount of sequence information, including whole genome sequences, has led to the creation of a new field of genomics reffered to as Comparative Genomics. Data management systems and analysis platforms can be used for comparison of subsets of genes or whole genomes. The Joint Genome Institute (JGI) provides such a platform, Integrated Microbial Genomes (IMG)

Comparative genomics examines both similarity & difference of genomes to-
• Draw function of particular gene
• Identify regulatory regions
• Find evidence of evolution
• Find genetic exchange by providing insights into the mobility of chromosomal sections and lateral gene transfer.
Bacterial and archeal thermophiles share same habitats and there is abundant evidence from genome analysis that lateral gene transfer is common in group
E.g. Thermotoga maritima genome have approx. 20% of genes that have homology to hyperthermophilic archaea Pyrococcus spp. (Nelson et al 1999)


Future consideration of soil microflora-

• Studying soil microflora, it has been reported that some microbes (Geobacter) can generate electricity. So used in production of microbial fuel cells to generate electricity.
• Some microbes can be used for the conversion of toxic compounds into non toxic forms or less toxic ones.
e.g. PAHs (polycyclic aromatic hydrocarbons) like pyrene is converted into less toxic or non toxic compounds by various microbes like Bacillus, Aeromonas and Pseudomonas

recombination

Contents:

• Definition
• Examples
-In prokaryotic system
-In eukaryotic system
• Types
• Meselson and Wiegle exp.
• Holliday model
• Potter and Dressler’s evidence
• Meselson and Raddding
• Enzymology
• Conclusion


Recombination
Definition- Recombination is a process or set of processes by which DNA molecules interact with one another to bring about a rearrangement of the genetic information or content in an organism.

Examples of recombination in prokaryotic systems are:• Integration of the bacteriophage lambda prophage,
• Recombination of bacterial DNA following conjugation between bacteria,
• Formation of plasmid multimers.
Examples of recombination in prokaryotic systems are:• Meiosis crossing over- also called gemetic recombination.
• At the time of expression of antibody genes so as to generate great potential diversity of antibody molecules.

Types of recombination- 4 general types of recombination:
Homologous genetic recombinationAlso known as general recombination or general homologous recombination involves an exchange of genetic material between homologous chromosomes, resulting in the arrangement of genes into new combinations.
It is also called general recombination because the enzymatic machinery that mediates the exchange can use essentially any pair of homologous DNA sequences as substrates.
This is the type of recombination that is required during meiotic crossing over, for recombination following bacterial conjugation, and during the formation of plasmid multimers
Site-specific recombination
As the name implies, this type of recombination involves the exchange of genetic material at very specific sites only. Examples include the integration of bacteriophage lambda into the host chromosome to form the prophage and the rearrangement of chromosomal DNA prior to expressing antibody genes.
DNA transpositionServes as mechanism by which particular DNA sequences called transposons may be moved from one location to another on chromosomes independently.
Illegitimate recombinationA very rare form of recombination, occurs between non homologous DNA independently of any unique sequence element.

Molecular mechanism of recombination-
The Meselson - Weigle Experiment
In the simplest sense, recombination is an exchange of both strands between two DNA molecules:

This representation implies that both strands of each molecule must be broken and then rejoined. This was first demonstrated by an experiment performed by Matt Meselson and Jean Weigle in 1961.
Meselson and Weigle infected E. coli cells at the same time with phage from two different stocks of bacteriophage lambda. One stock had been prepared by growing the bacteriophage lambda c-mi- in cells grown in medium containing heavy isotopes of carbon (13C) and nitrogen (15N). The other stock had been prepared by growing bacteriophage lambda c+mi+ in medium containing light isotopes of carbon and nitrogen.


After infection, the progeny phage were isolated and banded on a CsCl gradient.
A broad band of phage particles were found on the gradient.
• Nonrecombinant phage were found, as expected, at two well-defined densities corresponding to the parental light and heavy phages.
• Recombinant phage were found - surprisingly - at all intermediate densities between these two.



They also followed the course of the infection using two genetic markers, c and mi, which were located near one end of the lambda chromosome.

When the phenotypes of the intermediate density phage particles were analyzed, recombinant phage that were c-mi+ were found near the band of "heavy" phage while recombinant phage that were c+mi- were found near the band of "light" non-recombinant phage.
These results can only be explained if recombination between the two parental phage involves breakage and rejoining of both DNA strands (as shown above).

The Holliday Model of Genetic Recombination
This model of recombination was first proposed by Robin Holliday in 1964 and re-established by David Dressler and Huntington Potter in 1976 who demonstrated that the proposed physical intermediates existed.
The basic (simple) model
Align two homologous DNA molecules.

Nick the DNA at the same place on the two molecules.
This must happen in strands with the same polarity.

Exchange strands and ligate.The intermediate that is formed is called a Holliday intermediate or Holliday structure. The shape of this intermediate in vivo is similar to that of the greek letter chi, hence this is also called a chi form.

Visualization of the next step is made easier if one molecule is now rotated through 180š with respect to the other. This also helps to emphasize the chi-shape of the intermediate:


Resolve the structure.There are two ways in which this can happen:
• If the same strands are cleaved a second time then the original two DNA molecules are generated:

• If the other strands are cleaved, then recombinant molecules are generated:


A more realistic model
The above model is too simple and does not explain a number of genetic results, including the occurence of two different recombinant bacteriophage in a single plaque in the Meselson-Weigle experiment.
These can be explained by modifying the model slightly. As before, two homologous DNA molecules must be aligned and nicked at the same place. Following strand exchange the intermediate Holliday structure is formed. At this stage a new step is introduced:
Branch migration.
Migration of the branch can occur over many nucleotides in either direction. The result is a physical transfer of part of one of the strands of one molecule with that of the other:

Once again, visualization of the next step is made easier if one molecule is now rotated through 180š with respect to the other.

Resolve the structure.
There are still two ways in which this can happen, however, the consequences are different:

• If the same strands are cleaved a second time then nonrecombinant DNA molecules are generated but they each contain a region of heteroduplex DNA that spans the region of branch migration. Such heteroduplexes are called Patch recombinants.

• If the other strands are cleaved, then recombinant molecules are generated as before, however, each will also contain a region of heteroduplex DNA that spans the region of branch migration. Such heteroduplexes are called Splice recombinants.


Potter & Dressler's evidence for the Holliday ModelIn 1976, David Dressler and Hunt Potter published the results of a series of experiments that demonstrated the validity of the Holliday model of recombination.
They used E. coli cells containing the colicin E1 derived plasmid, pMB9. This plasmid was one of the very earliest plasmids developed for cloning in Herbert Boyer's laboratory. Normally, E. coli contain about 20 copies of this plasmid per cell. However, if the cells are exposed to chloramphenicol then, although chromosomal replication stops, plasmid replication does not and the number of plasmid molecules increases to 1000 copies per cell. With so many more copies of the plasmid in the cell, the chances of recombination increase as does the probability of observing a recombination intermediate.
When plasmid was isolated from the cells, purified by CsCl gradient centrifugation, and observed in the electron microscope, a number of candidates for intermediates were observed. These all had the appearance of "figure 8" structures. However, there are 3 possible ways such structures might arise:
• as a double-sized circular plasmid twisted over on itself.
• as two interlocking circular plasmid molecules.
• as a genuine recombination intermediate.
In order to distinguish between the three possibilities, Potter and Dressler digested their plasmid preparations with EcoRI. This enzyme will generate monomer sized linear molecules from either of the first two possible structures. However, it will generate unique chi-shaped structures from the third.
When they did this, Potter and Dressler found that between 0.5% and 3% of the molecules they observed were chi-shaped structures. The molecules were symmetrical in that the opposite arms were identical lengths and had identical denaturation patterns. Finally, they saw no such structures if they prepared their plasmids from recA- strains of E. coli.
From this evidence they concluded:
. . . the intermediates we have observed in the electron microscope provide physical evidence in support of the recombination intermediate postulated by Holliday on genetic grounds.


Matt Meselson And Charles Radding Models of Genetic RecombinationAlthough, the basic features of the Holliday model are well-established, the model does have flaws. For example, the mechanism by which two homologous regions of DNA are paired and then nicked is not well explained. In addition, the model does not explain all of the observed results in different recombination systems.
A central feature of the Holliday model as outlined above is that the heteroduplex regions in recombinant molecules will be identical in length and position. Experimental results suggest, however, that this is not necessarily so.



In order to explain such discrepancies, Matt Meselson and Charles Radding proposed a modified model. In their model, only one strand in one of the two paired homologous DNA molecules is nicked. This strand is then displaced by DNA polymerase I and invades the homologous chromosome. A Holliday intermediate is eventually formed and resolved as above. In this model, the extent of heteroduplex DNA in the resolved products, whether recombinant or nonrecombinant, will be different. Finally, gene conversion (which is a type of recombination event) in yeast follows a mechanism, proposed by Szostak, that requires double-strand breaks in one of the recombining molecules.

Enzymology of general recombination-Recombination Proteins in E. coli
The characterization of the structures and biochemical function of a number of the key proteins required for recombination is now helping us to understand details of the mechanism of recombination. The most important proteins are RecA, RecBCD, RuvA, RuvB and RuvC.

RecBCD The recB, recC & recD genes code for the three subunits of the RecBCD enzyme which has five activities: exonuclease V; a helicase activity; an endonuclease activity; an ATPase activity; and, an ssDNA exonuclease activity.
The RecBCD helicase activity can unwind DNA faster than it rewinds. Thus as it travels along a DNA molecule, it can generate ssDNA loops.
The RecBCD complex functions as a DNA exonuclease. It will bind to double-stranded breaks in DNA and degrade both strands simultaneously. However, when RecBCD encounters a Chi sequence, its activity changes. These Chi sites are recombinational Hot Spots. Characterized by 5’-GCTGGTGG-3’. Rec BCD cleaves DNA strand 4-6 nucleotides to 3’ side of Chi. The RecD subunit is released and the RecBC proteins act as a helicase to unwind the DNA in an ATP dependent reaction. This generates a ssDNA region that can serve (along with RecA) to initiate strand exchange and a recombination reaction.
the RecBCD pathway illustrates this:




RecAThe RecA protein is a multifunctional powerhouse! It has strand-exchange, ATPase and co-protease activities all packed into a compact 352 amino-acid, 38 kDa structure. It is required for all recombination pathways in E. coli.
The RecA protein will bind cooperatively to a ssDNA molecule with each monomer of RecA binding to a span of 4-6 nucleotides. Assembly of the nucleoprotein complex proceeds in a 5' -> 3' direction. The complex is both fairly stable (half-life is 30 min) and is the active species that will promote strand exchange.
The RecA protein monomer structure is shown above. Monomers associate with one another via the N-terminal alpha helix which interacts with the adjacent monomer -- forming a molecular conga line, if you will, with one helix on the shoulder of the molecule in front of it! The RecA filament that forms is helical with a pitch of 82.7 Å and it consists of 6 monomer units per turn.
RecA will promote strand exchange between DNA molecules as long as the following conditions apply:
• One of the two molecules must have a ssDNA region to which RecA can bind.
• The two molecules must share a region of homologous (i.e. nearly identical) DNA sequence - a minimum of 50 bp is required.
• There must be a free end within this region of homology which can initiate the strand exchange.
The strand exchange reaction probably involves the following steps:
• RecA binds to the ssDNA partner.
• The two molecules are aligned possible through the formation of a triple-stranded intermediate.
• Displacement of one of the old strands. This requires concurrent migration of the RecA nucleoprotein filament along the molecule - which proceeds in one direction only (5' -> 3') - and consequent winding/unwinding. ATP hydrolysis takes place during this step.
Proteins required for Resolving Holliday Junctions in E. coli

RuvARuvA is a small protein whose function is to recognize a Holliday junction thereby assisting the RuvB helicase to promote branch migration.
The crystal structure of the E. coli RuvA protein was solved at a resolution of 1.9 Å. The protein forms a tetramer in an unusual manner - though one that is ideally suited to its function. To quote from the summary part of the paper:
"Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction."
The RuvA protein is 203 amino acids in length but only 190 of them could be assigned in the crystal structure. Most of the missing assignments represent amino acids in a flexible part of the protein.





More recently, the crystal structure of RuvA complexed with DNA has been solved first at 6.0 A and then at 3.1 A.
Finally, examine these three model structures showing how a Holliday intermediate could interact with RuvA:






The RuvA-Holliday junction complexes have the predicted structure with a four-fold symmetry. The DNA bound at the junction is not flat but slightly concave to maximize the contact between the DNA and the protein and, as a result, the axes of the four helices are inclined at about 10 degrees. In the centre of the complex, eight residues corresponding to Glu-55 and Asp-56 from each of the four subunits, form an acidic central pin which may repel the DNA backbone away from the centre of the junction thereby, opening up the centre.
These refined structures have also shown that there are only 4 unpaired bases in the centre of the complex. This is a lot fewer than found for typical helicases.

RuvBThe RuvB protein is a helicase that catalyzes branch migration of Holliday junctions. By itself it cannot bind to DNA efficiently. It functions in combination with RuvA. Like other helicases, RuvB functions as a hexamer; but, unlike other helicases, RuvB encloses double-stranded DNA not ssDNA.
Electron microscopy has shown that RuvB is a heptamer in solution and that it converts to a hexamer ring when it binds to DNA. Electron microscopy has also shown that the two hexamer rings of RuvB lie contacting RuvA on the two opposite sides of a RuvAB-Holliday junction complex.
RuvCThe RuvC protein resolves the Holliday intermediate. It functions as a dimer to cleave two of the four strands that make up the central part of the intermediate. Since binding is symmetrical, RuvC can bind to the Holliday intermediate in two equally likely ways. Hence, Holliday intermediates can be resolved in two different, but equally likely, ways.
RuvC does have some sequence specificity. It cleaves DNA at the 3'-side of thymidine, preferentially at the consensus 5'-A/TTT|C/G -3' where | indicates the site of cleavage
The interaction of RuvC with Holliday junction is shown:


A Model for Resolving Holliday Junctions in E. coliThe crystal structures of RuvA and RuvC, as well as detailed electron microscope visualization of Holliday junction protein complexes allow for a reasonable model to be drawn showing how branch migration occurs and how the junction is resolved:

It i sknown now that RuvC functions with RuvA and RuvB in a higher order complex, termed the RuvABC resolvasome. However, it is also clear that the known structure of RuvC cannot be docked with the detailed RuvA-Holliday complex structure - steric conflicts are found. It is believed that there must be some other structural changes that occur within the resolvasome; but, these have yet to be identified.
The structure of RuvB at 1.9A has been determined recently. The proposed model of the complete resolvasome fits well with the hypothetical models have have been devised based on the structures of RuvA and RuvC. It has also been possible to devise a model to explain DNA translocation through RuvB as a result of ATP hydrolysis.

Conclusion-
• Chromosomal segregation, shuffles together the genetic material carried by different members of a sexual species.
• This genetic mixing unties the evolutionary fate of alles at one locus from the fate of alleles at neighboring loci and can increase the amount of genetic variation found within a population.
• In the process, however, recombination separates advantageous gene combinations, the very gene combinations that enabled the parents to survive and reproduce.
• Whether or not the adaptation of a population to an environment is more rapid in the presence of recombination, that is, whether or not recombination speeds up the evolutionary process, depends critically on the ways in which this process is modelled. As we shall see, the effect of recombination depends on the population size, the initial population composition, and the selection regime under consideration.

no inverter, no generator, own a powerhouse in basement

NO GENERATOR, NO INVERTER- OWN A POWERHOUSE
24 HOURS ELECTRICITY
(FUTURE APPROACH)



Power plant in basement-

• Within a decade or two, we find ourselves living in home whose electricity coming not from a generating plant many kms away but rather from a refrigerator sized power station in basement or backyards.
• Not just homes but shops, hotels, apartment buildings & possibly factories may all be powered in same way.

This would have been possible by use of microbes based fuel cell (MFCs).

CONTENTS:

• Microbes
• Electricity
• Sources for Electricity Generation
• Limitations of Different Sources
• Microbial Fuel Cells
• Types:
- Organic Waste Water Based
- Sugar Based
- Diesel Based

• Improved Performance In MFCs:
- By Addition of Sulphate Compounds
- Using Stacked MFCs

• Limitation in Commercialization Of MFCs
• Microbes Used In MFCs
• Conversion of Mud Into Electricity
• Mechanism of Electron Transfer
• Advantages of MFCs
• Conclusion
• References


What Are Microbes?

Living organisms that are usually too small to be seen clearly with naked eyes OR with a diameter of 1mm or less.

Mainly include bacteria, viruses, yeasts &molds, algae, protozoa &helminthes.


What Is Electricity?

Flow of electrons generates electricity.

In a closed circuit, electrons flow from positive pole to negative pole & current flows in opposite direction.



Sources for Electricity Generation-

• Water
• Coal
• Wind
• Atomic power


Limitations of Different Sources-


Water-
• Depletes natural flora.
• Processed water affects crop in nearby fields.
• Not available everywhere & so transportation of current makes it cost effective.

Coal-
• Nonrenewable & exhaustible.
• Creates pollution problems.

Wind-
• Not available everywhere & all the time.

Atomic power-
• Poses health hazard.


The latest research proves that Organic Waste when acted upon by microbes generates electric current.


So MICROBIAL FUEL CELLS have been constructed. In this a particular substrate is used as fuel and particular microbial species act on this, releases electrons exocellularly and throws electrons on electrode which flows through circuit & generates electricity.






Types of Microbial Fuel Cells-

1. Organic waste water based.
2. Sugar based.
3. Diesel based.

1. Organic waste water based-

• Fuel - organic waste water
• Current - 0.2 mA
• Resistance - 1k ohm
• Fall in COD - from 1700mg/l to 50mg/l
• Identification of microbes - by denaturing gradient gel electrophoresis.

Microbial population is found to be different from that in sludge used as inoculum.On electrode surface a thin biofilm is observed in which nanobacteria like particles were present.

2. Sugar based-

• Fuel - Sugar
• Current density - 158mA/m²
• Resistance - 510 Ω
• Microbe - Rhodoferax ferriducans

Following 20 days growth, large no. of bacterial cells attached to electrode surface. Planktonic cell conc. was 140mg/l & attached biomass of electrode surface was 1180mg/l. By analyzing voltage value it was proved that planktonic cell conc. play no role in electricity production & microbial electricity generation is attributed only to biologically active cells attached to electrode.

This kind of MFC is efficient in biomass utilization & energy restoration.

3. Diesel based-

Involves anaerobic biodegradation of diesel

• Power generated - 31mW/m²
• Identification of microbes - 16S rRNA gene sequencing.

Majority of microbes more than 98% show similarity to bacteria capable of denitrification such as Citrobacter spp., Pseudomonas & Stenotrophomonas. Remaining show similarity to Shewanella, Alishewanella.
Also observed that anaerobic biodegradation of diesel was enhanced in MFC (82% removal) as compared to an anaerobically incubated control cell (31% removal) over 21 days at 30ºC




Improved Performances In MFCs-

1. By addition of sulphate compounds
2. By using stacked MFCs


1. By addition of sulphate compounds- Mechanism of electron transfer from microbes found in anaerobic sludge to anode electrode in MFCs have been investigated. In doing so, both energy accumulation & improved performance were observed as a result of addition of exogenous sulphate compounds. Treatment of anaerobic sludge by washing & centrifugation can provide samples devoid of sulphide/ sulphate. Addition of exogenous sulphate can give matched samples of S-deplete & S-replete suspensions. When these are compared in exp MFCs power output of S-deplete is only 20 % of S-replete systems.


2. By using stacked MFCs- WILLY VERSTREATE & his colleagues at Ghent University in Belgium tested fuel cells in an array of configuration :

• in series
• in parallel
• individually

They observed that connecting several MFC units in series or parallel can increase voltage and current. Six individual MFC units in stacked configuration produced-

• Max power output - 258 W/m³
• Increase voltage - 2.02 V
• Increase current - 255mA

Also observed that with time, initial microbial community decreased in diversity & Gram positive species dominant. The shift of microbial community accompanied tripling of short time power output of individual MFC from 73 W/m³ to275 W/m³ and decrease of internal ohmic resistance from 6.5Ω to 3.5Ω
Among different configurations, parallel stack was found to be most efficient.




Limitations in Commercialization of MFCs-


Mainly due to two reasons-

• Power produced is limited
• High internal ohmic resistances

MFCs are not yet commercialized but they show great promises as a power sources for environmental sensors as well as a method of waste water treatment.
Improvements in system architecture will soon result in power generation dependant on capabilities of microorganisms.

Microbes Used In MFCs-

• Among different groups of microbes, mainly bacterial communities dominate in MFC.
• These show great diversity ranging from proteobacteria including several species of Geobacter & Shewanella to communities composed of proteobacteria, firmicutes & uncharacterized clones in other MFC. But by the end of experiment when performance was at its peak – one species, Brevibacillus brevi made up majority of electron producing microbes.
• Bacteria capable of exocellular electron transfer are called as Exoelectrogens.
• Also called as Electricigens because producing electricity.



Conversion of Mud into Electricity-

Certain microbes found at bottom of ocean can transform organic matter into electrical energy. These microbes could not only be used to produce power in subsurface settings but also have implications in industry & military.





Acc to DEREK K. LOVELY, UMass microbiologist & team leader, an understanding of how microbes generate & use electrical energy may also prompt development of new technologies.

EXP. SET UP- They used water & sediment from Boston Harbor, a collection of Mason jars, sterile graphite electrode, ordinary electrical wirings to determine science behind mechanics of simple sediment battery. They added a layer of common mud to water in jars, put one graphite electrode in mud & another in overlying water. The resulting electric current was strong enough to activate a light bulb or a simple computer.

“Even using a primitive electrode made from graphite, it is possible to produce current to power basic electronic marine instruments.”

Through more refined exp. Lovely’ group found that a family of energy harvesting microbes commonly referred to as Geobacters were key to production of electric current.

Characteristics of Geobacters- As most life forms get their energy oxidizing organic compounds, Geobacters can grow in envt lacking oxygen by using iron naturally present in soil, in place of oxygen. So Geobacters can also substitute an unnatural substance such as iron for electrode.

A large no. of Geobacter species Desulphoromonas acetoxidans were found on anode end of primitive batteries. When researchers destroyed D. acetoxidans in sediment, the current stopped. In mud community of microbes break down complex organic matter into acetate. Geobacters then transfer electron from acetate to electrode generating electrical energy.

Earlier studies had shown that bacteria could produce electricity under artificial conditions in which special chemicals were added, but UMass study was first to prove that microbes living in a typical marine envt. could produce electricity under natural conditions of environment.



Advantages of MFCs-


1. NASA has believed that such microbes could be used to recycle waste on long space flights.

2. Reliable source of electricity in poor countries-

• In poor countries like Uganda, use of a cell phone or MP3 player charger can be a struggle due to difficulty in accessing electrical grid.
• More than 99% of rural households in African nation are cut off from reliable source of electricity.
• So to solve this problem, a group of MIT students has devised a microbial fuel cell that run entirely on plant waste. The students’ BioVolt MFC prototype uses cellulose munching bacteria to generate electricity.
• But main challenge for students was to develop cheap, yet efficient, device one that use non platinum catalyst.
• Though they were not willing to share exact specifications for fuel cell but they claimed that they will provide such fuel cell at cost of as little as 2 US dollars.

3. Power plant in basement-

• Within a decade or two, we find ourselves living in home whose electricity coming not from a generating plant many kms away but rather from a refrigerator sized power station in basement or backyards.
• Not just homes but shops, hotels, apartment buildings & possibly factories may all be powered in same way.

4. Companies & industrial research laboratories in Belgium, Canada, Denmark, Italy, Japan, Korea & US have fuel cell development efforts under way & few are already selling units.
Some United Tech has sold about 170 units, which are being use for generation of both heat & power.

5. MFCs are also use for treatment of wastewater and in green facilities.



Conclusion-

Today’s world is highly commercialized & every aspect of our life depends on electricity. Various sources used for electricity generation suffer certain limitations. So alternative power sources are needed-

• To reduce nation’s dependence on foreign oil.
• To reduce global warming.
• To reduce pollution problems.

So use of organic waste material as a source for electricity generation could bring about revolution in commercialized world dependant on electrical energy. But this project is in its infancy & much research work is required to be done.

history of microbiology

HISTORY OF MICROBIOLOGY


Microorganisms were probably the first living things to appear on earth and the study of fossil remains indicate that microbial infections and epidemic diseases existed thousands of years ago.

• It was Rogen Bacon who in the 13 century postulated that disease is produced by invisible living creatures.

• Physician Girolamo Fracastoro of North Italy made this suggestion again in 1546. Fracastoro wrote a treatise De Contagione in which he said disease was caused by minute “seed” or “germ” and was spread from person to person.

• In 1665, Robert Hooke used a simple lens that magnified objects approx. 30X. He examined thin slices of cork, the bark of oak tree and found that cork was made of tiny boxes that Hooke referred to as cell.

• It was followed by Matthias Schleiden and Theodore Scwann who examined a variety of micro organisms and in 1838- 1839 reported that “all forms of life are composed of cells.” an observation that later became cell theory. The cell theory has since been modified to all life is composed of cells that originate from other cells.


THE DISCOVERY OF MICROBIOLOGY AS A DISCIPLINE COULD BE TRACED ALONG THE FOLLOWING HISTORICAL ERAS:

• Discovery Era
- Antony van Leeuwenhoek
- Robert Hooke

• Transition Era
- Francesco Redi
- John Needham
- Lazzaro Spallanzani
- Schulze and Scwann
- Schroeder and Von Dusch

• Golden Era
- Louis Pasteur
- Robert Koch

• Immuniztion Era
- Edward Jenner
- Lord joseph lister
- Elie metchnikoff

• Chemotherapy Era
- Alexender fleming

• Molecular Era
- Joshua lederberg
- Winogradsky and beijerenck




DISCOERY ERA-

This period concerns with the discovery of microbial world that has been dominated by Antony van Leeuwenhoek




1. Antony van Leeuwenhoek [1632- 1723]

Leeuwenhoek was born in Holland (Netherlands) delft on October 24, 1632. It was a cloth merchant only who discovered bacteria, free living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematode, and rotifers and much more.

His notable contributions to microbiology are:

• Discovery of microbial world- He was the first to report his observations with accurate descriptions and drawings. It was he only who described microorganisms accurately for very first time. He described thread like fungi as well as certain microscopic algae. He gave outlined structural details of familiar Paramecium and amoeba.

• Introduction of term animalcules- In 1676 he gave the term animalcules to microorganisms. His descriptions of protozoa were so accurate that many of the forms he described were easily recognized today.

• Formation of microscopes- He was a Dutch linen merchant but spent much of his time constructing simple microscopes composed of double convex lenses held between two silver plates. He constructed over 250 powerful microscopes that could magnify around 50 to 300 times and he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at an angle of 45° to specimen plane. This arrangement would have provided a form of dark field illumination and made microbes clearly visible

• Contribution to medicines- He contributed to medicines also. He was founder of animal histology. He discovered the existence of spermatozoa and red blood cells. He discovered and described the capillary circulation and completed the work on circulation of blood, the capillary connection between arteries and veins.

• Transmission of letters to Royal Society- He transmitted his observations to the royal society in 1676 in the form of long series of letters written in Dutch including numerous drawings. He astonished everything by claming that many oh the tiny things he saw with his lens alive because he saw them swimming in purposefully about. Most of these letters were translated and published in England in the proceedings of Royal Society. He got fellowship in 1680 from Royal Society.

Because of this extraordinary contribution to microbiology, he is considered as Father of Bacteriology and Protozoology.




2. Robert Hooke [1635- 1703]

Born on July 18, 1635 at freshwater on Isle of Wight. He was apparently largely educated at home by his father he was able to enter Westminster school and from there went to Oxford where some of the best scientists of England were working. Hooke impressed other scientists with his skills at designing experiments and building equipments.

In 1662, Hooke was named curator of experiments of the newly formed Royal Society of London meaning that he was responsible for demonstrating new experiments. He was an active, infatigable genius even almost to last. He died in London on March 3, 1703.

His notable contributions to microbiology are:

• Micrographia- Hooke’s reputation in history of biology largely rests on his book Micrographia published in 1665. He devised compound microscope and illumination system.

• Study of cell- In 1665, he used a simple lens that magnified objects approx. 30x he examined thin slices of cork, the bark of oak tree and found that cork was made of tiny boxes that Hooke referred to as cell. He discovered plant cell.



TRANSITION ERA-

During this period, noteworthy contribution was made: the controversy over spontaneous generation that said that living organisms could develop from non living matter i.e. abiogenesis.

• Francesco Redi- The ancient belief in spontaneous generation was first of all challenged by Redi, who carried out a series of experiments on decaying meat and its ability to produce maggots spontaneously. Redi in 1665 put the theory of spontaneous generation to rest by conducting a simple experiment in which he placed meats in three jars. One jar was covered with a fine gauze, second was covered with paper and third was left uncovered. Flies entered the jar that was open to air i.e. left uncovered and landed on meat where they laid their eggs that later developed into maggots. The other two pieces of meat did not produce maggots spontaneously. However, flies were attracted to the gauze covered jars and laid their eggs on the gauze and maggots subsequently developed access to the meat, indicating that maggots were the offspring of the flies and did not arise from some “vital source” in the meat as previously believed.

• John Needham- he was probably the greatest supporter of the theory of spontaneous generation. In 1749, he proposed that tiny organisms, the animalcules arose spontaneously on his mutton gravy. He had covered the flasks with cork as done by Redi and even heated some flasks. Still the microbes appeared on mutton broth.

• Lazzaro Spallanzani- Spallanzani attempted to refuse Needham’s work by performing extensive experiments. He boiled beef broth for longer period, removed the air from the flask and then sealed the container. Following incubation, no growth was observed in flasks. When he was accused of destroying the vegetative force of the nutrients by over heating, he showed that the heated nutrients could still grow animalcules when exposed to air by simply making a small crack in the neck. Thus, in Spallanzani in 1775 disproved the doctrine of spontaneous generation.

• Franze Schulze and Theodor Scwann- Needham and other critics countered the doctrine of Spallanzani and stated that life force had been killed when the flasks were from oxygen, a gas known to be required in the respiration of animals. Schulze and Scwann, who were of the view that air was the source of microbes and sought to prove this by passing air through hot glass tubes or strong chemicals into boiled infusions into flasks, answered this argument. The infusion in both the cases remained free from the microbes. However, the die hard advocates of spontaneous generation were still not convinced and said that the treatment of air by acid or heat had altered the air so that it would not support growth.

• Georg Schroeder and Theodor Von Dusch- In 1854, Schroeder and Von Dusch performed convincing experiments to disprove the theory of spontaneous generation by simply passing air through cotton into flasks containing heated broth. No growth of microbes was observed on the infusions due to the filtering out of microscopic organisms by cotton. Thus, Schroeder and Von Dusch were the first to introduce the idea of using cotton plugs for plugging microbial culture tubes in 1854.




GOLDEN AGE OF MICROBIOLOGY-

Golden age of microbiology began with the work of Louis Pasteur of France and Robert Koch of Germany who had their own research institutes. Here we find indisputable proof that microbes cause disease. More important, there was an acceptance of their work by the scientific community throughout the world and a willingness to continue and expand their work. During the period, we see the real beginning of microbiology as a discipline of biology.






3. Louis Pasteur [1822-1895]:

Pasteur was born on December 27,1822 in Dole, France. His name is forever cemented in history of medicines. From his young years, Pasteur was a talented artist, his portraits were so professional, that his name was listed in the compendiums of artists in xix century.

His notable contributions to microbiology are:

• Microbes as cause of disease- Pasteur in 1842, demonstrated that microbes could be cause of disease for if they could spoil the wine; perhaps they could also make the body sick. This leads to the development of germ theory of diseases.

• Crystallography – He studied the shapes of organic crystals and proved that they have two kinds of structure, later called isomers and formulated the law: asymmetry differentiates the organic world from mineral world. The external shape of crystal, its molecular composition and its action on polarized light are all linked. Polarized light is rotated by asymmetric crystals, not by crystals that have plane of symmetry.

• Alcoholic fermentation (1857)- Fermentation is a biological phenomenon. Each type of wine fermentation is caused by a specific microorganism, which can be cultivated. Also Pasteur found that the main cause of economic losses in France wine industry, recommended the correct type of micro organism to be used in winery (1864) and recommended to heat the wine up to 55°C killing the unwanted bacteria (process known as Pasteurization).

• Pasteur against spontaneous generation (1860)- Pasteur performed a series of experiments to prove that although micro organisms were present in air they were not spontaneously produced. To prove that air was source of microbes, he filled several round bottomed flask with nutrient solution and fashioned their openings into elongated, swan neck shaped tubes. The flask’s openings were freely open to air but curved so that gravity would cause any air borne dust particles to deposit in lower part of neck. The flask were heated to sterilize the broth and incubated. No growth occurred even though the contents of flasks were exposed to air. Pasteur pointed out that no growth took place because dust and germs had been trapped on the walls of the curved necks but if the necks were broken off so that dust fell directly down into flask, microbial growth commenced immediately. Some of the ingenious flasks are still on display at Pasteur Institute in Paris in their original sterile form. This experiment clearly showed that micro organisms present in or on non living material such as dust or water were responsible for the contamination of sterile solutions. PASTEUR thus in 1861 finally resolved the controversy of spontaneous generation vs biogenesis and proved that micro organisms are not spontaneously generated from inanimate matter but arise from other micro organisms.

• Disease of silk worms (1865-1870)- Healthy worms became infected when allowed to nest on leaves used by infected worms. He also noted that the susceptibility of the worms varied widely, some worms dying shortly after infection, some weeks later, some not at all. Pasteur isolated bacilli of pebrine and flacherie worked out hygienic rules for silkworms’ farmers.

• Pasteurization- Pasteur in 1867 suggested that, mild heating at 62.8°C for 30 minutes, rather than boiling, was enough to destroy the undesirable microorganisms without ruining the taste of product, the process was called Pasteurization. It was introduced in United States on a commercial basis in 1892.

• Germ theory of disease (1877) - The top of Pasteur‘s career were development of the germ theory of disease and the use of vaccines and other physicians antiseptic medicine and surgery.

• Anthrax (1877-1881) - Anthrax is transmitted from animals to humans caused by Bacillus anthracis discovered by Robert Koch in 1876. Pasteur isolated the germs and started-
-vaccinating sheep against anthrax in 1879
-vaccinating chickens against chicken cholera 1879

Discovery of immunization against disease using weakened bacteria.

• Rabies (1885) - The final and certainly most famous success Pasteur research was the development of a vaccine against rabies or hydrophobia on July 6, 1885, 9-year-old Joseph Mister was the first person to be successfully treated against rabies with Pasteur vaccine. After that, people started coming to Pasteur. Ill wolf bit a group of Russian peasants from Smolensk. There was little hope for any success, because 12 days passed after incident but miraculously 16 people from 19 survived due to Pasteur’s help.

• Pasteur’s effect- Pasteur was the first to introduce two terms: aerobic anaerobic. He told that more production of alcohol takes place in anaerobic conditions by anaerobic yeasts. This is called Pasteur’s effect.

Because of his notable contributions to the field of microbiology Pasteur is called as Father of Modern Microbiology and Father of Fermentation Technology.





4. Robert Koch [1843- 1910]

Robert Koch was a German scientist, born in Hanover in 1843. Koch read Louis Pasteur’s work and in 1872 began research in microbes affecting diseased animals and people. He first studied mathematics and natural sciences and then medicines.

• Koch’s postulates- The most notable contribution of Koch was the establishment of casual relationship between a microorganism and a specific disease by applying a set of criteria to as Koch’s postulates or “Standards of Proof.” These were established in 1884 are the milestones of germ theory of disease and are necessary for identifying etiological or specific cause of disease.

These are as follows:

- The suspected microorganism must always be present in diseased but never in healthy individuals.
- The microorganism must be isolated in a pure culture on nutrient medium.
- The same disease must result when the isolated microorganism is inoculated into a healthy host.
- The same microorganism must be re-isolated from the experimentally infected host.

• Experimentation with microbes- In 1872, Koch became district medical officer for a rural area in berlin. He started to experiment with microbes in a small lab. He had built for himself in surgery.

• Discovery of Baciilus anthracis- The discovery that bacteria can act as specific agents of diseases in animals was given by Robert Koch, who first of all isolated Bacillus anthracis, the cause of anthrax which is devastating disease of cattle. In 1870, Robert Koch saw a large bacterium in the blood of anthrax victims. He reasoned that it might be agent of disease, but he knew that he would have difficulty in getting such a proposal accepted. He had no laboratory and his experiments were conducted in his home, using very primitive and improvised equipments and developing basic microbiological techniques. He painstaking teased out the anthrax bacterium and purified it. He then inoculated the purified bacteria into healthy animals and produced the classical clinical disease. When he examined the blood of inoculated animals, he was able to re-isolate the same bacterium. He repeated the isolation, infection and disease cycle until he was certain that he had found the agent of anthrax. Because it was such an important commercial disease because his technique could be easily duplicated others quickly verified his findings and Koch became famous.

• Discovery of new microbiological technique- He soon had his own institute and other discoveries followed. Koch attracted other bright scientists and together they developed the basic techniques of microbiology lab we still use today. These include:

- Sterile culture techniques.
- Pure culture techniques.
- Use of Petri plates, inoculation needles and solid medium
- Use of agar and gelatin to produce a solid surface.

• Use of cover slips- He also used cover slips for maintaining permanent records.

• Use of Gelatin- In 1881, Koch struggled with the disadvantage of using liquid media for certain experiments. He looked out for other alternatives including slices of potatoes as a solid culture medium. Potato slices could not be used for his animal pathogens, since these organisms did not grow on this substrate. He therefore employed gelatin s a solidifying agent and as a result was able to obtain individual colonies well separated from each other on the surface of gelatin medium.

Gelatin used by Koch to prepare solid media was not an ideal solidifying agent because of two important reasons:

- Since gelatin is a protein, many bacteria capable of producing proteolytic exoenzyme gelatinase that hydrolyses the proteins to amino acids digest it.

- It has a very low melting point i.e. it melts when temperature rises above 25°C.

Later Fannie Angelina Hesse first proposed the use of agar in culture media. She explained that Agar agar, a complex polysaccharide extracted from sea weed can be used as a solidifying agent because of following reasons:

- Non toxic to microbes
- Melts at 95 C but solidifies at about 45°C ( a temp. at which most bacteria can survive)
- Non-toxic to other forms of life.
- Stable to sterilization temperature.
- Physiologically inert as very few bacteria have the enzymes for digesting it.

• While Koch’s plating procedures provided advantages for the study of microorganisms, there was still problem of a suitable culture vessel. In 1887, Richard Petri solved this problem by what was known as Petri plates, a culture dish that is extensively used today.

Thus, contribution of Robert Koch, Fannie Hesse and Richard Petri made possible the isolation of pure culture of microorganisms and directly stimulated progress in all areas of microbiology.

• He systematically investigated the efficiency of chemical disinfectants demonstrating that carbolic acid used by Lister was merely bacteriostatic and bactericidal.

• He first recognized that disinfection depended on chemical concentration and contact time. He dried anthrax spores on silk threads, exposed them to disinfectants, washed them with sterile distilled water and cultured them to evaluate the range of chemicals.

• In 1882, he discovered Mycobacterium tuberculosis from tissues of a workman, stained it with methylene blue and observed blue colored rods.

• He also discovered and isolated the Vibrio cholerae, a causative agent of cholera and the importance of water filtration in the control of cholera.

• He proposed the concept of disease “Carriers.”

Koch was awarded Noble Prize in 1905 for his work. He will be remembered for both his discovery of important disease causing microorganisms and his fundamental contribution to Bacterial Techniques.

His vital contribution to microbiological methodology also makes him the “Founder of the field bacteriology.”







IMMUNIZATION ERA



5. Edward Jenner [1749-1823]

An English physician born in 1749 at Berkley (England) and died in 1823. He was a country doctor who had studied nature and his natural surroundings since childhood.

His notable contributions to microbiology are:

• Vaccine for smallpox- His great gift to world was his vaccine for smallpox. This disease was a horror as it killed in one in three of those who caught it and badly disfigured those who were lucky enough to survive catching it. Rural old wives that milkmaids only got a weak version of smallpox – non-life threatening smallpox but did not get smallpox itself had always fascinated him. A milkmaid who got cowpox got blisters on her hands and Jenner concluded that it must be pus in blisters that somehow protected milkmaids. To confirm his conclusion Jenner experimented on James Phipps (11 year old boy) where he injected the boy subsequently with tiny doses of pus and after that with smallpox. Amazingly the boy did not die, not even disfigured, just became ill for few days and recovered soon without any side effects. So successful was Jenner’s discovery , that in 1840 the government of the day banned any other treatment for smallpox than Jenner’s one.

• Vaccination– Jenner in 1798, published his results of 23 successful vaccinations. Eventually the process was known as vaccination based on latin word ‘vacca’ meaning cow.






6. Lord Joseph Lister [1827-1912]

Lord Joseph Lister was born in 1827 and died in 1912. He was a famous English surgeon. He is known for his notable contribution to antiseptic environment, for the precaution and cure of wound infections. He did not discover a new drug but he did make the link between lack of cleanliness in hospitals and deaths after operation.

His notable contributions to microbiology are:

• Wound infection by micro organisms- Lister impressed with Pasteur’s studies on the involvement of microorganisms in fermentation, concluded that wound infections too were due to microorganisms. Lister decided that wound itself had to be thoroughly cleaned. He then covered the wound with piece of lint covered in carbolic acid. He used this treatment on patients who had a compound fracture. It was the reason that the broken bone had penetrated the skin thus leaving wound that was open to germs. Death was so common by gangrene after such accidents. Lister covered the wound made with lint soaked in carbolic acid. His success rate for survival was very high.

• Development of antiseptic surgery- In 1867, he developed a system of antiseptic surgery designed to prevent micro organisms from entering wounds by application of phenol on surgical dressings and at times it was sprayed over surgical area.

• Antiseptic treatment- He also devised a method to destroy the micro organisms in the operation theatres by spraying fine mist of carbolic acid into air, thus producing antiseptic environment.

• Introduction of aseptic techniques- He also heated or sterilized the instruments to be used during surgery. Thus, he was fist to introduced aseptic technique for the control of microorganisms by the use of physical and chemical agents, which are still in use today.

Because of notable contributions Joseph Lister is known as Father of Antiseptic Surgery.




7. Elie metchnikoff [1845-1916]

One of the Pasteur’s associates Elie metchnikoff was a native of Ukraine. He was a Russian embryologist and immunologist; born in Kharkov in 1845, died in Paris in 1916. he studied natural sciences at university of Kharkov, in Germany and became professor of zoology in Odessa in 1867.

His notable contributions to microbiology are:

• Phagocytosis- Credit for coining the term Phagocytosis goes to him which literally mean phagocytose (eating of cell). In 1884, he published the account of phagocytosis and formulated the basic theory on which the science of immunology is founded i.e. body is protected from infection by leucocytes that engulf bacteria & other invading microbes.

While studying starfish larvae, he observed that certain cells engulfed splinters that he had introduced into larvae. These are called phagocytes.

• He formulated the theory that the phagocytes were body’s first and most important line of defense against infection.

• He became administrator at Pasteur institute in 1888 and eventually director.

For his discovery He alongwith Paul Ehrlich received Noble Prize in 1908.





ERA OF CHEMOTHERAPY-


Chemotherapy- can be defined as the use of chemicals that selectively inhibit or kill the pathogen without causing any damage to victim.
Two scientists Paul Ehrlich and Sakaherohatta (Japanese) discovered a drug Salvarsan which is an arsenobenzol compound in 1910 for treatment of disease of syphilis caused by Treponema pallidum.

Paul Ehrlich was responsible for lading an important foundation of era of chemotherapy. It was followed by Gerhard Domagk of Germany who, in 1935, reported Prontosil, a red dye used for staining leather, was active against pathogenic Streptococci and Staphylococci in mice. Although it had no effect against same infectious agent in test tube.

Two French scientist Jacques and Trefonal in same year showed that prontosil is broken down within body of mice to sulphanilamide ( i.e. sulpha drug) which was true active factor and Domagk was awarded Noble Prize in 1939 for the discovery of first sulpha drug.


8. Sir Alexander Fleming

Credit for the discovery of first wonder drug penicillin goes to Scottish physician and bacteriologist Sir Alexander Fleming in 1929 from mold Penicilliun notatum. He was born in year 1881 that was physician by training but spent much of his time on studying bacteria. He died in 1955.

• Discovery of penicillin- is fascinating accident. FLEMING was actually interested in searching something that would kill pathogen ever since working on wound infection during world war I (1914- 1918). One day in September in year 1928 upon return from a week vacation he observed that a plate of Staphylococcus aureus ha d contaminated with a green mold Penicillium notatum. It was forming a clear zone which has accidentally fallen on the plate and observed the plates, he noticed that colonies of P. notatum evidently being destroyed by bacteria.

Rather than discarding these contaminated plates he speculated that mold was producing a diffusible substance that inhibited growth of bacterium and Fleming became interested in subculturing for further studies and he extracted a compound from fungus which he named penicillin after the name of mold P. notatum and in year 1929 he published his findings in paper ‘Penicillin and its effects on gram positive bacteria’ Fleming’s work remain unnoticed until 1940 when Florey and Chain in 1941 at oxford university develop methods for industrial production of penicillin in England. When penicillin was finally produced in major quantity in 1941 its power and availability launched the antibiotic era, a major revolution in public health and medicine.

Because of the discovery and commercial production of penicillin all three Fleming, Florey and Chain shared Noble Prize in 1945.

P. notatum has been replaced by P. chrysogenum for the commercial production of penicillin and the latest strain yields 85000units/ ml of medium.

• Discovery of lysozyme- In 1920, Fleming searched an effective antiseptic. He discovered an enzyme called lysozyme in many body fluids such as tears. It had a natural antibacterial effect but not against strongest infectious agents.





ERA OF MOLECULAR BIOLOGY-


9. Joshua lederberg

Microbiology in 20 th century got a new era of molecular biology. With the recognition of unity of biochemical life processes in micro organisms and higher forms of life including human beings the use of micro organisms as a tool to explore fundamental life processes became attractive.

During 1940s a closer relationship was established between microbiologists, physicists biochemists and biologists resulting in creation of a new discipline which is now called molecular biology. Joshua Lederberg is among such a few renowned scientists who did great discoveries during the sense era and same field i.e. molecular biology. Joshua Lederberg is an American Genetists and Microbiologists who received the noble prize in 1958 for his work in bacteria. He was born in Montclair, near New York on 23 may, 1925. He was brought up in Washington. He served as a professor of genetics at the university of Washington then at Stanford school of medicine.

He has been actively involved in Artificial Intelligence Research and NASA experimental programs seeking life on mars.

His notable contributions to microbiology are:

• Conjugation- In 1946, Joshua Lederberg and Edward Tatum proposed conjugation in bacteria. The proof is based on generation of daughter cells able to grow in media that cannot support growth of either of parental cells. Their experiments showed that this type of gene exchange requires direct contact between bacteria. At the time lederberg began studying with tatum scientists believed that bacteria reproduced asexually but from the work of beadle and tatum lederberg knew that fungi reproduced sexually and he suspected that bacteria did as wellederberg

• Introduction of term plasmid- Joshual in 1952 used the term plasmid to describe extra chromosomal genetic material that replicate autonomously.

• Transduction- along with Norton zinder, lederberg discovered that genetic information could be transferred between bacteria by bacteriophage known as transduction. They gave a report on transduction in which the transfer of genetic material takes place by viruses. They showed that a phage salmonella typhimurium can carry DNA from one bacterium to another.

• Replica plating- along with his wife esther, lederberg discovered or developed a unique method of studying bacterial mutants now known as replica plating. With this technique it is possible to detect and isolate rare mutants with difeering nutritional requirements. Lederberg with this technique showed that mutations in bacteria occur randomly and spontaneously.

• One gene one enzyme- Joshua Lederberg, Beadle and Tatum started the relationships between genes and enzymes in 1941 using mutants of the bread mold fungus Neurospora crassa and gave the concept of one gene one enzyme hypothesis.

Also Lederberg, Beadle and Tatum were awarded Noble Prize in 1958 for the discovery of one gene one enzyme hypothesis.






10. Winogradsky and Beijerinck

The establishment of cardinal roles that microbes play in the biologically important cycles of matter on earth – the cycles of carbon, nitrogen and sulphur was largely the work of Winogradsky and Beijerinck.

His notable contributions to microbiology are:

• Importance of soil bacteria- The history of soil microbiology was opened in late 1800 by Winogradsky. He showed the importance of bacteria present in soil taking nitrogen from atmosphere, combating it with other elements, and thus making it available as plant food and hence animal food.

• Discovery of Azotobacter- in 1901, William Beijerinck found free living nitrogen fixing bacteria Azotobacter and described its usefulness in promoting soil fertility.

• Role of microbes- In cycles of Carbon, Nitrogen and Sulphur was observed by Winogradsky and Beijerinck and they discovered that many groups of microbes are specialized for carrying chemical transformations in soil that can not be formed by plants and animals.

• Microbial physiological specialization- Winogradsky discovered chemoautotrophic bacteria. These can grow in completely inorganic environments, obtaining energy necessary for their growth by the oxidation of reduced inorganic compounds and use carbon dioxide as the source of their cellular carbon. He also found several physiologically distinct groups among autotrophic bacteria i.e. sulphur bacteria, nitrifying bacteria.

• Atmospheric nitrogen fixation- Winogradsky and Beijerinck both contributed to the discovery that microbes play an important role in fixation of atmospheric nitrogen. They showed that certain bacteria, symbiotic or free living can use gaseous nitrogen for the synthesis of their cell constituents. These microbes help to maintain the supply of combined nitrogen, upto which all other forms of life are dependent.

• Enrichment culture- Both these developed a new and profoundly important technique enrichment culture for the isolation and supply of various physiological types of microbes that exist in nature.

Because of his notable contributions, Beijerinck is called as the Father of Virology.

bioremediation

Bioremediation


Naturally occurring bioremediation and phytoremediation have been used for centuries. For example, desalination of agricultural land by phytoextraction has a long tradition.


CONTENTS:

• Invention
• Definition
• Types of bioremediation
o In situ
o Ex situ
• In situ
o Definition
o Advantages
o Disadvantages
o Types-
o Intrinsic
o Engineered

• Ex situ
o Definition
o Disadvantages
o Types
-Solid phase treatment
Composting
Composting process
-Slurry phase treatment
Aerated lagoons
Low shear airlift reactors
o Factors affecting
• Bioremediation of hydrocarbons
• Bioremediation of industrial waste
• Bioremediation of dyes
• Bioremediation of coal waste
• Bioremediation of heavy metals





Invention
Bioremediation technology using microorganisms was reportedly invented by George M. Robinson. He was the assistant county petroleum engineer for Santa Maria, California. During the 1960's, he spent his spare time experimenting with dirty jars and various mixes of microbes.

Definition
It is the use of living microorganisms to degrade environmental pollutants and to prevent pollution.
It is the technology to remove pollutants from environment restoring contaminated sites and preventing future pollution.

Types of Bioremediation-
Is the enormous natural capacity of microorganisms to organic compounds which could further be improved by genetic engineering.

The toxic waste material remain in vapour, liquid or solid phases, therefore, Bioremediation technology varies accordingly whether waste material involved is in its natural surroundings or is removed and transported into a fermenter. On the basis of removal and transportation of waste for treatment, basically there are are two methods-

• In situ bioremediation
• Ex situ. bioremediation

IN SITU BIOREMEDIATION-
In situ bioremediation involves treating the contaminated material at the site. It is the clean up approach which directly involves the contact between microorganisms and the dissolved and the sorbed contaminants for biotrasformation.

Advantages
• minimal site disruption
• simultaneous treatment of contaminated soil and ground water
• minimal exposure of public and site personnel
• low cost

Disadvantages
• time consuming
• seasonl variation of microbial activity resulting fron direct exposure to prevailing environmental factors and lack of control of these factors.
• Problematic applications if treatment additives.

The microorganisms work well only when waste material help them to generate energy and nutrients to build up more cells.

In situ bioremediation is of two types
• Intrinsic
• Engineered

Intrinsic bioremediation- Conversion of environmental pollutants into the harmless forms through the innate capabilities of naturally occurring microorganisms is called intrinsic bioremediation. The intrinsic i.e. inherent capacity of microbes to metabolize the contaminants should be tested at laboratory and field levels before use for intrinsic bioremediation. Through site monitoring programmes progress of intrinsic bioremediation should be recorded time to time.

Conditions of site that favour intrinsic bioremediation are-

• Ground water flow throughout year.
• Carbonate minerals to buffer acidity produced during biodegradation.
• Supply of electron acceptors and nutrients for microbial growth.
• Absence of toxic compounds.
• Other environmental factors such as pH, concentration, temperature and nutrient availability

Determine whether biotransformation takes place.

Bioremediation of waste mixtures containing metals such as Hg, Pb, As and cyanide at toxic concentrations can create problem.

The ability of surface bacteria to degrade a given mixture of pollutants in ground water is dependant on the type and concentration of compounds, electron acceptor and duration of bacteria exposed to contamination. Therefore, ability of indigenous bacteria degrading contaminants can be determined in laboratory by plate count and microcosm studies.

Limitations of intrinsic bioremediation-

• It is slow process due to poorly adapted microorganisms.
• Limited ability of electron acceptor and nutrients
• Cold temperature
• High concentration of contaminants.

Engineered in situ bioremediation- When site conditions are not suitable, bioremediation requires construction of engineered systems to supply materials that stimulate microorganisms. Engineered in situ bioremediation accelerates the desired biodegradation reactions by encouraging growth of more microorganisms via optimizing physico chemical conditions. Oxygen and electron acceptors (e.g. nitrate and sulphate ions) and nutrients (e.g. nitrogen and phosphorus) promote microbial growth in surface. When contamination is deeper, amended water is injected through wells. But in some in situ bioremediation systems both extraction and injection wells are used in combinations to control the flow of contaminated ground water combined with above ground bioreactor treatment and subsequent re-injection of nutrients spiked effluent are done.

EX SITU BIOREMEDIATION-
Ex situ bioremediation involves removal of waste materials and their collection at a place to facilitate microbial degradation.

Limitations- Suffers from the cost associated with solid handling process e.g. excavation, screening, and fractionation, mixing, homogenizing and final disposal.

On the basis of phases of contaminated materials under treatment Ex situ bioremediation is classified into two types:

• Solid phase system (including land treatment and soil piles) i.e. composting
• Slurry phase treatment ( involving treatment of solid liquid suspensions in bioreactors.

Solid phase treatment-

Solid phase system includes organic wastes (e.g. leaves, animal manures and agricultural wastes) and problematic wastes (e.g. domestic and industrial wastes, sewage sludge and municipal solid wastes). The traditional clean up practice involves the informal processing of the organic materials and production of composts which may be used as soil amendment.

Composting- Composting is self heating, substrate dense, managed microbial system, and one solid phase biological treatment technology which is suitable to the treatment of large amount of contaminated solid materials. However, many hazardous compounds are resistant to microbial degradation due to complex chemical structure, toxicity and compound concentration that hardly support growth. Microbial growth is also affected by moisture, pH, inorganic nutrients and particle size. Because composting of hazardous wastes typically involves the bioremediation of contaminated substrate sparse soils, support of microbial self heating needs incorporation of proper amount of supplements. The hazardous compounds reported to disappear through composting includes aliphatic and aromatic hydrocarbons and certain halogenated compounds. The possible routes leading to disappearance of hazardous compounds include volatilization, assimilation, adsorption, polymerization and leaching.

Composting can be done in -
• Open system i.e. land treatment and
• In closed system.
The open land system can be inexpensive treatment method, but the temperature fluctuates from summer to winter. Therefore, rate of biodegradation of waste materials declines. Secondly, land treatment system may become oxygen limited, depending on amount of substrate, depth of waste, application etc.

However, efficiency of open treatment system can be increased by passing air. This approach is referred to as engineered soil piles and forced aeration treatment. The closed treatment system is preferred over the open land treatment system because controlled air is supplied to maintain microbial activity. As a result of microbial growth and volatilization of hazardous compounds, internal temperature gradually rises. Therefore, use of blowers for air circulation and exhaust for removal of toxic volatiles are set up in closed treatment system. Ventilators supply oxygen and remove heat through evaporation of water.

Composting process- As composting is a solid phase biological treatment, target compound must be either solid or a liquid associated with a solid matrix. The hazardous compounds should be biologically transformed. To achieve this goal, the waste material should be suitably prepared so that biological treatment potential should maximize. This is done by adjustment of several physical, chemical and biological factors. The hazardous waste must be solubilized so that they may be bioavailable. The hazardous waste and soil organic matters serve the source of carbon and energy for microorganisms. Microbial enzymes secreted during growth phase degrade toxic compounds. However, proper maintenance of water, oxygen, inorganic nutrients and pH increase rate of decomposition.













































Outline of composting treatment sequence


If there is low substrate density or site specific conditions, analogue or non analogue, non hazardous carbon sources that can stimulate microbial growth and enzyme production can be added to compost. Organic amendment also stabilize microbial population in inhibitory environment. Secondly, the presence of sufficient amount of water enhances microbial growth. Addition of inorganic nutrients influences microbial growth and rate of decomposition of hazardous wastes. Under nitrogen limiting conditions Phanerochaete chrysosporium produces extracellular lignin peroxidase that degrades benzopyrene and 2,4,6-trinitotoluene. It has also been noted that a pH range of 5.0 to 7.8 promoted the highest rates of biodegradation of hazardous wastes. But lignin degradation has been found the most rapid at pH of 3.0-6.5. this shows that optimal pH levels can be species, site and waste specific.

Gradual colonization of organic materials is done by indigenous microflora, but hazardous chemicals may inhibit microbial growth. Therefore, bioaugmentation (i.e. use of commercial or GMMs) of wastes is also recommended.

To provide experimental proof of biodegradation during composting, a common hazardous contaminant pesticide, 14C labeled Carbaryl was added in sewage sludge wood chip mixture at 1.3 – 2.2 ppm concentration. After 18-20 days in laboratory composting apparatus, 1.6 – 4.9 % of Carbaryl was recovered as 14CO2 and remaining bound to soil organic matter.

Slurry Phase treatment- The contaminated solid materials (soil, degraded sediments etc) microorganisms and water formulated into slurry are brought within a bioreactor i.e. fermenter. Thus slurry phase treatment is a triphasic system involving three major components : water, suspended particulate matter and air. Here water serves as suspending medium where nutrients, trace elements, pH adjustment chemicals and desorbed contaminants are dissolved. Suspended particulate matter includes a biologically inert substratum consisting of contaminants and biomass attached to soil matrix or free in suspending medium. Air provides oxygen for bacterial growth. Slurry phase reactors are new design in bioremediation.

The objectives of bioreactor designing are to:

• Alleviate microbial growth limiting factors in soil environment such as substrate, nutrients and oxygen availability.
• Promote suitable environmental conditions for bacterial growth such as moisture, pH, temperature and
• Minimize mass transfer limitations and facilitate desorption of organic material from soil matrix.

Biologically there are three types of slurry phase bioreactors:

• Aerated lagoons
• Low shear airlift reactor
• Fluidized bed soil reactor

The first two types are in full use of full scale bioremediation while third is in developmental stage.

Aerated lagoons: the slurry phase lagoon system which is very similar to aerated lagoons used for the treatment of small common municipal waste water. Nutrients and aeration are supplied to reactor. Mixers are fitted to mix different components and form slurry whereas surface aerators provide air required for microbial growth. The process may be used as single stage or multi stage operation. If the wastes contains volatiles, the reactor is not appropriate.

Low shear airlift reactors (LSARs)- LSARs are useful when waste contains volatile components; tight process control and increased efficiency of bioreactors are required. LSARs are cylindrical tanks which is madeup of stainless steel. In this bioreactor, pH, nutrient addition, temperature mixing and oxygen can be controlled as desired. Shaft is equipped with impellers. It is driven by motor set up at top. The rake arms are connected with blades which is used for resuspension of coarse materials that tend to settle on bottom of bioreactor. Air diffusers are placed radially along rake arm. Airlft provides to bottom circulation of contents in bioreactor. Baffles make hydrodynamic behaviour of slurry phase bioreactors. Pretreatment process includes size fractionation of solids, soil washing, milling to reduce particle size and slurry preparation. Certain surfactants such as anthracene, pyrene, perylene etc are added to enhance rate of biodegradation. These act as co-substrate and utilizes as carbon source. Co-substrates also induce production of beneficial enzymes.

Factors affecting slurry phase biodegradation- Factors that affect slurry phase biodegradation are-
• pH (optimum 5.5-8.5)
• moisture content
• temperature (20-30°C)
• oxygen (aerobic metabolism preferred)
• aging
• mixing ( mechanical and air mixing)
• nutrients ( N,P and micronutrients)
• microbial population (naturally occurring microorganisms are satisfactory, genetically engineered microorganisms for layer compound may be added)
• reactor operation (batch and continuous cultures)

BIOREMEDIATION OF HYDROCARBONS-

Petroleum and its products are hydrocarbons. These two have much economic importance. Oil constitutes a variety of hydrocarbons viz., xylanes, naphthalenes, octanes camphor etc. if present in the environment these cause pollution. For example, during cold war between iraq and America, millions of gallons of petroleum was leaked into sea which resulted in fish mortality. In addition, leakage of oil and petrol in marine is usual phenomenon.
In toxic environment microbes act only if the conditions e.g. temperature, pH and nutrients are adequate. Oil is insoluble in water and less dense. It floats on water surface and forms slicks. It should be noted that in bulk storage tank microbial growth is not possible provided air and water are supplied. The microbes which are capable of degrading petroleum includes pseudomonads, various corynebacteria, mycobacteria and some yeasts.

However, there are two methods for bioremediation of hydrocarbons/ oil spills,:

• By using mixture of bacteria
• By using genetically engineered microbial strains.

Use of mixture of bacteria- A large number of bacteria resides in interfaces of water and oil droplets. Each strains of bacteria consumes a very limited range of hydrocarbons, therefore, methods have been devised to introduce mixture of bacteria. Moreover, mixture of bacteria have been successfully been used to control oil pollution in water supplies or oil spills from ships. Bacteria living in interface degrade oil at a very slow rate. The rate of degradation could not be accelerated without human intervention. Artificially well characterized mixture of bacterial strains along with inorganic nutrients such as phosphorus and nitrogen are pumped into ground or applied to oil spill areas as required. This increases the rate of bioremediation significantly. For example, in the Exxon Valdez spill, accelerated bioremediation of oil washed upon beaches was noticed after spraying bacteria with admixture of inorganic nutrients.

Swaranjit singh and his group (IMTECH, Chandigarh) have isolated both bacterial and fungal cultures from the petroleum sludge. The fungal culture could degrade 0.4% sludge in 3 weeks. Degradation of petroleum sludge occurred within two weeks when the bacterial culture (Bacillus circulans CI) was used. Moreover, significant degradation of petroleum sludge was noticed in 10 days when the fungus + B. circulans and a prepared surfactant were exogenously added to petroleum sludge.

Use of genetically engineered bacterial strains- In 1979, for the first time Anand Mohan Chakrabarty, an Indian borne American scientist obtained a strain of Pseudomonas putida that contained the XYL and NAH plasmid as well as a hybrid plasmid derived by recombinant parts of CAM and OCT (these are incompatible plasmid and can not coexist as separate plasmid in same bacterium). This strain could grew rapidly on crude oil because it was capable of metabolizing hydrocarbons more efficiently than any other single plasmid. In 1990, the USA Government allowed him to use this Super bug for cleaning up of an oil spill in water of State of Texas. Superbug was produced on large scale in laboratory, mixed with straw and dried. The bacteria laden straw can be stored until required. When the straw was spread over oil slicks, the straw soaked up the oil and bacteria broke up the oil into non polluting and harmless products.

BIOREMEDIATION OF INDUSTRIAL WASTES-

A variety of pollutants are discharged in the environment from a large number of industries/mills. For example, textile industry alone contributes a significant amount of pollutants to water bodies such as enzymes, acids, alkali, alcohols, phenols, dyes, heavy metals, radionucliods etc. traces of zinc, cadmium, mercury, copper, chromium, lead are found in dyes.
It has been reported that actinomycetes show a higher capacity to metal ions as compared to fungi and bacteria. In addition, uptake mechanism of living and dead cells differ. Due to these differences they have potential application in industries. The living microbial cells accumulate intracellularly at a higher concentration, whereas dead cells precipitate metals in around cell walls by several metabolic processes. Dead biomass immobilized on polymeric membrane absorbs Uranium well, and immobilized Aspergillus oryzae cells on reticulated foam particles have been used for Cd removal. Aspergillus niger biomass contains upto 30 % of chitin and glucan. Chitin phosphate and chitosan phosphate of fungi absorb greater amount of U than Cu, Cd, Mn, Co, Mg and Ca.

BIOREMEDIATION OF DYES-

There is limited supply on microbial degradation of azo and reactive dyes. Maximum number of dyes undergo degradation through reduction.

• Kulla discussed azo-reductase of Pseudomonas strains in the chemostat culture. This enzyme catalyses azo linkage of dye. During degradation process of azo, NAD(P) acts as electron donor.

• Srivastva et.al. (1995) observed degradation of black liquor pulp mill effluents by the strains of Psuedomonas putida. Some anaerobic bacteria, Streptomyces and fungi e.g. Phaenerochaete chrysosporium have been characterized for decolouration of chromogenic dyes. The enzymes involved in dye degradation are lignases (lignin peroxidase), Mn (II) dependant peroxidase and glyoxal oxidase. These enzymes are well associated with lignin degrading system.

BIOREMEDIATION OF COAL WASTES THROUGH VAM FUNGI-

Bioremediation of coal waste land through VAM fungi is gaining importance in recent years. Selected fungi are introduced through plants in coal mine areas. Extensive infection of most plant species colonizing coal waste has been observed in India and other countries. It has been found that VAM fungi improved the growth and survival of desirable revegetation species. Increased growth of red maple, maize, alfalfa and several other plants inoculated with VAM fungi growing in coal mine soil has been recorded.

BIOREMEDIATION OF HEAVY METALS-

Bacteria, algae, fungi, actinomycetes and higher plants accumulate high amount of heavy metals in their cells.

Algae- The species of Chlorella, Anabaena inaequalis, Weatiellopsis prolifica, Stigeoclonium tenue, Synechococcus sp. tolerate heavy metals. However, several species of Chlorella, Anabaena, marine algae have been used for the removal of heavy metals. But the operational conditions limit the practical application of these organisms. Rai.et.al. studied biosorption i.e. both adsorption and absorption of Cd++ by a capsulated nuisance cyanobacterium, Microcystis both from field and laboratory. The naturally occurring cells showed higher efficiency for Cd++ and Ni++ as compared to laboratory cells.

Fungi- Fungi are also capable of accumulating heavy metals in their cells. However, several mechanisms operate in them for the removal of heavy metals from the solution; a few of these have been discussed below:

• Metabolism independent accumulation- The positively charged ions in the solution are attracted to negatively charged ligands in cell materials. Biosorption of metal ion occur on microbial cell surface. But composition of biomass and other factors affect biosorption. For example, in Rhizopus arrhizus depends on ionic radius of Li3+, Mn2+, Cu2+, Zn2+, Cd2+, Ba2+, Hg2+, Pb2+. However, binding of Hg2+, Ag2+, Cd2+, Al3+, Ni2+, Cu2+ and Pb2+ strongly depends on concentration of yeast cells.

• Metabolism dependent accumulation- In fungi and yeast, heavy metals ions are transported into the cells through cell membrane. However, because of metabolic processes ions are precipitated around the cells, and synthesized intracellularly as metal binding proteins. Energy dependent uptake of Cu2+, Zn2+,Cd2+ Ni2+ by fungi has been demonstrated. Moreover, intracellular uptake is influenced by certain external factors such as pH, cations, anions and organic materials, growth phase etc. metal uptake by growing batch culture was found maximum during lag phase and early log phase in Aspergillus niger, Penicillium spinulosum and Trichoderma viride.

• Extracellular precipitation and complexation- Fungi produce several extracellular products which can complex or precipitate heavy metals. For example, many fungi and yeast release high affinity Fe binding compounds that chelate iron. It is called siderophores. The Fe3+ chelates which are formed outside cell wall are taken up into the cell. In Saccharomyces cerevisiae removal of metals is done by their precipitation as sulphides e.g. Cu2+ is precipitated as CuS.