BIOREMEDIATION: EMERGING TECHNOLOGIES TO RESCUE THE EARTH FROM POLLUTION

OVERVIEW FOR BIOREMEDIATION

A. Principles of Bioremediation

In The Merriam-Webster Online dictionary (1986), bioremediation is defined

as "the treatment of pollutants or waste (as in an oil spill, contaminated

groundwater, or an industrial process) by the use of microorganisms (as bacteria)

that break down the undesirable substances". Bonaventura (1996, in

Sasse,2007:1) added that bioremediation not only limited for bacteria but also

“uses living systems or biological products to biodegrade anthropogenic waste,

with the objective being reduction of waste to chemical forms that can be

assimilated into natural cycles”. Specifically, it also uses naturally occurring

bacteria and fungi or plants (Rani et al., 2007 in Prasad, 2012:13)

Mueller (1996, in Prasad, 2012: 13) defined bioremediation as the process

whereby organic wastes are biologically degraded under controlled conditions to

an innocuous state, or to levels below concentration limits established by

regulatory authorities. King (1998, in Ford,1999:3) sums up the idea of

bioremediation and define the term as “a treatability technology that uses

biological activity to reduce the concentration or toxicity of a pollutant. It

commonly uses processes by which microorganisms transform or degrade

chemicals in the environment”.

Based on those definition by expert we could define some keywords about

the principle bioremediation, they are: Biodegradation, Anthropogenic Toxic

Waste, Biological System (Organism, Processes and Products) and acceptable

level of waste.

B. Brief History of Bioremediation

This use of microorganisms (mainly bacteria) to destroy or transform

hazardous contaminants is not a new idea. Microorganisms have been used

Review Paper on Bioremediation 1

since 600 B.C. by the Romans and others to treat their wastewater. Although this

same technology is still used today to treat wastewater it has been expanded to

treat an array of other contaminants. In fact, bioremediation has been used

commercially for almost 30 years. The first commercial use of a bioremediation

system was in 1972 to clean up a Sun Oil pipeline spill in Ambler, Pennsylvania

(National Research Council, 1993:47 in Ford,1999: 6). Since then, bioremediation

has become a well-developed way of cleaning up different contaminants.

On the past time studies on the molecular mechanisms behind the

contaminant transformation processes received less attention largely due to

technical difficulties. Although using traditional molecular techniques, some

functional genes involved in the microbial degradation of a specific contaminant

have been discovered. Nowadays, the advancement in modern molecular

biology, system biology, and availability of whole genome sequence data, fosters

new techniques including genomics, transcriptomics, proteomics, and

metabolomics, which might potentially be applied in bioremediation of organic

chemicals in the environment (Ma & Zhai, 2012).

C. Factors to Consider When Applying Bioremediation

Prasad (2012: 15) present the idea of a complex system of factors that

control the optimization of bioremediatio. These factors include: the existence of

a microbial population capable of degrading the pollutants; the availability of

contaminants to the microbial population; the environment factors (type of soil,

temperature, pH, the presence of oxygen or other electron acceptors, and

nutrients).Those factors include:

1. The existence of a microbial population capable of degrading the pollutants

(Vidali, 2001, in Zeyaullah et al. 2009:2)

2. The availability of contaminants to the microbial popuation (Vidali, 2001, in

Zeyaullah et al. 2009:2).


3. The environment factors (type of soil, temperature, pH, the presence of

oxygen or other electron acceptors and nutrients) (Vidali, 2001, in Zeyaullah

et al. 2009:2).

4. Types of the contaminants compounds (natural or synthethic), effect of

halogenation, contaminant mixtures (US. Environmental Protection Agency:

1998).

D. Strategies for the Application Bioremediation

Based on the place where the bioremediation occur, bioremediation could

be divided into two strategies. Those strategies are: the In-Situ Bioremediation

and Ex-Situ Bioremediation (Prasad, 2012:8). In situ techniques are defined as

techniques that are applied to soil and groundwater at the site with minimal

disturbance. Ex situ techniques are those that are applied to soil and

groundwater at the site which has been removed from the site via excavation

(soil) or pumping (water).

While based on the origin of the process, bioremediation could be divided

into: Intrinsic Bioremediation and Synthetic Bioremediation (US. Environmental

Protection Agency: 1998). Intrinsic bioremediation applies for the biore-

mediation that is accomplished without human intervention by microorganisms

that are naturally found in the contaminated site. While Engineered

bioremediation applies for bioremediation that techniqeus that use engineered

systems to supply nutrients, electron acceptors or other materials that enhance

the rate or extent of contaminant degradation.

Nonbiological treatment technologies or source removal may be used to

reduce the total amount of contaminant present at the site before, or

concurrent with, bioremediation. The following papers of this collection will then

describe and expose techniques that are widely in bioremediation. Those


techniques will be described subsequently are Biosensor, Ex-Situ Bioremediation

and In-Situ Bioremediation.

E. References

Ford, N. (1999). Bioremediation Termpaper (Online). Available at: http://ce540.

groups. et.byu.net/syllabus/termpaper/1999-W/ford.pdf (29th October 2012).

Ma, Jincai; Zhai,Guangshu. (2012). Microbial Bioremediation in Omics Era:

Opportunities and Challenges. Bhatt, J Bioremed Biodeg 2012, 3:9

http://dx.doi.org/10.4172/2155-6199.1000e120

Merriam-Webster Online Dictonary. (1986). Bioremediation (Online). Available

at: http://www.merriam-webster.com/dictionary/bioremediation (29th

October 2012)

Prasad, M. (2012). Decontamination of Polluted Water Employing Bioremedia

tion Processes: A Review. Int. J. LifeSv. Bt & Pharm. Res. 2012. ISSN 2250-3137.

Vol.1, No.3, July 2012 (page:14)

US. Enviromental Protection Agency. (1998). FUNDAMENTAL PRINCIPLES OF

BIOREMEDIATION (An Aid to the Development of Bioremediation Proposals)

(Online). Available at: http://www.deq.state.ms.us/MDEQ.nsf/pdf/GARD

_Bioremediation/$File/Bioremediation.pdf?OpenElement

Sasse, V. (2007). Essay on Bioremediation (Online). Available at: http://nvwater

loo.weebly.com/uploads/1/0/8/8/108809/1.bioremediation.pdf (29th October

2012)

Zeyaullah, Md., Atif, M., Islam, B., Abdelkafe, A.S., Sultan, P., Elsaady, M.A, Ali, A.

(2009). Bioremediation:A Tool for Environmental Clearning (Online). Available at:

http://www.academicjournals.org/ajmr/abstracts/abstracts/abstracts2009/Jun/

Zeyaullah%20et%20al.htm (27th October 2012)


http://www.deq.state.ms.us/MDEQ.nsf/pdf/GARD

1ST PAPER

BIOSENSOR: AN AID FOR BIOREMEDIATION

By Group 5

A. Introduction of Biosensor

The increasing number of potentially harmful pollutants in the environment call for

fast and cost-effective analytical techniques to be used in extensive monitoring

programs. Additionally, over the last few years, a growing number of initiatives and

legislative actions for environmental pollution control have been adopted in parallel

with increasing scientific and social concern in this area (Silva,2011).

Though traditional analytical tools provide accurate, reproducible and sensitive

determination of contaminant concentrations. Nevertheless, their use requires to take

samples on the contaminated sites and to transport the samples to laboratory for

analysis. Such handling of samples is time-consuming and expensive. This situation is

constitute an important impediment for their application on a regular basis (Malandain

et al., 2005).

The unique characteristics of biosensors will allow these devices to complement

current field screening and monitoring methods such as immunoassay test kits and

chemical sensors. Further, since certain of these devices can operate in high

concentrations of organics such as methanol and acetonitrile, these biosensors show

promise for in situ monitoring of mixed organic wastes. Other potential applications

include down-hole or perimeter groundwater surveillance as well as process stream

monitoring for remediation procedures (Rogers, 2003)

In this review paper we provide an overview of biosensor systems for

environmental applications, and in the following sections we describe the various

biosensors that have been developed for environmental monitoring, considering the

pollutants. We also provide description about the principles of biosensor, the

application of biosensor and we also provide an analysis about the benefit and

limitation of biosensor based on literature review.


B. Problem Formulation

Based on the above background we formulated a few problem that will be

the focus of our paper. Here we present those formulated problems:

1. What is the principle of biosensor in bioremediation?

2. What are the techniques of biosensor in bioremediation?

3. What are the benefits of biosensor in bioremediation?

4. What are the limitations of biosensor in bioremediation?

C. Aim of the Paper

These are the aim of the paper that we presented:

1. To provide a reference about an alternative techniques of bioremediation

2. To remind ourselves to be actively involve in the effort to remediate the

environment.

3. To propose the most recent successful applications of bioremediation

4. To promote public and government awareness about the emerging

technology of bioremediation.

D. Principles of Biosensor

A biosensor is a device that detects, transmits and records information regarding

physiological or biochemical change. Technically, it is a probe that integrates a biological

component with a electronic transducer thereby converting a biochemical signal into

quantifiable electrical response (Souza,2001). Each biosensor, therefore, has a biological

component that acts as the sensor and an electronic component to transduce and

detect the signal.The biosensor system was first invented by Leland Clark in 1956 who

was later known as the father of biosensor, he invented the biosensor system for

glucose (Chauhan et al., 2004).

Malandain (2005) describe three modules that comprised a biosensor system as we

can see on figure 1.1:


Figure 1.1 Principles of Biosensor

1. Recognition module, which can be biological or biomimetic; This module

comprised with the sensitive biological element (biological material (e.g.

tissue, microorganisms, organelles, cell receptors, enzymes, antibodies,

nucleic acids, etc.), a biologically derived material or biomimic component

that interacts (binds or recognises) the analyte under study. The biologically

sensitive elements can also be created by biological engineering.

2. Transduction module, which tranforms the recognition event into a

measurable signal; the transducer or the detector element (works in a

physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms

the signal resulting from the interaction of the analyte with the biological element


into another signal (i.e., transduces) that can be more easily measured and

quantified;

3. Module of data evaluation. The data evaluation module will sometime

includes a biosensor reader device with the associated electronics or signal

processors that are primarily responsible for the display of the results in a

user-friendly way. This sometimes accounts for the most expensive part of

the sensor device, however it is possible to generate a user friendly display

that includes transducer and sensitive element.

Kumar and D’Souza (2012) describe biosensor as a compact analytical device,

incorporating a biological or biologically derived sensing element, either closely

connected to, or integrated within a transducer system. The principle of

detection is the specific binding of the analyte of interest to the complementary

biorecognition element immobilized on a suitable support matrix (Fig. 1.2). The

specific interaction results in a change in one or more physico-chemical

properties which can be detected and measured by the transducer. The usual

aim is to produce an electronic signal, which is proportional to the

concentration of a specific analyte or group of analytes, to which the biosensing

element binds.

Figure 1.2 Detection Principle of Biosensor


E. Types of Biosensor

1. Based on Biological Elements

D’Souza (2011) and Gautam (2012) explained that biosensors can be

classified according to the biological elements that are used in biosensor

technology which is described as follow:

a. Plant and Animal Tissue based Biosensor

Nerve cells in animals and phloem cells in plants share one

fundamental similarity that they possess excitable membranes through

which electrical excitations can propagate in the form of action

potentials. It is conceivable that action potentials are the mediators for

intercellular and intracellular communication in response to

environmental irritants. Plants quickly respond to changes. Once initiated,

electrical impulses can propagate to adjacent excitable cells. The change

in transmembrane potential creates a wave of depolarization or action

potential, affecting the adjoining resting membrane. Most plant

tissuebased biosensors are based on electrochemical detection.

b. Microbial Whole Cell Biosensor

Whole cells can be used as biosensors if they have transducer

property along with the bio receptor element. Generally, cells capable

of sensing are modified to incorporate the transducer capacity. Certain

parameter such as bioavailability, toxicity and genotoxicity can be assayed

using whole cells only. They provide estimation for pollutant bioavailability.

The use of whole cells as biocatalysts has several advantages as compared

to isolated enzymes, the most important being increased stability and

protection from interfering substances. Consequently, microbial

biosensors are preferred for measurements in contaminated samples.

Whole cell bioassays can be classified as turn off assay- degree of


inhibition of a cellular activity that is continuous; or turn on assay –

active.tion of a certain process by the target pollutant.

c. Immunosensor

Immunosensors are based on highly selective antibody (Ab) - antigen

(Ag) reactions. The immobilized sensing element can be either an Ab or an

Ag which can be chemically modified (hapten). In the first case, analyte

binding is measured directly. In the second case, the method is based on the

competition between immobilized Ag, the analyte (Ag) and a fixed amount

of Ab. All types of immunosensors can either be run as nonlabeled or

labeled immunosensors. Label free immunosensors rely on the direct

detection of antigen-antibody complex formation by measuring variations in

electrical properties using electrochemical impedance spectroscopy (EIS), or

changes in optical properties using SPR (Lagarde and Renault, 2010).

The second type of immunosensors use signal-generating labels

which allow more sensitive and versatile detection modes. Peroxidase,

glucose oxidase, alkaline phosphatase, catalase enzymes and electroactive

compounds such as ferrocene are the most common labels used for

electrochemical detection, while fluorescent labels (rhodamine, fluorescein,

Cy5, etc…) are employed for optical detection (Lagarde and Renault, 2010).

d. Nucleic-Acid based Biosensor

Nucleic acid-based biosensors are finding increasing use for the

detection of environmental pollution and toxicity. A nucleic acid-based

biosensor employs as the sensing element an oligonucleotide, with a

known sequence of bases, or a complex structure of DNA or RNA. Nucleic

acid biosensors can be used to detect DNA/RNA fragments or either

biological or chemical species. In the first application, DNA/RNA is the

analyte and it is detected through the hybridization reaction (this kind of


biosensor is also called a genosensor). In the second application, DNA/RNA

plays the role of the receptor of specific biological and/or chemical

species, such as target proteins, pollutants or drugs.

2. Based on Transductor

Depending on the method of signal transduction, biosensors can also

be divided into different groups (Chauhan et al., 2004):

a. Electrochemical Sensors

In this configuration, sensing molecules are either coated onto or

covalently bonded to a probe surface. A membrane holds the sensing

molecules in place, excluding interfering species from the analyte

solution. The sensing molecules react specifically with compounds to be

detected, sparking an electrical signal proportional to the concentration

of the analyte. The bio-molecules may also respond to an entire class of

compounds such as opiates and their metabolites. The most common

detection method for electrochemical biosensors involves measurement

of current, voltage, conductance, capacitance and impedance.

b. Optical Biosensor

In optical biosensors, the optical fibers allow detection of analytes on

the basis of absorption, fluorescence or light scattering. Since they are

non-electrical, optical biosensors have the advantages of lending

themselves to in vivo applications and allowing multiple analytes to be

detected by using different monitoring wavelengths. The versatility of

fiber optics probes is due to their capacity to transmit signals that reports

on changes in wavelength, wave propagation, time, intensity, distribution

of the spectrum, or polarity of the light.


c. Piezoelectric Sensors

In this mode, sensing molecules are attached to a piezoelectric

surface –a mass to frequency transducer – in which interactions between

the analyte and the sensing molecules set up mechanical vibrations that

can be translated into an electrical signal proportional to the amount of

the analyte. Example of such a sensor is quartz crystal micro or nano

balance.

d. Field Effect Transistor (FET)

This method makes use of an ion-sensitive field effect transistor

(ISFET) built on standard technology that produces source, drain and gate

regions. The gate uses an ion sensitive membrane that renders ISFET

capable of biochemical recognition in the presence of the analyte with

the increase in local ion concentra-tion. Microelectrodes are created on a

silicon nitride surface using vapour deposition method and partially

insulated by titanium oxide. The hardware component consists of an

electrode system that could either be a conventional platinum or silver–

silver chloride microelectrode and a field effect transistor with an ion

sensitive gate or gas sensing electrode.

F. Mechanism of Biosensor

The essence of the biosensor is matching the appropriate biological and

electronic components to produce a relevant signal during analysis. Isolation of

the biological component is necessary to ensure that only the molecule of

interest is bound or immobilized on the electronic component or the transducer.

The stability of the biological component is critical, since it is being used outside

its normal biological environment. Attachment of the biological component to

the electronic component is vital for the success of these devices. If the

biological component is destroyed in the process of binding or if it binds with the


active site unavailable to the analyte, the biosensor will not function.

Attachment can be accomplished in a variety of ways, such as covalent binding

of the molecule to the detector (usually through a molecular cross-bridge),

adsorption onto the surface entrapment in porous material, or micro

encapsulation. As seen in the figure below:

Figure. 1.3 Immobilization of the Biocomponents

(Lagarde and Jaffrezic-Renault,2011)

G. Benefit and Limitation of Biosensor

For environmental applications, the main advantages offered by biosensors

over conventional analytical techniques are the possibility of portability,

miniaturization, work on-site, and the ability to measure pollutants in complex

matrices with minimal sample preparation. Although many of the developed

systems cannot compete yet with conventional analytical methods in terms of

accuracy and reproducibility, they can be used by regulatory authorities and by

industry to provide enough information for routine testing and screening of

samples (Rogers and Gerlach, 1996 in Silva, 2011).


H. Conclusion

To summarize, biosensors, the technology of the future, may increasingly

rely on the structure and function- specificity of the biological component.

Through the use of biosensors it is hoped that the cost to manage an

environment monitoring could be cut up, but still pertaining the low-risk aspect

of biotechnology.

I. References

Belkin,S. (2003). Microbial Whole-Cell Sensing Systems of Environmental

Pollutants. Current Opinion in Microbiology, 2003, 6:206-2012

Kumar, J., Souza, S.F.D (2012). Biosensors for Environmental and Clinical

Monitoring (Online). Available at: http://www.barc.gov.in/publications/

nl/2012/2012010210.pdf (27th October 2012)

Silva, L.M. (2011). Biosensors for Environmental Applications (Online).Available

at:http://www.intechopen.com/books/environmental-biosensors/ biosensor

-for-environmentalapplications (27th October 2012)

Tecon, R. and van der Meer, J.R. (2008). Bacterial Biosensors for Measuring

Availability of Environmental Pollutants.Sensors 2008, 8, 4062-4080; DOI:

10.3390/s8074062

2ND PAPER

BIOREACTORS: AN EX-SITU BIOREMEDIATION STRATEGY


http://www.intechopen.com/books/environmental-biosensors/

http://www.barc.gov.in/publications/

By Group 6

A. Introductionvof Bioreactors

Enormous quantities of organic and inorganic compounds are released into

the environment each year as a result of human activities. In some cases, these

releases are deliberate and well regulated (e.g., industrial emissions) while in

other cases they are accidental (e.g., chemical or oil spills). Many of these

compounds are both toxic and persistent in terrestrial and aquatic

environments. The contamination of soil, surface and groundwater is simply the

result of the accumulation of these toxic compounds in excess of permissible

levels. The quality of life on earth is linked inextricably to overall quality of the

environment. Contaminated lands generally result from past industrial activities

when awareness of the health and environmental effects connected with the

production, use and disposal of hazardous substances were less well recognized

than today (Prasad et al, 2012).

Thus the recent advances in bioremediation techniques for the treatment of

toxic waste will be of high significance. Bioremediation techniques are typically

more economical than traditional methods of waste treatment such as

incineration, absorbent/adsorbent techniques, catalytic destruction, etc.

Bioremediation technologies are improving as greater knowledge and

experience are being gained in the field (Singh and Fulekar, 2010). Ex-situ

bioremediation using reactors involves the processing of contaminated solid

material (soil, sediment, sludge) or water through an engineered containment

system. On this paper techniques, principles and factors to be considered when

applying ex situ bioremediation especially bioreactor is explained thoroughly

based on literature review.





1. What is the principle of bioreactors?

2. What are the techniques of bioreactors?

3. What are the benefits of bioreactors?

4. What are the limitations of bioreactors?

C. Aim

1. To identify the techniques of bioremediation using bioreactors.

2. To explain the types of bioreactors.

3. To explain the benefits of bioreactors.

4. To expalin the limitation of bioreactors.

5. To identify the factors to consider in uses bioreactors

D. Principles of Bioreactors

The term "bioreactor" in the context of soil and water bioremediation refers

to any vessel or container where biological degradation of contaminants is

isolated and controlled. Bioreactors can range from crude devices such as lined

depressions in the ground to advanced metal containers where environmental

conditions can be monitored and controlled. The essential treatment mechanism

in a bioreactor is natural degradation by existing and/or added populations of

microorganisms. Bioreactors have proven to be effective in remediating soil, and

in some cases water, polluted with fuel hydrocarbons (oil, gasoline, and diesel)

and organics (Lalli and Russell, 1996).

The aerated bioreactor for solids processing is a 3-phase (solid–liquid–gas)

multiphase system. The solids phase contains the adsorbed contaminants, the

liquid phase (process water) provides the medium for microbial growth, aeration

complicates the system. Nutrients and adapted bio-mass may be added to

enhance breakdown. Furthermore, process conditions (temperature, pH, O2


level, etc.) can be monitored and to some extent controlled (Kleijntjens and

Luyben, 2008).

Bioreactor design is dependent on the contaminant to be remediated, the

media that is contaminated, and cost constraints. The two major types of soil

reactors are dry and slurry. Dry bioreactors treat soil with no other amendments

other than microbes and nutrients. Adequate moisture is maintained for

microbial growth by sprinkler system or by rainfall. Physical mixing of the soil

keeps it aerated. A liner can be fitted over the soil to collect vapors volatilizing

from the soil. After the remediation process is complete the soil can be

transported to a desired location (Lalli and Russell, 1996).

This system is only applicable in highly specific situations. Only soils that

are contaminated at a shallow depth are practical to treat with a soil pile

reactor. It also frequently results in soil/microbe pellet formation which hinder

remediation by reducing the availability of pollutants to microbes. Slurry

reactors have proven more effective and efficient against a wider range of

pollutants. In a slurry reactor the soil is mixed with equal or greater amounts

of water and mixed with microbes and nutrients to form a soil slurry.

Conditions in a slurry reactor are easier to maintain than dry reactors and

result in faster treatment rates. This design offers many advantages such as

relatively rapid treatment, reduced pellet formation, increased slurry

homogenization, and increased bioavailability. Soil-water separation can

become a problem, especially with high clay soils. Also, there is a need for

wastewater treatment after the soil is dewatered (Lalli and Russell, 1996).


Figure 2.1. The drawing of

a simple pile reactor

shows its relative size (Lalli

and Russell, 1996).

Bioreactors for groundwater treatment are usually fixed film or some form of

activated sludge reactors. Fixed film reactors contain high surface area media that

support microbial growth. Activated sludge reactors are aerated basins where

microbes are mixed with the wastewater and nutrients. Bioreactors can be

operated in batch or steady state flow regimes (Lalli and Russell, 1996).

Figure 2.2. A

full-scale

system

diagram (Lalli

and Russell,

1996).


E. Techniques of Bioreactors

A slurry bioreactor may be defined as a containment vessel and apparatus

used to create a three phase (solid, liquid, and gas) mixing condition to hasten

the biodegradation of soil-bound and water-soluble contamination as a water

slurry of the contaminated soil, sediment, or sludge and biomass (usually

indigenous bacteria) capable of degrading targeted contaminants (McCauley and

Glaser, 1996).

Kleijntjens and Luyben (2008) explain that regarding the bioreactor

configuration there are two major topics:

1. Physical state of the multiphase system:

a) Bioreactors with a restricted solids hold-up: slurry reactors

b) Bioreactor with restricted humidity: solid state fermentation

2. Operation mode:

a) Batch operation; no fresh material is introduced to the bioreactor during

processing, the composition of the content changes continuously

b) Continuous operation (plug flow); fresh material is introduced and

treated material removed during processing, the composition in the

reactor remains unchanged with time, in practice semi-continuous

operation is often used (interval-wise feeding and removal giving small

fluctuations in the reactor).

Three basic reactor configurations exist:

1. Slurry bioreactors

2. Solid state fixed bed bioreactors

3. Rotating drum dry solid bioreactors.


Characteristic for all types of slurry bioreactors is the need of energy input

to sustain a 3-phase system in which the solid particles are suspended; the gravity

forces acting on the solids have to be compensated by the drag forces executed

by the liquid motion. In a properly designed slurry system the energy input is

used to establish three phenomena:

1. Suspension,

2. Aeration,

3. Mixing.

Figure 3. Common Configuration of Slurry Bioreactors

(Kleijntjens and Luyben, 2008)


Figure 4. Bioreactors for Solid State Processing

(Kleijntjens and Luyben, 2008)

A slurry bioreactor can only work properly if these three measures are

balanced. For each reactor configuration, the appropriate process conditions

depend on parameters such as the reactor scale, particle size distribution, slurry

density, slurry viscosity, oxygen demand of the biomass, and the solids hold-up

(Kleijntjens and Luyben, 2008).

For solid state fermentations there is no need to maintain a solids–liquid

suspension; a compact moist solid phase determines the system. Both the fixed

bed reactor as well as the rotating drum bioreactor are suited for solid state

fermentation. In the fixed bed reactor the contaminated solids rest on a drained

bottom as a stationary phase. Forced aeration and the supply of water are mostly

applied as a continuous phase. Fixed bed reactors are mostly batch operated.

Although land farming might be considered as a solid state batch treatment

under fixed bed conditions. This technique offers limited control options (in


comparison to other solid state treatment) and, therefore, is not considered to be

a bioreactor within the present context (Kleijntjens and Luyben, 2008).

Continuous solid state processing is possible in the rotating system. Here

the solid phase (as a compact moist material) is “screwed and pushed” through

the reactor. In line with slurry processing energy is required to maintain the

transport of the solids through the system (Kleijntjens and Luyben, 2008).

F. Benefit and Limitation of Ex-Situ Bioreactors

1. Benefits of Bioreactors

McCauley and Glaser (1996) explain that bioremediation of contaminated

soils, sludge, and sediments using slurry bioreactors offers several advantages

over other remediation technologies:

a. Intimate contact between micro biota and contaminants combined with

process controls such as (but not limited to) pH, temperature, and

nutrients provide conditions favorable for rapid remediation of targeted

contaminants.

b. Since most reactor vessels fully contain the contaminated solid and liquid

fractions, they offer almost unlimited treatment flexibility. Nutrient

amendments, which in some cases may not be permitted in situ (such as

ammonium and nitrate), may be used in a slurry bioreactor. Other

amendments that can be used in slurry bioreactors include designer

bacteria, surfactants, and enzyme inducers. Slurry bioreactors may be

fitted to provide sequential anaerobic/aerobic treatment conditions.

Slurry bioreactors may fit into various treatment trains, which must

include particle size separation (most slurry bioreactors do not accept

particles larger than ¼ inch in diameter) and commonly include soil


washing. Slurry bioreactors can be operated in batch mode (at least 10

percent of the slurry should be reserved for seeding subsequent batches),

or several bioreactors can be sequentially linked for continuous or semi

continuous operation.

c. Most bioreactor vessels fully contain the contaminated solid and liquid

fractions and can be designed to contain volatile contaminants; they offer

a high degree of safety as related to contaminant containment.

d. Slurry bioreactors require a relatively small space compared to

technologies such as land treatment, biopiles, and composting. Many

slurry bioreactors may be mounted on trailers and transported for use at

several sites.

e. Contaminants that have been successfully remediated using slurry

bioreactors include wood treating waste, oil separator sludge, munitions,

pesticides (not including highly chlorinated pesticides), and halogenated

aromatic hydrocarbons. Slurry bioreactors have been used most

frequently to remediate creosote.

2. Limitations of Bioreactors

Prasad et al (2012) explain that the limitations of bioreactors are soil

requires excavation, relatively high cost capital, relatively high operating

cost, toxicity of amendments, and toxic concentration of contaminants.

McCauley and Glaser (1996) also explain that slurry bioreactors have

limitations:

a. Bioslurry is an ex situ process, which by definition requires excavation

and transport (even if only a few feet) of the contaminated waste.

b. Reactor mixers consume energy.

c. Slurry bioreactors generally will not accept particles larger than ¼ inch in

diameter, requiring soil sieving or some other type of particle size


separation. Sand particles are highly abrasive in slurry bioreactors,

shorten their operating life, and generally contain a small fraction of the

contamination. Operators often choose hydrocycloning for sand fraction

rejection.

d. Bioslurrys require dewatering after remediation is terminated.

e. There is a limited history of full-scale bioslurry operations. Although there

are many pilot studies, slurry bioreactors are not easily scaled upward in

size. Some investigation or experimentation may be required to achieve

optimal operating conditions in a full-scale operation. These limitations

will increase the cost of remediation by slurry bioreactors.

G. Conclusion

Bioreactor in the context of soil and water bioremediation refers to any

vessel or container where biological degradation of contaminants is isolated and

controlled. The essential treatment mechanism in a bioreactor is natural

degradation by existing and/or added populations of microorganisms. A slurry

bioreactor mat be defined as a containment vessel and apparatus used to create

a three-phase (solid, liquid and gas) mixing condition to increase the

bioremediation rate of soil bound and water soluble pollutants as a water slurry

of the contaminated soil and biomass (usually indigenous microorganism)

capable of degrading target contaminants. Bioremediation of contaminated

soils, sludge, and sediments using slurry bioreactors offers several advantages

over other remediation technologies but also have the limitation in application.


H. References

Kleijntjens R.H., Luyben K.CH.A.M. (2008). Chapters 14 Bioreactor. Willey

Online Library: http://www.wiley-vch.de/books/biotech/pdf/v11b_ bior.pdf

(31 October 2012)

Lalli C., Russell M. (1996). Soil and Water Bioremediation Using Bioreactors.

[Online]. Available:http://www.webapps.cee.vt.edu/ewr/environmental/

teach/gwprimer/bioreact/bior.html (2 November 2012)

McCauley P., Glaser J. (1996). Slurry Bioreactors for Treatment of

Contaminated Soils, Sludges, and Sediments. Seminar Series on

Bioremediation of Hazardous Waste Sites: Practical Approaches to

Implementation. [Online]. Available:http://wvlc.uwaterloo.ca/biology447/

modules/module8/epadocs/slurry.pdf (2 November 2012)

Prasad M., Garg A., Maheswari R. (2012). Decontamination of Polluted Water

Employing Bioremeditaion Processes: A Review. International Journal of Life

Sciences Biotechnology and Pharma Research. 1 (3), pp 11-21. [Online].

Available:http://www.ijlbpr.com/jlbpradmin/upload/ijlbpr_4ff32665bb73c.p

df (24 October 2012)

Singh D., Fulekar M.H. (2010). Benzene Bioremediation Using Cow Dung

Microflora in Two Phase Partitioning Bioreactor. Journal of Hazardous

Materials. 175 (2010), pp 336-343. [Online]. Available: http://ipac.kacst.

edu.sa/eDoc/2011/191920_1.pdf (1 November 2012)


http://ipac.kacst/

http://www.wiley-vch.de/books/biotech/pdf/v11b_

3RD PAPER

VAROUS TECHNIQUES OF IN-SITU BIOREMEDIATION

By Group 5

A. Introduction of In-Situ Bioremediation

As stated before bioremediation is the use of microorganisms plants or

biological enzymes to achieve treatment of hazardous waste. Treatment can

target a variety of media (wastewater, groundwater, soil/sludge, gas) with

several possible objectives (e.g., mineralization of organic compounds,

immobilzation of contaminants). In situ bioremediation (ISB) is the application

of bioremediation in the subsurface – as compared to ex situ bioremediation,

which applies to media readily accessible aboveground (e.g., in treatment

cells/soil piles or bioreactors). In situ bioremediation may be applied in the

unsaturated/vadoze zone (e.g., bioventing) or in saturated soils and

groundwater (US. Department of Energy, 2012).

In situ bioremedation technology was originally developed as a less costly,

more effective alternative to the standard pump-and-treat methods used to

clean up aquifers and soils contaminated with organic chemicals (e.g., fuel

hydrocarbons, chlorinated solvents), but has since expanded in breadth to

address explosives, inorganics (e.g., nitrates), and toxic metals (e.g., chromium).

ISB has the potential to provide advantages such as complete destruction of the

contaminant(s), lower risk to site workers, and lower equipment/operating costs

(US. Department of Energy, 2012). Here in this paper we present principles,

techniques and a few consideration when applying in situ bioremediation.





1. What is the principle of In-Situ bioremediation?

2. What are the techniques of In-Situ bioremediation?

3. What are the benefits of In-Situ Bioremediation?

4. What are the limitations of Bioremediation?

C. Aim

These are the aim of the paper that we presented:

1. To provide a reference about an alternative techniques of bioremediation

2. To remind ourselves to be actively involve in the effort to remediate the

environment.

3. To propose the most recent successful applications of bioremediation

4. To promote public and government awareness about the emerging

technology of bioremediation

D. Principles of In-Situ Bioremediation

By definition, In situ can refer to where a clean up or remediation of a

polluted site is performed using and simulating the natural processes in the soil,

contrary to ex situ where contaminated soil is excavated and cleaned elsewhere,

off site. The in-Situ bioremediation technology process consists of the following

activities (Sims, et al., 1992 in Cauwenberghe and Roote, 1998:

1. A site investigation to determine the transport and fate characteristics of

organic waste constituents in the contaminated site

2. Performance of treatability studes (using batch or flow-through microcosms)

to determine the potential for bioremediation and to define required

operating and management practices

3. Removal of the source of the contaminant and recovery of free products.

4. Design and Implementation of a bioremediation plan based on fundamental

engineering principles


5. Establishment of a monitoring program to evaluate performance of the

remediation effort.

E. Techniques of In-Situ Bioremediation

1. Bioaccumulation

Originally bioaccumulation refers to the accumulation of substances, such

as pesticides, or other organic chemicals in an organism. Bioaccumulation

refers to the accumulation of substances, such as pesticides, or other organic

chemicals in an organism (Waldichuck, 1979). But in this paper,

bioacumulation refers to bioremediation technique that uses organism to

accumulate pollutant for further removal. There are two known

bioacumulation that are widely used:

a. Mycoremediation

Mycoremediation, is a form of bioremediation, the process of using

fungi to degrade or sequester contaminants in the environment.

Stimulating microbial and enzyme activity, mycelium reduces toxins in-

situ. Some fungi are hyperaccumulators, capable of absorbing and

concentrating heavy metals in the mushroom fruit bodies. One of the

primary roles of fungi in the ecosystem is decomposition, which is

performed by the mycelium. The mycelium secretes extracellular

enzymes and acids that break down lignin and cellulose, the two main

building blocks of plant fiber. These are organic compounds composed of

long chains of carbon and hydrogen, structurally similar to many organic

pollutants. The key to mycoremediation is determining the right fungal

species to target a specific pollutant (Stamets, 1998). Similar terms

mycofiltration, refer to the process of using mushroom mycelium mats as

biological filters.


b. Phytoremediation

Phytoremediation is a general term for several ways in which plants

are used to remediate sites by removing pollutants from soil and water.

Plants can degrade organic pollutants or contain and stabilize metal

contaminants by acting as filters or traps as we can see on figure 3.1

below.

Some of the methods of phytoremediation is described below (US.

Environmental Agency, 1999:7-8):

1) Phytoextraction — uptake and concentration of substances from the

environment into the plant biomass.

2) Phytostabilization — reducing the mobility of substances in the

environment, for example, by limiting the leaching of substances from

the soil.

3) Phytotransformation — chemical modification of environmental

substances as a direct result of plant metabolism, often resulting in their

inactivation, degradation (phytodegradation), or immobilization

(phytostabilization).

4) Phytostimulation — enhancement of soil microbial activity for the

degradation of contaminants, typically by organisms that associate with

roots. This process is also known as rhizosphere degradation.


Figure 3.1 Basic Principles of Phytoremediation

Phytostimulation can also involve aquatic plants supporting active

populations of microbial degraders.

5) Phytovolatilization — removal of substances from soil or water with

release into the air, sometimes as a result of phytotransformation to

more volatile and/or less polluting substances.

6) Rhizofiltration — filtering water through a mass of roots to remove toxic

substances or excess nutrients. The pollutants remain absorbed in or

adsorbed to the roots.

2. Bioaugmentation

Bioaugmentation is the introduction of a group of natural microbial

strains or a genetically engineered variant to treat contaminated soil or

water. Usually the steps involve studying the indigenous varieties present in

the location to determine if biostimulation is possible. If the indigenous

variety do not have the metabolic capability to perform the remediation

process, exogenous varieties with such sophisticated pathways are

introduced (Leeson, 2001).

Bioaugmentation is commonly used in municipal wastewater

treatment to restart activated sludge bioreactors. Most cultures available

contain a research based consortium of Microbial cultures, containing all

necessary microorganisms. Whereas activated sludge systems are generally

based on microorganisms like bacteria, protozoa, nematodes, rotifers and

fungi capable to degrade bio degradable organic matter (Leeson, 2001).

3. Bioventing

Bioventing is a technology that stimulates the natural in situ

biodegradation of any aerobically degradable compounds in soil by providing

oxygen and possibly heat to existing soil micro-organisms. Oxygen is

delivered to contaminated unsaturated soil zones by forced air movement


trough either injection or extraction of air to increase oxygen concentrations

and stimulate biodegradation (Langenhoff,2007) as you can see on the figure

3.2 below.

Figure 3.2 Schematic Overview ot the Biosparging System

Oxygen can also be supplied through direct air injection into residual

contamination in soil. In addition to degradation of adsorbed pollutants,

volatile compounds are biodegraded as vapours move slowly through

biologically active soil. Bioventing is a frequently applied technique for the

remediation of aerobically degradable organic compounds, such as fuel

residuals, in the unsaturated zone of the soil (Langenhoff,2007).

The effectiveness of the technique increases with the permeability of

the soil. In heterogeneous soils, bioventing is less effective since

contaminants remain in the less permeable layers. Oxidation of organic

matter or iron, present in the soil, is a process that competes with the

oxidation of the pollutants. Therefore, bioventing is less effective for very

heterogeneous soils and for soils with high contents of organic matter and

iron. Remediation times are considerably longer compared to SVE and vary Review Paper on Bioremediation 31

from a few years in homogeneous sandy soils up to several decades in

heterogeneous soils (Langenhoff,2007).

4. Biosparging

Biosparging is a technology that stimulates the natural in situ

biodegradation of any aerobically degradable compounds in soil by providing

oxygen to soil micro-organisms. Oxygen is delivered to the contaminated

saturated soil zone by injection of air to increase oxygen concentrations and

stimulate biodegradation (See figure 3.3) (Langenhoff,2007)..

Figure 3.3 Schematic View of Biosparging

The pollutants are degraded to harmless compounds within the soil,

and extraction and treatment of air is not needed. Biosparging uses low

airflow rates in order to provide enough oxygen to sustain microbial

activity.Biosparging is an established and frequently applied technique for

the remediation of aerobically degradable organic compounds, such as fuel

residuals in the saturated zone of the soil. The effectiveness of the technique

increases with the permeability of the soil. Biosparging is less effective in

heterogeneous soils, since contaminants remain in the less permeable layers.


Oxidation of organic matter in the soil competes with the oxidation of the

pollutants. As a result, biosparging is less efficient for very heterogeneous

soils or soils with high organic matter and iron contents (Langenhoff,2007)..

5. Bioslurping

Bioslurping is an in situ remediation technology, adapted from

vacuum dewatering techniques used in construction projects, that is being

developed and tested for the cleanup of light non-aqueousphase liquid

(LNAPL) contamination to recover free-product from the groundwater and

soil, and to bioremediate soils. The bioslurper system uses a “slurp” tube

that extends into the free-product layer. Much like a straw in a glass draws

liquid, the pump draws liquid (including free-product) and soil gas up the

tube in the same process stream (Miller, 1996).

Figure 3.3 Schematic view of Bioslurping

Pumping lifts LNAPLs, such as oil, off the top of the water table and

from the capillary fringe (i.e., an area just above the saturated zone, where

water is held in place by capillary forces). The LNAPL is brought to the

surface, where it is separated from water and air. The biological processes in


the term “bioslurping” refer to aerobic biological degradation of the

hydrocarbons when air is introduced into the unsaturated zone (Miller,

1996).

F. Benefit and Limitation of In-Situ Bioremediation

1. Benefit of In-Situ Bioremediation

It may be possible to completely transform organic contaminants to

innocuous substances (e.g., carbon dioxide, water, ethane). Accelerated ISB

can provide volumetric treatment, treating both dissolved and sorbed

contaminant. The time required to treat subsurface pollution using

accelerated in situ bioremediation can often be faster than pump-and-treat

processes. In situ bioremediation often costs less than other remedial

options. The areal zone of treatment using bioremediation can be larger

than with other remedial technologies because the treatment moves with

the plume and can reach areas that would otherwise be inaccessible (US.

Department of Energy, 2012).

As an in situ (versus ex situ) technology, there is typically little secondary

waste generated . As an in situ (versus ex situ) technology, there is reduced

potential for cross-media transfer of contaminants As an in situ (versus ex

situ) technology, there is reduced risk of human exposure to contaminated

media With ISB, there is less intrusion because few surface structures are

required ISB can be used in conjunction with, or as a follow-up to, other

(active) remedial measures. ISB has lower overall remediation costs than

those associated with active remediation (US. Department of Energy, 2012).

2. Limitation of In-Situ Bioremediation

Depending on the particular site, some contaminants may not be

completely transformed to innocuous products.n If biotransformation halts

at an intermediate compound, the intermediate may be more toxic and/or


mobile than the parent compound.bSome contaminants cannot be

biodegraded (i.e., they are recalcitrant).b When inappropriately applied,

injection wells may become clogged from profuse microbial growth resulting

from the addition of nutrients, electron donor, and/or electron acceptor (US.

Department of Energy, 2012)..

Accelerated In situ bioremediation relies on appropriate distribution

of amendments and thus, may be difficult to implement completely in low-

permeability or heterogeneous aquifers.bHeavy metals and toxic

concentrations of organic compounds may inhibit activity of indigenous

microorganisms.bIn situ bioremediation usually requires an acclimatized

population of microorganisms, which may not develop for recent spills or for

recalcitrant compounds (US. Department of Energy, 2012)..

With ISB, longer time frames may be required to achieve remediation

objectives, compared to active remediation. With ISB, sit echaracterization/

monitoring may be more complex and costly; long-term monitoring and

periodic re-evaluation of the remedy effectiveness will generally be

necessary With ISB, institutional controls may be necessary to ensure long

term protectiveness. With ISB, hydrologic and/or geochemical conditions

may change over time and could result in renewed mobility of previously

stabilized contaminants, adversely impacting remedial effectiveness. With

ISB, more extensive education and outreach efforts may be required to gain

public acceptance of the remedy (US. Department of Energy, 2012).

G. Conclusion

By definition the in-situ bioremediation is a techniques of remediation that

employs biological agent in the place where the pollution occurred. This

technique is often viewed to be more beneficial and effective than the ex-situ

technique. At a comparison of costs between conventional methods and


bioremediation, it should be kept in mind, that in case of in situ remediation

costs for transport and excavation cease. In terms of sustainability,

bioremediation has priority, because it leads to real reduction of waste and not

only storage or displacement of pollutants.

H. References

Cauwenberghe L.V. (1998). In Situ Bioremediation-Environmental Expert

(Online). Available at: www.environmental-expert.com/Files/0/ …/insbio_o.pdf –

United States (27th October 2012)

Langenhoff, A. (2007). In Situ Bioremediation Technologies-Experiences in the

Netherlands and Future Eropean Challenges (Online). Available at: http://www.clu-

in.org/download/contaminantfocus/dnapl/Treatment_

Technologies/Eurodemo_TNO_Summary_Bioremediation_web.pdf (27th October

2012)

Leeson, A. Alleman,B.C., Alvarez, P.J.J.,Magar, V.S. (2001). Bioaugmentation,

biobarriers, and biogeochemistry: the Sixth International In Situ and On-Site

Bioremediation Symposium. An Diego, California, June 4-7, Battelle Press

U.S. Department of Energy. (2012). In Situ Bioremediation (Online). Available

at: http://bioprocess.pnnl.gov/resour/rt3d.in.situ.bioremediation.htm (27th October

2012)

U.S. Environmental Protection Agency. (1999). Phytoremediation Resource

Guide (Online). Available at: http://www.epa.gov/tio/download/remed/

phytoresgude.pdf (27th October 2012)

Stamets. P. (1999). Helping the Ecosystem through Mushroom Cultivation.

Whole Earth Magazine, Fall 1999.

Waldichuk M., Bryan, G.W., Pentreath, R.J., Darracott, A. (1979).

Bioaccumulation of Marine Pollutants. London: The Royal Society


http://www.epa.gov/tio/download/remed/

http://bioprocess.pnnl.gov/resour/rt3d.in.situ.bioremediation.htm

http://www.clu-in.org/download/contaminantfocus/dnapl/Treatment_

http://www.clu-in.org/download/contaminantfocus/dnapl/Treatment_

http://www.environmental-expert.com/Files/0/

GENERAL CONCLUSION

Bioremediation is a powerful tool available to clean up contaminated sites

and it occurs when there are availability of microorganisms that can biodegrade the

given contaminant and the necessary nutrients. Regardless of which aspect of

bioremediation that is used, this technology offers an efficient and cost effective

way to treat contaminated ground water and soil. Its advantages generally outweigh

the disadvantages, which is evident by the number of sites that choose to use this

technolog and its increasing popularity.

RECOMMENDATION

With the given proof of the ease and benefit that provided by

bioremediation, it is highly recommended that the government use this techniques

to face pollution. Since it is relatively low in cost while stil providing multitude

benefit, and the most important thing is that this technique is eco-friendly and will

keep the sustainability of our environment.


BIOREMEDIATION: EMERGING TECHNOLOGIES TO RESCUE THE EARTH FROM POLLUTION

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