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CONTENTS
I NTRODUCTION
C OMPARISON OF B IOTECHNOLOGICAL T REATMENT AND OTHER M ETHODS
A EROBIC T REATMENT OF WASTES
A NAEROBIC T REATMENT OF WASTES
T REATMENT OF H EAVY M ETALS -C ONTAINING WASTES
E NHANCEMENT OF B IOTECHNOLOGICAL T REATMENT OF WASTES
B IOSENSORS
R EFERENCES
1
2 V. Ivanov and Y.-T. Hung
is not sufficient to achieve the treatment goal within the prescribed duration; when it is
necessary to direct the biodegradation to the best pathway of many possible pathways; and
to prevent growth and dispersion in waste treatment system of unwanted or nondetermined
microbial strain which may be pathogenic or opportunistic one. Biosensors are essential tools
in biomonitoring of environment and treatment processes. Combinations of biosensors in array
can be used to measure concentration or toxicity of a set of hazardous substances. Microarrays
for simultaneous qualitative or quantitative detection of different microorganisms or specific
genes in the environmental sample are also useful in the monitoring of environment.
1. INTRODUCTION
Environmental biotechnology is a system of sciences and engineering knowledge related to
the use of microorganisms and their products in the prevention, treatment, and monitoring of
environmental pollution through solid, liquid, and gaseous wastes biotreatment, bioremedia-
tion of polluted environments, and biomonitoring of environmental and treatment processes.
Biotechnological agents used in environmental biotechnology include Bacteria and
Archaea, Fungi, Algae, and Protozoa. Bacteria and Archaea are prokaryotic microorganisms.
Prokaryotes are the most active organisms participating in the biodegradation of organic mat-
ter and are used in all areas of environmental biotechnology. Fungi are eukaryotic organisms
that assimilate organic substances. Fungi are important degraders of biopolymers and are used
in solid waste treatment, especially in composting, or in soil bioremediation. Fungal biomass
can also be used as an adsorbent of heavy metals. Algae are eukaryotic microorganisms
that assimilate light energy and are used in environmental biotechnology for the removal of
organic matter and nutrients from water exposed to light. Protozoa are unicellular animals that
absorb and digest organic food. Protozoa play an important role in the treatment of industrial
hazardous solid, liquid, and gas wastes by grazing on bacterial cells, thus maintaining adequate
bacterial biomass levels in the treatment systems and helping to reduce cell concentrations in
the waste effluents.
The main application of environmental biotechnology is the biodegradation of organic
matter of municipal wastewater and biodegradation/detoxication of hazardous substances in
industrial wastewater. It is known that approximately two-thirds of the hazardous substances
of oil polluted soil and sludges, sulfur-containing wastes, paint sludges, halogenated organic
solvents, non-halogenated organic solvents, galvanic wastes, salt sludges, pesticide-containing
wastes, explosives, chemical industry wastewaters, and gas emissions can be treated by
different biotechnological methods. Organic substances, synthesized in the chemical indus-
try, are often difficult to biodegrade. Substances that are not produced naturally and are
slowly/partially biodegradable are called xenobiotics. The biodegradability of xenobiotics can
be characterized by biodegradability tests such as rate of CO2 formation (mineralization rate),
rate of oxygen consumption (respirometry test), ratio of BOD to COD (oxygen used for bio-
logical or chemical oxidation), and the spectrum of intermediate products of biodegradation.
Applications of Environmental Biotechnology 3
little space and takes some days for treatment, but its cost is higher than that of open systems.
To intensify the composting of solid waste, the following pre-treatments can be used:
1. Mechanical disintegration and separation or screening to improve bioavailability of substances
2. Thermal treatment
3. Washing of waste using water or solution of surfactants to diminish toxic substances in waste
4. Chemical pre-treatment by H2 O2 , ozone, or Fenton’s reagent to oxidize and cleave aromatic rings
of hydrocarbons
Soil bioremediation is used in or on the sites of post-accidental wastes. There are many options
in the process design described in the literature (4–6). The main options tested in the field are
as follows:
1. In situ bioremediation (in-place treatment of a contaminated site)
2. On-site bioremediation (the treatment of a percolating liquid or eliminated gas in reactors placed
on the surface of the contaminated site). The reactors used for this treatment are suspended
biomass stirred-tank bioreactors, plug-flow bioreactors, rotating-disk contactors, packed-bed
fixed biofilm reactors (biofilter), fluidized bed reactors, diffused aeration tanks, airlift bioreactors,
jet bioreactors, membrane bioreactors, and upflow bed reactors (7)
3. Ex situ bioremediation (the treatment of contaminated soil or water that is removed from a
contaminated site)
The first option is used when the pollution is weak, treatment time is not a limiting factor,
and there is no groundwater pollution. The second option is usually used when the pollution
level is high and there is secondary pollution of groundwater. The third option is usually
used when the pollution level is so high that it diminishes the biodegradation rate due to the
toxicity of substances or a low mass transfer rate. Another reason for using this option might
be that the conditions in situ or on site (pH, salinity, dense texture or high permeability of
soil, high toxicity of substance, and safe distance from public place) are not favorable for
biodegradation.
Preventing hazardous substances from dispersing from the accident site into the environ-
ment is an important task of environmental biotechnology. This goal can be achieved by
creating physical barriers in the migration pathway with microorganisms capable of bio-
transformation of intercepted hazardous substances, e.g., in polysaccharide (slime) viscous
barriers in the contaminated subsurface. Another approach, which can be used to immobilize
heavy metals in soil after pollution accidents, is the creation of biogeochemical barriers.
These biogeochemical barriers could comprise gradients of H2 S, H2 , or Fe2+ concentrations,
created by anaerobic sulfate-reducing bacteria (in absence of oxygen and presence of sulfate
and organic matter), fermenting bacteria (after addition of organic matter and in absence of
oxygen), or iron-reducing bacteria (in presence of Fe(III) and organic matter), respectively.
Other bacteria can form a geochemical barrier for the migration of heavy metals at the
boundary between aerobic and anaerobic zones. For example, iron-oxidizing bacteria oxidize
Fe2+ and its chelates with humic acids in this barrier and produce iron hydroxides that can
diminish the penetration of ammonia, phosphate, organic acids, cyanides, phenols, heavy
metals, and radionuclides through the barrier.
6 V. Ivanov and Y.-T. Hung
Anaerobic respiration is more effective in terms of output of energy per mole of trans-
ferred electrons than fermentation. Anaerobic respiration can be performed by different
groups of prokaryotes with such electron acceptors as NO3 − , NO2 − , Fe3+ , SO4 2− , and CO2 .
Therefore, if the concentration of one such acceptor in the hazardous waste is sufficient
for the anaerobic respiration and oxidation of the pollutants, the activity of the related
bacterial group can be used for the treatment. CO2 -respiring prokaryotes (methanogens)
are used for methanogenic biodegradation of organic hazardous wastes. Sulfate-reducing
bacteria can be used for anaerobic biodegradation of organic matter or for the precipita-
tion/immobilization of heavy metals of sulfate-containing hazardous wastes. Iron-reducing
bacteria can produce dissolved Fe2+ ions from insoluble Fe(III) minerals. Anaerobic
biodegradation of organic matter and detoxication of hazardous wastes can be signifi-
cantly enhanced as a result of precipitation of toxic organics, acids, phenols, or cyanide
by Fe(II). Nitrate-respiring bacteria can be used in denitrification, i.e., reduction of nitrate
to gaseous N2 . Nitrate can be added to the hazardous waste to initiate the biodegradation
of different types of organic substances, for example polycyclic aromatic hydrocarbons
(9). Nitrogroups of hazardous substances can be reduced by similar pathway to related
amines.
Anaerobic fermenting bacteria (e.g., from genus Clostridium) perform two important
functions in the biodegradation of hazardous organics: (a) they hydrolyze different nat-
ural polymers and (b) ferment monomers with production of alcohols, organic acids,
and CO2 . Many hazardous substances, for example chlorinated solvents, phthalates, phe-
nols, ethyleneglycol, and polyethylene glycols can be degraded by anaerobic microor-
ganisms (4, 10–12). Fermenting bacteria perform reductive anaerobic dechlorination, thus
enhancing further biodegradation of xenobiotics. Different biotechnological systems perform
anaerobic biotreatment of wastewater: biotreatment by suspended microorganisms, anaer-
obic biofiltration, and biotreatment in upflow anaerobic sludge blanket (UASB) reactors
(4, 5).
Organic and inorganic wastes can be slowly transformed by anaerobic microorganisms in
landfills (13). Organic matter is hydrolyzed by bacteria and fungi. Amino acids are degraded
using ammonification with formation of toxic organic amines and ammonia. Amino acids,
nucleotides, and carbohydrates are fermented or anaerobically oxidized with formation of
organic acids, CO2 , and CH4 . Xenobiotics and heavy metals may be reduced, and subse-
quently dissolved or immobilized. These bioprocesses may result in the formation of toxic
landfill leachate, which can be detoxicated by aerobic biotechnological treatment to oxidize
organic hazards and to immobilize dissolved heavy metals.
A combined anaerobic/aerobic biotreatment can be more effective than aerobic or anaerobic
treatment alone. The simplest approach for this type of treatment is the use of aerated stabi-
lization ponds, aerated and non-aerated lagoons, and natural and artificial wetland systems,
whereby aerobic treatment occurs in the upper part of these systems and anaerobic treatment
occurs at the bottom end. A typical organic loading is 0.01 kg BOD/m3 day and the retention
time varies from a few days to 100 days (7). A more intensive form of biodegradation can
be achieved by combining aerobic and anaerobic reactors with controlled conditions, or by
Applications of Environmental Biotechnology 9
integrating anaerobic and aerobic zones within a single bioreactor. Combination or alteration
of anaerobic and aerobic treatments is useful in the following situations:
1. Biodegradation of chlorinated aromatic hydrocarbons including anaerobic dechlorination and
aerobic ring cleavage
2. Sequential nitrogen removal including aerobic nitrification and anaerobic denitrification
3. Reduction of sulfate or Fe(III) with production of H2 S or Fe(II) which are active reagents for the
precipitation of heavy metals, organic acids, and nutrients
fatty acids but with straw as an organic substrate, the direct reduction of chromium has been
observed without sulfate reduction (18).
in low concentrations in the waste. The best sources of essential metals are their dissolved
salts or chelates with organic acids. The source of metals for the bioremediation of oil spills
may be lipophilic compounds of iron and other essential nutrients that can accumulate at
the water–air interface where hydrocarbons and hydrocarbon-degrading microorganisms can
also occur (19). In some biotreatment cases, growth factors must also be added to the treated
waste. Growth factors are organic compounds, such as vitamins, aminoacids, and nucleosides,
that are required in very small amounts and only by some strains of microorganisms called
auxotrophic strains. Usually, those microorganisms that are commensals or parasites of plants
and animals require growth factors. However, sometimes these microorganisms may have the
unique ability to degrade some xenobiotics.
Substances may be protected from microbial attack by physical or chemical envelopes.
These protective barriers must be destroyed mechanically or chemically to produce fine parti-
cles or waste suspensions to increase the surface area for microbial attachment and subsequent
biodegradation. Another way to increase the bioavailability of hydrophobic substances is
through the washing of waste or soil by water or a solution of surface-active substances.
The disadvantage of this technology is the production of secondary hazardous waste due to
the resistance of chemically produced surfactants to biodegradation. Therefore, only easily
biodegradable or biotechnologically produced surfactants can be used for the pretreatment of
hydrophobic hazardous substances.
Extracellular enzymes produced by microorganisms are usually expensive for large-scale
biotreatment of organic wastes. However, enzyme applications may be cost-effective in certain
situations. Toxic organophosphate waste can be treated using the enzyme parathion hydro-
lase produced and excreted by a recombinant strain of Streptomyces lividans. The cell-free
culture fluid contains enzymes that can hydrolyze organophosphate compounds (20). Future
applications may involve cytochrome-P450-dependent oxygenase enzymes that are capable of
oxidizing different xenobiotics (21).
Low concentrations of dissolved oxygen (0.01–10 mg/L) can be rapidly depleted during
waste biotreatment with oxygen consumption rates ranging from 10 to 2,000 g O2 /Lxh.
Therefore, oxygen must be supplied continuously in the system. The air supply in liquid waste
treatment systems is achieved by aeration and mechanical agitation. Different techniques are
employed to supply sufficient quantities of oxygen in fixed biofilm reactors, in viscous solid
wastes, in underground layers of soil or in aquifers polluted by hazardous substances. Very
often the supply of oxygen is the critical factor in the successful scaling-up of bioremediation
technologies from laboratory experiments to full-scale applications (22). Air sparging in
situ is a commonly used bioremediation technology, which volatilizes and enhances aerobic
biodegradation of contamination in groundwater and saturated soils. Successful case studies
include a 6–12 month bioremediation project that targeted both sandy and silty soils polluted
by petroleum products and chlorinated hydrocarbons (23). The application of pure oxygen can
increase the oxygen transfer rate by up to five times, and this can be used in situations with a
strong acute toxicity of hazardous wastes and low oxygen transfer rates, to ensure sufficient
oxygen transfer in polluted waste.
In some cases, hydrogen peroxide has been used as an oxygen source because of the limited
concentrations of oxygen that can be transferred into the groundwater using above-ground
12 V. Ivanov and Y.-T. Hung
aeration followed by reinjection of the oxygenated groundwater into the aquifer or sub-
surface air sparging of the aquifer. However, because of several potential interactions of
H2 O2 with various aquifer material constituents, its decomposition may be too rapid, making
effective introduction of H2 O2 into targeted treatment zones extremely difficult and costly
(24). Pre-treatment of wastewater by ozone, H2 O2 , by TiO2 -catalized UV-photooxidation,
and electrochemical oxidation can significantly enhance the biodegradation of halogenated
organics, textile dyes, pulp mill effluents, tannery wastewater, olive-oil mills, surfactant-
polluted wastewater and pharmaceutical wastes, and diminish the toxicity of municipal landill
leachates. In some cases, oxygen radicals generated by Fenton’s reagent (Fe2+ + H2 O2 at
low pH), and iron peroxides (Fe (VI) and Fe(V)) can be used as oxidants in the treatment of
hazardous wastes.
Many microorganisms can produce and release to the environment such toxic metabolites of
oxygen as hydrogen peroxide (H2 O2 ), superoxide radical (O− 2 ), and hydroxyl radical (OH ·).
Lignin-oxidizing “white rot” fungi can degrade lignin and all other chemical substances
due to intensive generation of oxygen radicals which oxidize the organic matter by random
incorporation of oxygen into molecule. Not much is known about the biodegradation ability
of H2 O2 -generating microaerophilic bacteria.
Dissolved acceptors of electrons such as NO− − −
3 , NO2 , Fe , SO4 , and HCO3 can be
3+ 2−
used in the treatment system when oxygen transfer rates are low. Selection of the accep-
tor is determined by economical and environmental reasons. Nitrate is often proposed for
bioremediation (9) because it can be used by many microorganisms as an electron acceptor.
However, it is relatively expensive and its supply to the treatment system requires strict control
because it can pollute the environment. Fe3+ is an environmentally friendly electron acceptor.
It is naturally abundant in clay minerals, magnetite, limonite, goethite, and iron ores, but
its compounds are usually insoluble and it diminishes the rate of oxidation in comparison
with dissolved electron acceptors. Sulfate and carbonate can be applied as electron acceptors
only in anaerobic environments. Another disadvantage of these acceptors is that these anoxic
oxidations generate toxic and foul-smelling H2 S or “greenhouse” gas CH4 .
The addition of microorganisms (inoculum) to start up or to accelerate a biotreatment
process is a reasonable strategy under the following conditions:
1. If microorganisms, that are necessary for hazardous waste treatment, are absent or their concen-
tration is low in the waste
2. If the rate of bioremediation performed by indigenous microorganisms is not sufficient to achieve
the treatment goal within the prescribed duration
3. If the acclimation period is too long
4. To direct the biotreatment to the best pathway from many possible pathways
5. To prevent growth and dispersion in waste treatment system of unwanted or non-determined
microbial strains such as pathogenic or opportunistic organisms. The application of defined and
safe microbial strain(s) as a starter culture is especially important for biotechnological systems
using aggregated bacterial cells in biofilms, flocs, or granules for two reasons: a) aggregation
can be facilitated and enhanced; and b) self-aggregated or co-aggregated bacterial cells often are
pathogens or opportunistic pathogens
Applications of Environmental Biotechnology 13
Currently, a common environmental engineering practice is to use part of the treated waste
or enrichment culture as an inoculum. However, applications of defined pure starter cultures
have the following advantages:
1. Greater control over desirable processes
2. Lower risk of release of pathogenic or opportunistic microorganisms during biotechnological
treatment
3. Lower risk of accumulation of harmful microorganisms in the final biotreatment product. Pure
cultures that are most active in biodegrading specific hazardous substances can be isolated by
conventional microbiological methods, quickly identified by molecular–biological methods, and
tested for pathogenicity and biodegradation properties
4. Inoculum can be produced industrially
5. Regular additions of active microbial culture may be useful to maintain a constant rate of
biodegradation of toxic substances in case of high death rates of microorganisms during treatment
Microorganisms suitable for the biotreatment of hazardous substances can be isolated from the
natural environment. However, their ability for biodegradation can be modified and amplified
by artificial alterations of their genetic (inherited) properties. The description of the methods is
given in many books on environmental microbiology and biotechnology (4, 5). Natural genetic
recombination of the genes (units of genetic information) occurs during DNA replication and
cell reproduction, and includes the breakage and rejoining of chromosomal DNA molecules
(separately replicated sets of genes) and plasmids (self-replicating mini-chromosomes con-
taining several genes).
Recombinant DNA techniques or genetic engineering can create new, artificial combina-
tions of genes, and increase the number of desired genes in the cell. Genetic engineering
of recombinant microbial strains suitable for the biotreatment usually involves the following
steps:
1. DNA is extracted from cells and cut into small sequences by specific enzymes
2. Small sequences of DNA can be introduced into DNA vectors
3. A vector (virus or plasmid) is transferred into the cell and self-replicated to produce multiple
copies of the introduced genes
4. Cells with newly acquired genes are selected based on activity (e.g., production of defined
enzymes, biodegradation capability) and stability of acquired genes
Genetic engineering of microbial strains can create (transfer) the ability to biodegrade xenobi-
otics or amplify this ability through the amplification of related genes. Another approach is the
construction of hybrid metabolic pathways to increase the range of biodegraded xenobiotics
and the rate of biodegradation (25). The desired genes for biodegradation of different xenobi-
otics can be isolated and then cloned into plasmids. Some plasmids have been constructed con-
taining multiple genes for the biodegradation of several xenobiotics simultaneously. Strains
containing such plasmids can be used for the bioremediation of sites heavily polluted by a
variety of xenobiotics. The main problem in these applications is maintaining the stability
of the plasmids in these strains. Other technological and public concerns include the risk of
application and release of genetically modified microorganisms in the environment.
14 V. Ivanov and Y.-T. Hung
Self-aggregated microbial cells of biofilms, flocs, and granules, and artificially aggregated
cells immobilized on solid particles are often used in the biotreatment of hazardous wastes.
The advantages of microbial aggregates in hazardous waste treatment are as follows:
1. Upper layers and matrix of aggregates protect cells from toxic pollutants due to adsorption or
detoxication; therefore microbial aggregates or immobilized cells are more resistant to toxic
xenobiotics than suspended microbial cells
2. Different or alternative physiological groups of microorganisms (aerobes/anaerobes, het-
erotrophs/nitrifiers, sulfate-reducers/sulfur-oxidizers) may co-exist in aggregates and increase the
diversity of types of biotreatments, leading to higher treatment efficiencies in one reactor
3. Microbial aggregates may be easily and quickly separated from treated water. Microbial cells
immobilized on carrier surfaces such as granulated activated carbon that can adsorb xenobiotics
will degrade xenobiotics more effectively than suspended cells (26)
However, dense microbial aggregates may encounter problems associated with diffusion
limitation, such as slow diffusion of both nutrients into, and the metabolites out of, the
aggregate. For example, dissolved oxygen levels can drop to zero at some depth below the
surface of microbial aggregates so that obligate anaerobic bacteria can grow inside the biofilm
of an aerated reactor (27). This distance clearly depends on factors such as the specific
rate of oxygen consumption and the density of biomass in the microbial aggregate. When
environmental conditions within the aggregate become unfavorable, cell death may occur in
zones that do not receive sufficient nutrition or that contain inhibitory metabolites. Channels
and pores in aggregate can facilitate transport of oxygen, nutrients and metabolites. Channels
in microbial spherical granules have been shown to penetrate to depths of 900 µm (28) and
a layer of obligate anaerobic bacteria was detected below the channeled layer (27). This
demonstrates that there is some optimal size or thickness of microbial aggregates appropriate
for application in the treatment of hazardous wastes.
7. BIOSENSORS
An important application of environmental biotechnology is biomonitoring, including
monitoring of biodegradability, toxicity, mutagenicity, concentration of hazardous substances,
and monitoring of concentration and pathogenicity of microorganisms in wastes and in the
environment. Simple or automated off-line or on-line biodegradability tests can be performed
by measuring CO2 or CH4 gas production or O2 consumption (29). Biosensors may utilize
either whole bacterial cells or enzyme to detect specific molecules of hazardous substances.
Toxicity can be monitored specifically by whole cell sensors whose bioluminescence may be
inhibited by the presence of hazardous substance.
The most popular approach uses cells with an introduced luminescent reporter gene
to determine changes in the metabolic status of the cells following intoxication (30).
Nitrifying bacteria have multiple-folded cell membranes, which are sensitive to all membrane-
disintegrating substances: organic solvents, surfactants, heavy metals, and oxidants. There-
fore, respirometric sensors measuring the respiration rates of these bacteria can be used for
toxicity monitoring in wastewater treatment (31). Biosensors measuring concentrations of
hazardous substances are often based on the measurement of bioluminescence (32). This
Applications of Environmental Biotechnology 15
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