Aplication s Environmental Biotech

1Applications of Environmental Biotechnology

Volodymyr Ivanov and Yung-Tse Hung

CONTENTS

INTRODUCTION

COMPARISON OF BIOTECHNOLOGICAL TREATMENT AND OTHER METHODS

AEROBIC TREATMENT OF WASTES

ANAEROBIC TREATMENT OF WASTES

TREATMENT OF HEAVY METALS-CONTAINING WASTES

ENHANCEMENT OF BIOTECHNOLOGICAL TREATMENT OF WASTES

BIOSENSORS

REFERENCES

Abstract Environmental biotechnology is a system of scientific and engineering knowledgerelated to the use of microorganisms and their products in the prevention of environmentalpollution through biotreatment of solid, liquid, and gaseous wastes, bioremediation of pollutedenvironments, and biomonitoring of environment and treatment processes. The advantages ofbiotechnological treatment of wastes are as follows: biodegradation or detoxication of a widespectrum of hazardous substances by natural microorganisms; availability of a wide range ofbiotechnological methods for complete destruction of hazardous wastes; and diversity of theconditions suitable for biodegradation. The main considerations for application of biotechnol-ogy in waste treatment are technically and economically reasonable rate of biodegradabilityor detoxication of substances during biotechnological treatment, big volume of treated wastes,and ability of natural microorganisms to degrade substances. Type of biotreatment is based onphysiological type of applied microorganisms, such as fermenting anaerobic, anaerobicallyrespiring (anoxic), microaerophilic, and aerobically respiring microorganisms. All types ofbiotechnological treatment of wastes can be enhanced using optimal environmental factors,better availability of contaminants and nutrients, or addition of selected strain(s) biomass.Bioaugmentation can accelerate start-up or biotreatment process in case microorganisms,which are necessary for hazardous waste treatment, are absent or their concentration islow in the waste; if the rate of bioremediation performed by indigenous microorganisms

From: Handbook of Environmental Engineering, Volume 10: Environmental BiotechnologyEdited by: L. K. Wang et al., DOI: 10.1007/978-1-60327-140-0_1 c© Springer Science + Business Media, LLC 2010

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2 V. Ivanov and Y.-T. Hung

is not sufficient to achieve the treatment goal within the prescribed duration; when it isnecessary to direct the biodegradation to the best pathway of many possible pathways; andto prevent growth and dispersion in waste treatment system of unwanted or nondeterminedmicrobial strain which may be pathogenic or opportunistic one. Biosensors are essential toolsin biomonitoring of environment and treatment processes. Combinations of biosensors in arraycan be used to measure concentration or toxicity of a set of hazardous substances. Microarraysfor simultaneous qualitative or quantitative detection of different microorganisms or specificgenes in the environmental sample are also useful in the monitoring of environment.

Key Words Environmental biotechnology � wastes � biotreatment � biodegradation � bio-augmentation � biosensors � biomonitoring.

1. INTRODUCTION

Environmental biotechnology is a system of sciences and engineering knowledge related tothe use of microorganisms and their products in the prevention, treatment, and monitoring ofenvironmental 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 andArchaea, 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 organismsthat assimilate organic substances. Fungi are important degraders of biopolymers and are usedin solid waste treatment, especially in composting, or in soil bioremediation. Fungal biomasscan also be used as an adsorbent of heavy metals. Algae are eukaryotic microorganismsthat assimilate light energy and are used in environmental biotechnology for the removal oforganic matter and nutrients from water exposed to light. Protozoa are unicellular animals thatabsorb and digest organic food. Protozoa play an important role in the treatment of industrialhazardous solid, liquid, and gas wastes by grazing on bacterial cells, thus maintaining adequatebacterial biomass levels in the treatment systems and helping to reduce cell concentrations inthe waste effluents.

The main application of environmental biotechnology is the biodegradation of organicmatter of municipal wastewater and biodegradation/detoxication of hazardous substances inindustrial wastewater. It is known that approximately two-thirds of the hazardous substancesof oil polluted soil and sludges, sulfur-containing wastes, paint sludges, halogenated organicsolvents, non-halogenated organic solvents, galvanic wastes, salt sludges, pesticide-containingwastes, explosives, chemical industry wastewaters, and gas emissions can be treated bydifferent biotechnological methods. Organic substances, synthesized in the chemical indus-try, are often difficult to biodegrade. Substances that are not produced naturally and areslowly/partially biodegradable are called xenobiotics. The biodegradability of xenobiotics canbe 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

Other applications of environmental biotechnology are the prevention of pollution andrestoration of water quality in reservoirs, lakes and rivers, coastal area, in aquifers of ground-water, and treatment of potable water.

Areas of environmental biotechnology also include tests of toxicity and pathogenicity,biosensors, and biochips to monitor quality of environment, prevent hazardous waste pro-duction using biotechnological analogs, develop biodegradable materials for environmentalsustainability, produce fuels from biomass and organic wastes, and reduce toxicity by bioim-mobilization of hazardous substances.

2. COMPARISON OF BIOTECHNOLOGICAL TREATMENT AND OTHERMETHODS

The pollution of water, soil, solid wastes, and air can be prevented or removed by physical,chemical, physicochemical, or biological (biotechnological) methods. The advantages ofbiotechnological treatment of wastes are as follows:

1. Biodegradation or detoxication of a wide spectrum of hazardous substances by natural microor-ganisms

2. Availability of a wide range of biotechnological methods for complete destruction of hazardouswastes

3. A diverse set of conditions that are suitable for biotechnological methods

However, there are also many disadvantages of biotechnological methods for the preventionof pollution and treatment of environment and wastes:

1. Nutrients and electron acceptors must be added to intensify the biotreatment2. Optimal conditions must be maintained in the treatment system3. There may be unexpected or negative effects of applied microorganisms, such as emission of

cells, odors or toxic gases during the biotreatment, presence or release of pathogenic, toxigenic,opportunistic microorganisms into the environment

4. There may be unexpected problems in the management of the biotechnological system becauseof the complexity and high sensitivity of the biological processes

The main considerations for application of biotechnology in waste treatment are as follows:

1. Technically and economically reasonable rate of biodegradability or detoxication of waste sub-stances during biotechnological treatment

2. Large volume of treated wastes3. A low concentration of pollutant in water or waste is preferred4. The ability of natural microorganisms to degrade waste substances5. Better public acceptance of biotechnological treatment

The efficiency of actual biotechnological application depends on its design, process opti-mization, and cost minimization. Many failures have been reported on the way from benchlaboratory scale to field full-scale biotechnological treatment because of the instability anddiversity of both microbial properties and conditions in the treatment system (1).

In some cases, a combination of biotechnological and chemical treatments may be moreefficient than one type of treatment (2, 3). Efficient pre-treatment schemes, used prior tobiotechnological treatment, include homogenization of the particles of solid or undissolved


wastes in water, chemical oxidation of hydrocarbons by H2O2, ozone, or Fenton’s reagent,photochemical oxidation, and preliminary washing of wastes using surfactants.

3. AEROBIC TREATMENT OF WASTES

Aerobic microorganisms require oxygen as a terminal acceptor of electrons donated byorganic or inorganic substances. The transfer of electrons from donor to acceptor is a sourceof biologically available energy. Xenobiotics such as aliphatic hydrocarbons and derivatives,chlorinated aliphatic compounds (methyl-, ethyl, methylene and ethylene chlorides), aromatichydrocarbons and derivatives (benzene, toluene, phthalate, ethylbenzene, xylenes and phenol),polycyclic aromatic hydrocarbons, halogenated aromatic compounds (chlorophenols, poly-chlorinated biphenyls, dioxins and relatives, DDT and relatives), AZO dyes, compounds withnitrogroups (explosive-contaminated waste and herbicides), and organophosphate wastes canbe treated effectively by aerobic microorganisms.

3.1. Aerobic Treatment of Solid Wastes

Composting is the simplest way to treat solid waste aerobically. Composting convertsbiologically unstable organic matter into a more stable humus-like product that can be usedas a soil conditioner or organic fertilizer. Additional benefits of composting of organic wastesinclude the prevention of odors from rotting wastes, destruction of pathogens and parasites(especially in thermophilic composting), and the retention of nutrients in the end products.There are three main types of composting technology: windrow system, static pile system,and in-vessel system. Composting in windrow systems involves mixing an organic waste withinexpensive bulking agents (wood chips, leaves, corncobs, bark, peanut, and rice husks) tocreate a structurally rigid matrix, to diminish heat transfer from the matrix to the ambientenvironment, to increase the treatment temperature and to increase the oxygen transfer rate.The mixed matter is stacked in rows 1–2 m high called windrows. The mixtures are turnedover periodically (two to three times a week) by mechanical means to expose the organicmatter to ambient oxygen. Aerobic and partially anaerobic microorganisms, which are presentin the waste or were added from previously produced compost, will grow in the organicwaste. Due to biooxidation and release of energy, the temperature in the pile will rise. Thisis accompanied by successive changes in the dominant microbial communities, from lessthermoresistant to more thermophilic ones. This composting process ranges from 30 to 60days in duration.

The static pile system is an intensive biotreatment because the pile of organic waste andbulking agent is intensively aerated using blowers and air diffusers. The pile is usually coveredwith compost to remove odors and to maintain high internal temperatures. The aerated staticpile process typically takes 21 days, after which the compost is cured for another 30 days,dried, and screened to recycle the bulking agent.

In-vessel composting results in the most intensive biotransformation of organic wastes.In-vessel composting is performed in partially or completely enclosed containers in whichmoisture content, temperature, oxygen content in gas can be controlled. This process requires


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 substances2. Thermal treatment3. Washing of waste using water or solution of surfactants to diminish toxic substances in waste4. Chemical pre-treatment by H2O2, 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 optionsin the process design described in the literature (4–6). The main options tested in the field areas 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 suspendedbiomass stirred-tank bioreactors, plug-flow bioreactors, rotating-disk contactors, packed-bedfixed 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 acontaminated 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 pollutionlevel is high and there is secondary pollution of groundwater. The third option is usuallyused when the pollution level is so high that it diminishes the biodegradation rate due to thetoxicity of substances or a low mass transfer rate. Another reason for using this option mightbe that the conditions in situ or on site (pH, salinity, dense texture or high permeability ofsoil, high toxicity of substance, and safe distance from public place) are not favorable forbiodegradation.

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 bycreating physical barriers in the migration pathway with microorganisms capable of bio-transformation of intercepted hazardous substances, e.g., in polysaccharide (slime) viscousbarriers in the contaminated subsurface. Another approach, which can be used to immobilizeheavy metals in soil after pollution accidents, is the creation of biogeochemical barriers.These biogeochemical barriers could comprise gradients of H2S, H2, or Fe2+ concentrations,created by anaerobic sulfate-reducing bacteria (in absence of oxygen and presence of sulfateand organic matter), fermenting bacteria (after addition of organic matter and in absence ofoxygen), 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 theboundary between aerobic and anaerobic zones. For example, iron-oxidizing bacteria oxidizeFe2+ and its chelates with humic acids in this barrier and produce iron hydroxides that candiminish the penetration of ammonia, phosphate, organic acids, cyanides, phenols, heavymetals, and radionuclides through the barrier.


3.2. Aerobic Treatment of Liquid Wastes

Wastewater can be treated aerobically in suspended biomass stirred-tank bioreactors, plug-flow bioreactors, rotating-disk contactors, packed-bed fixed biofilm reactors (or biofilters),fluidized bed reactors, diffused aeration tanks, airlift bioreactors, jet bioreactors, membranebioreactors, and upflow bed reactors (4, 7). Secondary wastes include polluted air and sed-iments produced in the bioreactor. Wastewater with low concentrations of hazardous sub-stances may reasonably be treated using biotechnologies such as granular activated carbon(GAC) fluidized-bed reactors or co-metabolism. GAC or other adsorbents ensure sorptionof hydrophobic hazardous substances on the surface of GAC or other adsorbent particles.Microbial biofilms can also be concentrated on the surface of these particles and can biode-grade hazardous substances with higher rates compared to situations when both substrate andmicrobial biomass are suspended in the wastewater.

Cometabolism refers to the simultaneous biodegradation of hazardous organic substances(which are not used as a source of energy) and stereochemically similar substrates, whichserve as a source of carbon and energy for microbial cells. Biooxidation of the hazardoussubstance is performed by the microbial enzymes due to stereochemical similarity betweenthe hazardous substance and the substrate. The best-known applications of cometabolismare the biodegradation/detoxication of chloromethanes, chloroethanes, chloromethylene, andchloroethylenes by enzyme systems of bacteria for the oxidization of methane or ammonia as amain source of energy. In practice, bioremediation is achieved by adding methane or ammonia,oxygen (air), and biomass of methanotrophic or nitrifying bacteria to soil and groundwaterpolluted by toxic chlorinated substances.

To intensify the biotreatment of liquid waste, the following pre-treatments can be used:

1. Mechanical disintegration/suspension of the particles and hydrophobic substances to improve thereacting surface in the suspension and increase the rate of biodegradation

2. Removal from wastewater or concentration of hazardous substances by sedimentation, centrifuga-tion, filtration, flotation, adsorption, extraction, ion exchange, evaporation, distillation, freezing,and separation

3. Preliminary oxidation by H2O2, ozone, or Fenton’s reagent to produce active oxygen radicals;preliminary photo-oxidation by UV and electrochemical oxidation of hazardous substances

3.3. Aerobic Treatment of Gaseous Wastes

The main applications of biotechnology for the treatment of gaseous wastes includethe bioremoval of biodegradable organic solvents, odors, and toxic gases, such as hydro-gen sulfide and other sulfur-containing gases from the exhaust ventilation air in industryand farming. Industrial ventilated air containing formaldehyde, ammonia, and other lowmolecular weight substances can also be effectively treated in a bioscrubber or biofil-ter. Gaseous xenobiotics, which can be treated biotechnologically, include the follow-ing: chloroform, trichloroethylene, 1,2-dibromoethane, 1,2-dibromo-3-chloropropane, carbontetrachloride, xylenes, dibromochloropropane, toluene, methane, methylene chloride, 1,1-dichloroethene, bis(2-chloroethyl) ether, 1,2-dichloroethane, chlorine, 1,1-trichloroethane,ethylbenzene, 1,1,2,2-tetrachloroethane, bromine, methylmercury, trichlorofluoroethane,


1,1-dichloroethane, 1,1,2-trichloroethane, ammonia, trichloroethane, 1,2-dichloroethene,carbon disulfide, chloroethane, p-xylene, hydrogen sulfide, chloromethane, 2-butanone,bromoform, acrolein, bromodichloroethane, nitrogen dioxide, ozone, formaldehyde, chlorodi-bromomethane, ethyl ether, and 1,2-dichloropropane.

Gaseous pollutants of gas or air streams must pass through bioscrubbers containing sus-pensions of biodegrading microorganisms or through a biofilter packed with porous carrierscovered by biofilms of degrading microorganisms. Depending on the nature and volume ofpolluted gas, the biofilm carriers may be cheap porous substrates, such as peat, wood chips,compost, or regular artificial carriers, such as plastic or metal rings, porous cylinders andspheres, fibers, and fiber nets. The bioscrubber’s contents must be stirred to ensure a highmass transfer between gas and microbial suspension. The liquid that has interacted with thepolluted gas is collected at the bottom of the biofilter and recycled to the top part of the biofilterto ensure adequate contact of polluted gas and liquid and optimal humidity of biofilter. Theaddition of nutrients and fresh water to a bioscrubber or biofilter must be made regularly orcontinuously. Fresh water can be used to replace water that has evaporated in the bioreactor.If the mass transfer rate is higher than the biodegradation rate, the absorbed pollutants mustbe biodegraded in an additional suspended bioreactor or biofilter connected in series to thebioscrubber or absorbing biofilter.

4. ANAEROBIC TREATMENT OF WASTES

There are anaerobic (living without oxygen), facultative anaerobic (living under anaerobicor aerobic conditions), microaerophilic (preferring to live under low concentrations of dis-solved oxygen) and obligate aerobic (living only in the presence of oxygen), microorganisms.Some anaerobic microorganisms, called tolerant anaerobes, have mechanisms protecting themfrom exposure to oxygen. Others, called obligate anaerobes, have no such mechanisms andmay die after several seconds of exposure to aerobic conditions. Obligate anaerobes produceenergy from: a) fermentation (destruction of organic substances without external acceptor ofelectrons); b) anaerobic respiration using electron acceptors such as CO2, NO3

−NO2−, Fe3+,

SO42−; 3) anoxygenic (H2S → S) or oxygenic (H2O → O2) photosynthesis. The advantage

of anaerobic treatment is that there is no need to supply oxygen in the treatment system. Thisis useful in cases such as bioremediation of clay soil or high-strength organic waste. However,anaerobic treatment may be slower than aerobic treatment, and there may be significantoutputs of dissolved organic products of fermentation or anaerobic respiration.

The following sequence arranges respiratory processes according to increasing energeticefficiency of biodegradation (per mole of transferred electrons): fermentation → CO2 respi-ration (“methanogenic fermentation”) → dissimilative sulfate-reduction → dissimilative ironreduction (“iron respiration”) → nitrate respiration (“denitrification”) → aerobic respiration.

Facultative anaerobes can produce energy from these reactions or from the aerobic oxida-tion of organic matter and may be useful when integrated together with aerobic and anaerobicmicroorganisms in microbial aggregates. However, this function is still not well studied. Oneinteresting and useful feature in this physiological group is the ability in some representatives(e.g., Escherichia coli) to produce an active oxidant, hydrogen peroxide, during normalaerobic metabolism (8).


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 differentgroups 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 sufficientfor the anaerobic respiration and oxidation of the pollutants, the activity of the relatedbacterial group can be used for the treatment. CO2-respiring prokaryotes (methanogens)are used for methanogenic biodegradation of organic hazardous wastes. Sulfate-reducingbacteria can be used for anaerobic biodegradation of organic matter or for the precipita-tion/immobilization of heavy metals of sulfate-containing hazardous wastes. Iron-reducingbacteria can produce dissolved Fe2+ ions from insoluble Fe(III) minerals. Anaerobicbiodegradation of organic matter and detoxication of hazardous wastes can be signifi-cantly enhanced as a result of precipitation of toxic organics, acids, phenols, or cyanideby Fe(II). Nitrate-respiring bacteria can be used in denitrification, i.e., reduction of nitrateto gaseous N2. Nitrate can be added to the hazardous waste to initiate the biodegradationof different types of organic substances, for example polycyclic aromatic hydrocarbons(9). Nitrogroups of hazardous substances can be reduced by similar pathway to relatedamines.

Anaerobic fermenting bacteria (e.g., from genus Clostridium) perform two importantfunctions 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, thusenhancing further biodegradation of xenobiotics. Different biotechnological systems performanaerobic 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 inlandfills (13). Organic matter is hydrolyzed by bacteria and fungi. Amino acids are degradedusing ammonification with formation of toxic organic amines and ammonia. Amino acids,nucleotides, and carbohydrates are fermented or anaerobically oxidized with formation oforganic 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 toxiclandfill leachate, which can be detoxicated by aerobic biotechnological treatment to oxidizeorganic hazards and to immobilize dissolved heavy metals.

A combined anaerobic/aerobic biotreatment can be more effective than aerobic or anaerobictreatment 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 treatmentoccurs at the bottom end. A typical organic loading is 0.01 kg BOD/m3 day and the retentiontime varies from a few days to 100 days (7). A more intensive form of biodegradation canbe achieved by combining aerobic and anaerobic reactors with controlled conditions, or by


integrating anaerobic and aerobic zones within a single bioreactor. Combination or alterationof anaerobic and aerobic treatments is useful in the following situations:

1. Biodegradation of chlorinated aromatic hydrocarbons including anaerobic dechlorination andaerobic ring cleavage

2. Sequential nitrogen removal including aerobic nitrification and anaerobic denitrification3. Reduction of sulfate or Fe(III) with production of H2S or Fe(II) which are active reagents for the

precipitation of heavy metals, organic acids, and nutrients

5. TREATMENT OF HEAVY METALS-CONTAINING WASTES

Liquid and solid wastes containing heavy metals may be successfully treated by biotech-nological methods. Some metals can be reduced or oxidized by specific enzymes of microor-ganisms. Microbial metabolism generates products such as hydrogen, oxygen, H2O2, whichcan be used for oxidation/reduction of metals. Reduction or oxidation of metals is usuallyaccompanied by metal solubilization or precipitation. Solubilization or precipitation of metalsmay also be mediated by microbial metabolites. Microbial production of organic acids infermentation or inorganic acids (nitric and sulfuric acids) in aerobic oxidation will promoteformation of dissolved chelates of metals. Microbial production of phosphate, H2S, and CO2

will stimulate precipitation of non-dissolved phosphates, carbonates, and sulfides of heavymetals such as arsenic, cadmium, chromium, copper, lead, mercury, nickel; production of H2Sby sulfate-reducing bacteria is especially useful to remove heavy metals and radionuclidesfrom sulfate-containing mining drainage waters, liquid waste of nuclear facilities, drainagefrom tailing pond of hydrometallurgical plants; wood straw or saw dust. Organic acids,produced during the anaerobic fermentation of cellulose, may be used as a source of reducedcarbon for sulfate reduction and further precipitation of metals.

The surface of microbial cells is covered by negatively charged carboxylic and phosphategroups, and positively charged amino groups. Therefore, depending on pH, there may besignificant adsorption of heavy metals onto the microbial surface (5). Biosorption, for exampleby fungal fermentation residues, is used to accumulate uranium and other radionuclides fromwaste streams.

Metal-containing minerals such as sulfides can be oxidized and metals can be solubilized.This approach is used for the bioleaching of heavy metals from sewage sludge (14, 15) beforelandfilling or biotransformation. Some metals, arsenic and mercury for example, may bevolatilized by methylation due to the activity of anaerobic microorganisms. Arsenic can bemethylated by methanogenic Archaea and fungi to volatile toxic dimethylarsine and trimethy-larsine or can be converted to less toxic non-volatile methanearsonic and dimethylarsinic acidsby algae (16). Hydrophobic organotins are toxic to organisms because of their solubility in cellmembranes. However, many microorganisms are resistant to organotins and can detoxicatethem by degrading the organic part of organotins (17).

In some cases, the different biotechnological methods may be combined. Examples wouldinclude the biotechnological precipitation of chromium from Cr (VI)-containing wastes fromelectroplating factories by sulfate reduction to precipitate chromium sulfide. Sulfate reductioncan use fatty acids as organic substrates with no accumulation of sulfide. In the absence of


fatty acids but with straw as an organic substrate, the direct reduction of chromium has beenobserved without sulfate reduction (18).

6. ENHANCEMENT OF BIOTECHNOLOGICAL TREATMENT OF WASTES

Several key factors are critical for the successful application of biotechnology for thetreatment of hazardous wastes:

1. Environmental factors, such as pH, temperature, and dissolved oxygen concentration, must beoptimized

2. Contaminants and nutrients must be available for action or assimilation by microorganisms3. Content and activity of essential microorganisms in the treated waste must be sufficient for the

treatment

Optimal growth temperatures ranging from 10 to 90◦C must be maintained for effectivebiotreatment by certain physiological groups of microorganisms. The heating of the treatedwaste can come from microbial oxidation or fermentation activities, providing sufficient heatgeneration and good thermal isolation of treated waste from the cooler surroundings. Thebulking agent added to solid wastes may also be used as a thermal isolator.

The pH of natural microbial biotopes vary from 1 to 11: volcanic soil and mine drainagehave pH values between 1 and 3; plant juices and acid soils have pH values between 3 and5; fresh water and sea water have pH values between 7 and 8; alkaline soils and lakes,ammonia solutions, and rotten organics have pH values between 9 and 11. Most microbesgrow most efficiently within the pH range from 5 to 9 and are called neutrophiles. Speciesthat have adapted to grow at pH values lower than 4 are called acidophiles. Species that haveadapted to grow at pH values higher than 9 are called alkaliphiles. Therefore, the pH of atreatment medium must be maintained at optimal values for effective biotreatment by certainphysiological groups of microorganisms. The optimum pH may be maintained physiologicallyby the addition of a pH buffer or pH regulator in the following ways: (a) control of organicacid formation in fermentation; (b) prevention of formation of inorganic acids in aerobicoxidation of ammonium, elemental sulfur, hydrogen sulfide or metal sulfides; (c) assimilationof ammonium, nitrate, or ammonium nitrate, leading to decreased pH, increased pH, or neutralpH, respectively; (d) pH buffers such as CaCO3 or Fe(OH)3 can be used in large-scale wastetreatment; and (e) solutions of KOH, NaOH, NH4OH, Ca(OH)2, HCl, or H2SO4 can be addedautomatically to maintain the pH of liquid in a stirred reactor. Maintenance of optimum pH intreated solid waste or bioremediated soil may be especially important if there is a high contentof sulfides in waste or acidification/alkalization of soil in the bioremediation process.

The major elements found in microbial cells are C, H, O, N, S, and P. An approximateelemental composition corresponds to the formula CH1.8O0.5N0.2. Therefore, nutrient amend-ment may be required if the waste does not contain sufficient amounts of these macroelements.The waste can be enriched with carbon (depending on the nature of the pollutant that istreated), nitrogen (ammonium is the best source), phosphorus (phosphate is the best source)and/or sulfur (sulfate is the best source). Other macronutrients (K, Mg, Na, Ca, and Fe) andmicronutrients (Cr, Co, Cu, Mn, Mo, Ni, Se, V, and Zn) are also essential for microbialgrowth and enzymatic activities and must be added into the treatment systems if present


in low concentrations in the waste. The best sources of essential metals are their dissolvedsalts or chelates with organic acids. The source of metals for the bioremediation of oil spillsmay be lipophilic compounds of iron and other essential nutrients that can accumulate atthe water–air interface where hydrocarbons and hydrocarbon-degrading microorganisms canalso occur (19). In some biotreatment cases, growth factors must also be added to the treatedwaste. 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 calledauxotrophic strains. Usually, those microorganisms that are commensals or parasites of plantsand animals require growth factors. However, sometimes these microorganisms may have theunique 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 subsequentbiodegradation. Another way to increase the bioavailability of hydrophobic substances isthrough 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 tothe resistance of chemically produced surfactants to biodegradation. Therefore, only easilybiodegradable or biotechnologically produced surfactants can be used for the pretreatment ofhydrophobic hazardous substances.

Extracellular enzymes produced by microorganisms are usually expensive for large-scalebiotreatment of organic wastes. However, enzyme applications may be cost-effective in certainsituations. Toxic organophosphate waste can be treated using the enzyme parathion hydro-lase produced and excreted by a recombinant strain of Streptomyces lividans. The cell-freeculture fluid contains enzymes that can hydrolyze organophosphate compounds (20). Futureapplications may involve cytochrome-P450-dependent oxygenase enzymes that are capable ofoxidizing different xenobiotics (21).

Low concentrations of dissolved oxygen (0.01–10 mg/L) can be rapidly depleted duringwaste 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 wastetreatment systems is achieved by aeration and mechanical agitation. Different techniques areemployed to supply sufficient quantities of oxygen in fixed biofilm reactors, in viscous solidwastes, in underground layers of soil or in aquifers polluted by hazardous substances. Veryoften the supply of oxygen is the critical factor in the successful scaling-up of bioremediationtechnologies from laboratory experiments to full-scale applications (22). Air sparging insitu is a commonly used bioremediation technology, which volatilizes and enhances aerobicbiodegradation of contamination in groundwater and saturated soils. Successful case studiesinclude a 6–12 month bioremediation project that targeted both sandy and silty soils pollutedby petroleum products and chlorinated hydrocarbons (23). The application of pure oxygen canincrease the oxygen transfer rate by up to five times, and this can be used in situations with astrong acute toxicity of hazardous wastes and low oxygen transfer rates, to ensure sufficientoxygen transfer in polluted waste.

In some cases, hydrogen peroxide has been used as an oxygen source because of the limitedconcentrations of oxygen that can be transferred into the groundwater using above-ground


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 ofH2O2 with various aquifer material constituents, its decomposition may be too rapid, makingeffective introduction of H2O2 into targeted treatment zones extremely difficult and costly(24). Pre-treatment of wastewater by ozone, H2O2, by TiO2-catalized UV-photooxidation,and electrochemical oxidation can significantly enhance the biodegradation of halogenatedorganics, textile dyes, pulp mill effluents, tannery wastewater, olive-oil mills, surfactant-polluted wastewater and pharmaceutical wastes, and diminish the toxicity of municipal landillleachates. In some cases, oxygen radicals generated by Fenton’s reagent (Fe2+ + H2O2 atlow pH), and iron peroxides (Fe (VI) and Fe(V)) can be used as oxidants in the treatment ofhazardous wastes.

Many microorganisms can produce and release to the environment such toxic metabolites ofoxygen as hydrogen peroxide (H2O2), superoxide radical (O−

2 ), and hydroxyl radical (OH ·).Lignin-oxidizing “white rot” fungi can degrade lignin and all other chemical substancesdue to intensive generation of oxygen radicals which oxidize the organic matter by randomincorporation of oxygen into molecule. Not much is known about the biodegradation abilityof H2O2-generating microaerophilic bacteria.

Dissolved acceptors of electrons such as NO−3 , NO−

2 , Fe3+, SO2−4 , and HCO−

3 can beused 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 forbioremediation (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 controlbecause 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, butits compounds are usually insoluble and it diminishes the rate of oxidation in comparisonwith dissolved electron acceptors. Sulfate and carbonate can be applied as electron acceptorsonly in anaerobic environments. Another disadvantage of these acceptors is that these anoxicoxidations generate toxic and foul-smelling H2S or “greenhouse” gas CH4.

The addition of microorganisms (inoculum) to start up or to accelerate a biotreatmentprocess 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 achievethe treatment goal within the prescribed duration

3. If the acclimation period is too long4. To direct the biotreatment to the best pathway from many possible pathways5. 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 andsafe microbial strain(s) as a starter culture is especially important for biotechnological systemsusing aggregated bacterial cells in biofilms, flocs, or granules for two reasons: a) aggregationcan be facilitated and enhanced; and b) self-aggregated or co-aggregated bacterial cells often arepathogens or opportunistic pathogens


Currently, a common environmental engineering practice is to use part of the treated wasteor enrichment culture as an inoculum. However, applications of defined pure starter cultureshave the following advantages:

1. Greater control over desirable processes2. Lower risk of release of pathogenic or opportunistic microorganisms during biotechnological

treatment3. 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 byconventional microbiological methods, quickly identified by molecular–biological methods, andtested for pathogenicity and biodegradation properties

4. Inoculum can be produced industrially5. 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 thenatural environment. However, their ability for biodegradation can be modified and amplifiedby artificial alterations of their genetic (inherited) properties. The description of the methods isgiven in many books on environmental microbiology and biotechnology (4, 5). Natural geneticrecombination of the genes (units of genetic information) occurs during DNA replication andcell 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 engineeringof recombinant microbial strains suitable for the biotreatment usually involves the followingsteps:

1. DNA is extracted from cells and cut into small sequences by specific enzymes2. Small sequences of DNA can be introduced into DNA vectors3. A vector (virus or plasmid) is transferred into the cell and self-replicated to produce multiple

copies of the introduced genes4. 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 theconstruction of hybrid metabolic pathways to increase the range of biodegraded xenobioticsand 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. Strainscontaining such plasmids can be used for the bioremediation of sites heavily polluted by avariety of xenobiotics. The main problem in these applications is maintaining the stabilityof the plasmids in these strains. Other technological and public concerns include the risk ofapplication and release of genetically modified microorganisms in the environment.


Self-aggregated microbial cells of biofilms, flocs, and granules, and artificially aggregatedcells 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 ordetoxication; therefore microbial aggregates or immobilized cells are more resistant to toxicxenobiotics 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 thediversity 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 cellsimmobilized on carrier surfaces such as granulated activated carbon that can adsorb xenobioticswill degrade xenobiotics more effectively than suspended cells (26)

However, dense microbial aggregates may encounter problems associated with diffusionlimitation, such as slow diffusion of both nutrients into, and the metabolites out of, theaggregate. For example, dissolved oxygen levels can drop to zero at some depth below thesurface of microbial aggregates so that obligate anaerobic bacteria can grow inside the biofilmof an aerated reactor (27). This distance clearly depends on factors such as the specificrate of oxygen consumption and the density of biomass in the microbial aggregate. Whenenvironmental conditions within the aggregate become unfavorable, cell death may occur inzones that do not receive sufficient nutrition or that contain inhibitory metabolites. Channelsand pores in aggregate can facilitate transport of oxygen, nutrients and metabolites. Channelsin microbial spherical granules have been shown to penetrate to depths of 900 µm (28) anda layer of obligate anaerobic bacteria was detected below the channeled layer (27). Thisdemonstrates that there is some optimal size or thickness of microbial aggregates appropriatefor application in the treatment of hazardous wastes.

7. BIOSENSORS

An important application of environmental biotechnology is biomonitoring, includingmonitoring of biodegradability, toxicity, mutagenicity, concentration of hazardous substances,and monitoring of concentration and pathogenicity of microorganisms in wastes and in theenvironment. Simple or automated off-line or on-line biodegradability tests can be performedby measuring CO2 or CH4 gas production or O2 consumption (29). Biosensors may utilizeeither whole bacterial cells or enzyme to detect specific molecules of hazardous substances.Toxicity can be monitored specifically by whole cell sensors whose bioluminescence may beinhibited by the presence of hazardous substance.

The most popular approach uses cells with an introduced luminescent reporter geneto 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 fortoxicity monitoring in wastewater treatment (31). Biosensors measuring concentrations ofhazardous substances are often based on the measurement of bioluminescence (32). This


toxicity sensor is a bioluminescent toxicity bioreporter for hazardous wastewater treatment. Itis constructed by incorporating bioluminescence genes into a microorganism. These whole-cell toxicity sensors are very sensitive and may be used on-line to monitor and optimize thebiodegradation of hazardous soluble substances.

Similar sensors can be used for the measurement of the concentration of specific pollutants.A gene for bioluminescence has been fused to the bacterial genes coding for enzymes thatmetabolize the pollutant. When this pollutant is degraded, the bacterial cells will producelight. The intensity of biodegradation and bioluminescence depend on the concentration ofpollutant and can be quantified using fiber-optics on-line. Combinations of biosensors in arraycan be used to measure concentration or toxicity of a set of hazardous substances.

The mutagenic activity of chemicals is usually correlated with their carcinogenic prop-erties. Mutant bacterial strains have been used to determine the potential mutagenicity ofmanufactured or natural chemicals. The most common test, proposed by Ames in 1971 (33),utilizes back mutation in auxotrophic bacterial strains that are incapable of synthesizingcertain nutrients. When auxotrophic cells are spread on a medium that lacks the essentialnutrients (minimal medium), no growth will occur. However, cells that are treated with a testedchemical that causes a reversion mutation can grow in a minimal medium. The frequency ofmutation detected in the test is proportional to the potential mutagenicity and carcinogenicityof the tested chemical. Microbial mutagenicity tests are used widely in modern research(34–36).

Cell components or metabolites capable of recognizing individual and specific moleculescan be used as the sensory elements in molecular sensors (37). Sensors may be enzymes,sequences of nucleic acids (RNA or DNA), antibodies, polysaccharides or other “reporter”molecules. Antibodies, specific for a microorganism used in the biotreatment, can be coupledwith fluorochromes to increase sensitivity of detection. Such antibodies are useful in moni-toring the fate of bacteria released into the environment for the treatment of a polluted site.Fluorescent or enzyme-linked immunoassays have been derived and can be used for a varietyof contaminants, including pesticides and chlorinated polycyclic hydrocarbons. Enzymesspecific for pollutants and attached to matrices detecting interactions between enzymes andpollutants are used in on-line biosensors of water and gas biotreatment (38, 39).

A useful approach to monitor microbial populations in the biotreatment of hazardous wastesinvolves the detection of specific sequences of nucleic acids by hybridization with comple-mentary oligonucleotide probes. Radioactive labels, fluorescent labels, and other kinds of thelabels are attached to the probes to increase sensitivity and simplicity of the hybridizationdetection. Nucleic acids which are detectable by the probes include chromosomal DNA, extra-chromosomal DNA such as plasmids, synthetic recombinant DNA such as cloning vectors,phage or virus DNA, rRNA, tRNA and mRNA transcribed from chromosomal or extra-chromosomal DNA. These molecular approaches may involve the hybridization of wholeintact cells, or extraction and treatment of targeted nucleic acids prior to probe hybridization(40–42). Microarrays for simultaneous semi-quantitative detection of different microorgan-isms or specific genes in the environmental sample have also been developed (43–45).


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