• In situ bioremediation is the toxic removal technology where micro-organisms feed on contaminants and dissolve contaminants for biotransformation.
•  Biotransformation is a very difficult process.
• The potential advantages of in situ bioremediation are:
• Minimal site disruption
• Continuous treatment of contaminated soil and ground water
• Minimal exposure of public, site personnel
• Economical

• There are two types of in situ bioremediation:
  • i) Intrinsic in situ bioremediation
  • ii) Engineered in situ bioremediation.

i. Intrinsic in situ bioremediation:

• Intrinsic bioremediation is the process of converting environmental pollutants into the non-toxic forms through the inherent abilities of naturally occurring microbial population.
• This process is most effective in the soil and water as these biomes always have high chance of being fully contaminated by contaminants and toxins.
• This process is usually employed in underground places as such underground petroleum tanks.
• There is escalating attention on intrinsic bioremediation for control of all or some of the contamination at waste sites.
•  The natural ability of micro-organisms to degrade the contaminants should be examined and tested at laboratory and in field trails prior its use for intrinsic bioremediation.
• There are several conditions of site that promotes intrinsic bioremediation.
• These conditions are:-
  • Flow of ground water throughout the year
  • Carbonate minerals to buffer acidity produced during biodegradation
  • Dispense of electron acceptors and nutrients for microbial growth
  • Absence of toxic compounds
  • The other environmental factors such as pH, concentration, temperature and nutrient availability decides whether or not biotransformation takes place.
  •  The microbial growth during bioremediation of the waste is hindered by presence of metals such as Hg, Pb, As and cyanide at toxic concentration.
  •  Degradation of pollutants using bacteria in ground water relies on the type and concentration of compounds, electron acceptor and time period for which bacteria was exposed to contamination.
  • Therefore, capacity of bacteria used to degrade contaminants must be determined in laboratory by microbial studies prior to use.

ii. Engineered/accelerated in situ bioremediation:

• Despite the good results, intrinsic bioremediation may not be suitable when site conditions are not matching with the microbial growth requirement.
• It is because, intrinsic bioremediation is a slow process as growth and availability of micro-organisms are not adequate, there is limited capacity of electron acceptor and nutrients, cold temperature and high concentration of contaminants.
• In these conditions, engineered in situ bioremediation is employed.
• Engineered in situ bioremediation accelerates the desired biodegradation reactions by enhancing growth of more micro-organisms under optimum physico-chemical growth conditions. Oxygen and electron acceptors (e.g., NO31– and SO42–) and nutrients (e.g., nitrogen and phosphorus) increase microbial growth in surface.

Limitations of in situ bioremediation:

• Following are limitations of in situ bioremediation.
  • Tedious as compared to other remedial methods.
  • Direct exposure to existing environmental factors results seasonal variation of microbial activity and lack of control of these factors.
  • Difficulty in utilisation of treatment additives such as nutrients, surfactants and oxygen. The micro-organisms act properly only when the waste materials serves to generate more cells. If the native micro-organisms fails in executing biodegradation, genetically engineered micro-organisms may be added to the site during in situ bioremediation.

Ex-situ bioremediation:

• Ex situ bioremediation includes elimination of waste materials and their collection from the contaminated site or place to assists microbial degradation.
• Ex situ bioremediation technology includes most of demerits and limitations as it is expensive process due to costs associated with solid handling process, such as excavation, screening and fractionation, mixing, homogenising and final disposal.
• Contaminated material may be either in liquid or solid form.

• On the basis of phases of contaminated materials under treatment ex situ bioremediation is classified into two part as per following :
  • i) Solid-phase system (involving land treatment and soil piles), i.e., composting.
  • ii) Slurry-phase systems (including treatment of solid-liquid suspensions in bioreactors).

i. Solid-phase treatment:

• Solid-phase system includes organic wastes present in solid form (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 method involves the processing of the organic materials and production of composts which may be used as soil conditioning.
• example of solid phase treatment system is composting

Composting process:

• Composting is a solid-phase biological treatment process thus target compounds must be either solid or a liquid related with a solid matrix.
• The hazardous compounds needs to be biologically transformed. For this, the waste material should be treated prior so that biological treatment potential should boost.
• This is done by adaptation of several physical, chemical and biological factors.
• The hazardous wastes must be solubilised for the easy availability to the micro-organisms.
• The hazardous compounds and soil organic matters act as source of carbon and energy for micro-organisms.
•  Enzymes secreted by micro-organisms during growth phase are responsible to degrade toxic compounds.
•  Availability of water, O2, inorganic nutrients and pH, enhance the rate of decomposition of hazardous compounds.
• If there is site-specific conditions or low substrate-density, non-hazardous carbon sources that can enhance microbial growth and enzyme production can be added to compost.
•  Presence of sufficient amount of water stimulates microbial growth. Addition of inorganic nutrients enhances microbial growth and rate of decomposition of hazardous wastes.
• It has also been noted that a pH range of 5.0–7.8 enhanced the highest rates of degradation of hazardous wastes. But lignin degradation has been recorded the most rapid at pH of 3.0–6.5. This shows that optimal pH levels can be species, site and waste specific.

i. Slurry-phase treatment:

a) Aerated lagoons:

• Slurry-phase lagoon system which is almost identical to aerated lagoon is used for treatment of small common municipal wastewater.
•  For boosting growth of micro-organisms, nutrients and aeration are dispensed to the reactor.
•  Mixers are fitted to mix different components and form slurry, whereas surface aerators supplies air needed for microbial growth.
• Depending upon requirement, the process may be used as single-stage or multistage operation.
• The limitation of slurry phase lagoon system is that this reactor is not suitable for treatment of waste containing volatile materials or components.

b) Low-shear airlift reactors (LSARs):

• Low shear airlift reactor has been developed in order to overcome the limitations of slurry phase lagoon in case of volatiles containing waste.
•  The LSARs are employed when waste consists of volatile components; tight process control and  when increased efficiency of bioreactors are needed.
• LSARs are like cylindrical tank which is made up of stainless steel.
• In this bioreactor pH, temperature, nutrient addition, mixing and oxygen can be regulated as per need.
•  Impellers are mounted on shaft to accomplish the need and driven by motor set up at the top.
• The rake arms are linked with blades which is used for resuspension of coarse materials that seem to settle on the bottom of the bioreactor.
• Air diffusers are also arranged along the rake arm. Airlift serves to bottom circulation of contents in reactor.
• Baffles sustains the hydrodynamic behaviour of slurry-phase bioreactors.
• Contaminated material should be pre-treated using size fractionation of solids, soil washing, milling to decrease particle size and slurry preparation.
• To stimulate the rate of biodegradation, some surfactants such as anthracene, pyrene, perylene, etc., are added to waste. These act as co-substrate and are utilised as carbon and energy source. Co-substrates also increase the production of beneficial enzymes.

Factors affecting slurry-phase biodegradation:

• Following factors play vital role in slurry phase biodegradation:
  • pH (optimum 5.5–8.5)
  • Moisture content
  • Temperature (20–30°C)
  • Ageing
  • Mixing
  • Nutrients (N, P, micronutrients)
  • Microbial population.
  • Reactor operation (batch and continuous cultures).

In situ and Ex situ bioremediation

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• Bioremediation is a biotechnical process which involves the removal of complex material or contamination by degrading environmental pollutants using living micro-organisms.
•  It is a technique to eliminate pollutants from the environment, restoring contaminated sites and avoiding future pollution.
• Bioremediation activity relies on natural capacity of micro-organisms to degrade organic compounds.
• The ability of micro-organisms could be enhanced by providing optimum growth conditions to micro-organism or by employing the genetically modified micro-organisms (GMMs). This technology has been implemented to eradicate environmentally hazardous chemicals and detoxify them into nontoxic forms.
• Micro-organisms play vital role in bioremediation, several members of microbial group like algae, fungi and bacteria are able to solubilise, transport and deposit the metals, and detoxify dyes and complex chemicals.
•  The toxic waste materials are present in form of vapour, liquid or solid phases; therefore, bioremediation technology differs depending upon nature of toxic material.

Types of bioremediation:

• Basically there are two methods of bioremediation based on removal and transportation of wastes for treatment:
  • In situ bioremediation
  • Ex situ bioremediation

Advantages of bioremediation:

• Natural process:
  • Bioremediation being a natural process is accepted by the public as waste treatment method for contaminated material such as soil.
  • Microbes capable of degrading the contaminant, increase in numbers and produce harmless products.
  • The residues for the treatment are normally harmless products such as carbon dioxide, water, and cell biomass.
• Complete destruction:
  • Bioremediation is employed for the complete destruction of a wide variation of contaminants.
  • Many hazardous compounds can be transformed to non-toxic products. This reduces the chance of future responsibility related with treatment and disposal of contaminated material.
  • Bioremediation can be performed on site treatment, without causing a major disturbance of normal activities. This removes the requirement to transport huge quantities of waste off site and thus decreases potential hazards to human health and the environment that can arise during transportation.
• Economic process:
  • Bioremediation is cost effective in comparison to other methods that are used for removal of hazardous waste.

Limitations of bioremediation:

• Limited up to biodegradable compounds:
  • Bioremediation is limited to biodegradable compounds.
  • This method is prone to rapid and complete degradation.
  • Products of biodegradation may be more lasting or toxic than the parent compound.
• Specificity:
  • Biological processes are largely specific.
  • The presence of metabolically capable microbial populations, suitable environmental growth conditions, and adequate levels of nutrients and contaminants are the important sites factors required for successful bioremediation.
• Scale up limitation:
  • It is tough to scale up from bench and pilot scale studies to full scale field operations.
• Technological advancement :
  • Research is required to develop and advance bioremediation technologies that are appropriate for sites with complex mixtures of contaminants that are not evenly distributed in the environment i.e. it may be present as solids, liquids, and gases.
• Tedious process:
  • Bioremediation consumes much time compared to other treatment options, such as excavation and removal of soil from contaminated site.
• Regulatory uncertainty:
  • We are not sure to say that remediation is 100% completed, as there is no known definition of clean.
  • Due to that performance evaluation of bioremediation is complex, and there is no fixed endpoint for bioremediation treatments.

Bioremediation: Concept, Types, Advantages and Limitations

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Microbial interaction

• Microorganisms interacts with each other and can be physically associated with another organisms in a variety of ways.
• One organism can be located on the surface of another organism as an ectobiont or located within another organism as endobiont.
• Microbial interaction may be positive such as mutualism, proto-cooperation, commensalism or may be negative such as parasitism, predation or competition

Types of microbial interaction

1. Positive interaction: mutualism, proto-cooperation, commensalism
2. Negative interaction: Ammensalism (antagonism), parasitism, predation, competition

I. Mutualism:

• It is defined as the relationship in which each organism in interaction gets benefits from association. It is an obligatory relationship in which mutualist and host are metabolically dependent on each other.
• Mutualistic relationship is very specific where one member of association cannot be replaced by another species.
• Mutualism require close physical contact between interacting organisms.
• Relationship of mutualism allows organisms to exist in habitat that could not occupied by either species alone.
• Mutualistic relationship between organisms allows them to act as a single organism.

Examples of mutualism:

• • i. Lichens:
    • Lichens are excellent example of mutualism.
    • They are the association of specific fungi and certain genus of algae. In lichen, fungal partner is called mycobiont and algal partner is called
    • Phycobiont is member of cycanobacteria ad green algae (Trabauxua).
    • Because phycobionts are photoautotrophs, the fungus get its organic carbon directly from algal partner, in turn fungi protects the phycobiont from extreme conditions and also provide water and minerals to algae.
    • Lichen grow very slowly but are able to colonies habitat that do not permit the growth of other organisms.
    • Most lichens are resistant to high temperature and drying.
  • ii. Protozoan-termite:
    • Protozoan-termite relationship is the classical example of mutualism in which flagellated protozoan lives in the gut of termites.
    • Theses flagellated protozoan feeds on diet of carbohydrates acquired as cellulose or lignin by their host termites, metabolize into acetic acid which is utilized by termites.
  • iii. Paramecium-Chlorella:
    • Paramecium (protozoa) can host Chlorella (algae) within its cytoplasm.
    • The algae Chlorella provide the protozoan partner with organism carbon and O2, in turn protozoa provide protection, mortility, CO2 and other growth factors.
    • The presence of Chlorella within Paramecium helps to survive protozoa in anaerobic condition as long as there is sufficient light.

II. Syntrophism:

• It is an association in which the growth of one organism either depends on or improved by the substrate provided by another organism.
• In syntrophism both organism in association gets benefits.
  • Compound A
    • Utilized by population 1
  • Compound B
    • Utilized by population 2
  • Compound C
    • utilized by both Population 1+2
  • Products

• In this theoretical example of syntrophism, population 1 is able to utilize and metabolize compound A, forming compound B but cannot metabolize beyond compound B without co-operation of population 2. Population 2is unable to utilize compound A but it can metabolize compound B forming compound C. Then both population 1 and 2 are able to carry out metabolic reaction which leads to formation of end product that neither population could produce alone.

Examples of syntrophism:

• • i. Methanogenic ecosystem in sludge digester
    • Methane produced by methanogenic bacteria depends upon interspecies hydrogen transfer by other fermentative bacteria.
    • Anaerobic fermentative bacteria generate CO2 and H2 utilizing carbohydrates which is then utilized by methanogenic bacteria (Methanobacter) to produce methane.
  • ii. Lactobacillus arobinosus and Enterococcus faecalis:
    • In the minimal media, Lactobacillus arobinosus and Enterococcus faecalis are able to grow together but not alone.
    • The synergistic relationship between E. faecalis and L. arobinosus occurs in which E. faecalis require folic acid which is produced by L. arobinosus and in turn lactobacillus require phenylalanine which is produced by Enterococcus faecalis.

III.  Protocooperation:

• It is a relationship in which organism in association is mutually benefited with each other.
• This interaction is similar to mutualism but the relationships between the organisms in protocooperation is not obligatory as in mutualism.

Examples of Protocooperation:

• i. Association of Desulfovibrio and Chromatium: it is a protocooperation between carbon cycle and sulfur cycle.
• ii. Interaction between N2-fixing bacteria and cellulolytic bacteria such as Cellulomonas

IV. Commensalism:

• It is a relationship in which one organism (commensal) in the association is benefited while other organism (host) of the association is neither benefited nor harmed
• It is an unidirectional association and if the commensal is separated from the host, it can survive.

Examples of commensalism:

• i. Non-pathogenic coli in intestinal tract of human:
  • E. coli is a facultative anaerobe that uses oxygen and lower the O2 concentration in gut which creates suitable environment for obligate anaerobes such as Bacteroides. E. coli is a host which remains unaffected by Bacteroides.
• ii. Flavobacterium (host) and Legionella pneumophila (commensal):
  • Flavobacterium excrete cystine which is used by Legionella pneumophila and survive in aquatic habitat.
  • Association of Nitrosomonas (host) and Nitrobacter (commensal) in Nitrification:
  • Nitrosomonas oxidize Ammonia into Nitrite and finally Nitrobacter uses nitrite to obtain energy and oxidize it into Nitrate.

V. Amensalism (antagonism):

• When one microbial population produces substances that is inhibitory to other microbial population then this inter population relationship is known as Ammensalism or Antagonism.
• It is a negative relationship.
• The first population which produces inhibitory substances are unaffected or may gain a competition and survive in the habitat while other population get inhibited. This chemical inhibition is known as antibiosis.

Examples of antagonism (amensalism):

• • i. Lactic acid produced by lactic acid bacteria in vaginal tract:
    • Lactic acid produced by many normal floras in vaginal tract is inhibitory to many pathogenic organisms such as Candida albicans.
  • ii. Skin normal flora:
    • Fatty acid produced by skin flora inhibits many pathogenic bacteria in skin
  • iii. Thiobacillus thiooxidant:
    • Thiobacillus thioxidant produces sulfuric acid by oxidation of sulfur which is responsible to lowering of pH in the culture media which inhibits the growth of most other bacteria.

VI. Competition:

• The competition represents a negative relationship between two microbial population in which both the population are adversely affected with respect to their survival and growth.
• Competition occurs when both population uses same resources such as same space or same nutrition, so, the microbial population achieve lower maximum density or growth rate.
• Microbial population competes for any growth limiting resources such as carbon source, nitrogen source, phosphorus, vitamins, growth factors etc.
• Competition inhibits both population from occupying exactly same ecological niche because one will win the competition and the other one is eliminated.

Examples of competition:

• • i. Competition between Paramecium cadatum and Paramecium aurelia: 
    • Both species of Paramecium feeds on same bacteria population when these protozoa are placed together.
    • P. aurelia grow at better rate than P. caudatum due to competition.

VII. Parasitism:

• It is a relationship in which one population (parasite) get benefited and derive its nutrition from other population (host) in the association which is harmed.
• The host-parasite relationship is characterized by a relatively a long period of contact which may be physical or metabolic.
• Some parasite lives outside host cell, known as ectoparasite while other parasite lives inside host cell, known as endoparasite.

Examples of parasitism:

• • i. Viruses:
    • Viruses are obligate intracellular parasite that exhibit great host specificity.
    • There are may viruses that are parasite to bacteria (bacteriophage), fungi, algae, protozoa etc.
  • ii. Bdellovibrio:
    • Bdellavibrio is ectoparasite to many gram negative bacteria.
    • The parasite Bdellovibrio penetrate the outer membrane of its host and enters periplasmic space but not inside host cytoplasm.

VIII. Predation:

• It is a wide spread phenomenon when one organism (predator) engulf or attack other organism (prey).
• The prey can be larger or smaller than predator and this normally results in death of prey.
• Normally predator-prey interaction is of short duration.

Examples of Predation:

• • i. Protozoan-bacteria in soil:
    • Many protozoans can feed on various bacterial population which helps to maintain count of soil bacteria at optimum level
  • ii. Bdellovibrio, Vamparococcus, Daptobacter etc are examples of predator bacteria that can feed on wide range of bacterial population.

Microbial interaction and types (Mutualism, Syntropism, Proto-cooperation, Commensalism, Antagonism, Parasitism, Predation, Competition)

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Microbial ecology

• It is the relationship of microorganisms with one another and with their environment. Microorganism impacts the entire biosphere. They are present in virtually in all part of planet earth including some in extreme conditions such as acidic lakes to deep ocean and from frozen environment to hydrothermal vents.
• Environmental microbiology includes all over microbial processes that occurs in soil, water or air. It is not concern with particular microenvironment where the microorganisms actually functioning but with the broader effect of microbial presence and activity.
• Environmental microbiology includes microbial biodegradation of domestic, agricultural and industrial wastes and also sub-surface pollution in soil, sediments and marine environments.

Role of microorganism in ecosystem

1. Generate Oxygen in atmosphere:

• Almost all of the oxygen produced in earth today occurs by bacteria in prehistoric period.
• Most of the oxygen producers are cyanobacteria (blue green algae) in ocean.

2. Recycle nutrition stored in organic matters to inorganic form:

• Microbial decomposition of organic matters releases the minerals sucha s N, P, K etc and these minerals are bound up and made available for producers (green plants) to use. Without this recycling primary productivity of ecosystem would stop.
• In soil, fungi are the most important decomposers of plant materials and are followed by bacteria.

3. Fix atmospheric Nitrogen into useable form:

• The only organism capable of fixing atmospheric nitrogen into useable form are bacteria. These nitrogen fixing bacteria are Rhizobium, Cyanobacteria etc

4. Microorganisms give plant roots excess to nutrition in soil:

• Plant root creates a zone of nutrition depletion around their surface by using available nutrition.
• A group of fungi called mycorrhiza found associated with rood hairs of plants helps in effective absorption of nutrition in soil.

5. Allows herbivore animals to get nutrition from poor quality food:

• In ruminants animals the ingested food especially cellulose, lignin etc cannot be digested by digestive enzymes in gut but the rumen (stomach) of these animals contains numerous bacteria, fungi and protozoa that carry out fermentation where these complex carbohydrates are digested.

Microbial ecology and Role of microorganism in ecosystem

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