Natural and Enhanced Remediation Systems - Chapter 4 pot

Another study in the 1980s demonstrated that dechlori-nation of PCE to VC in a methanogenic column was achievable.5 Similar studies using 13C-TCE, showed that TCE was dechlorinated exclu

Suthersan, Suthan S “Chapter 4: In Situ Reactive Zones”

Natural and Enhanced Remediation Systems

Edited by Suthan S SuthersanBoca Raton: CRC Press LLC, 2001

4.2.1.1.1 Biostimulation vs Bioaugmentation4.2.1.2 Mechanisms of Reductive Dechlorination

4.2.1.11 Derivation of a Completely Mixed System for

Groundwater Solute Transport of Chlorinated Ethenes

4.2.1.12 IRZ Performance Data4.2.2 In SituMetals Precipitation 4.2.2.1 Principles of Heavy Metals Precipitation4.2.2.2 Aquifer Parameters and Transport Mechanisms4.2.2.3 Contaminant Removal Mechanisms

4.2.3 In SituDenitrification4.2.4 Perchlorate Reduction4.3 Engineered Aerobic Systems

4.3.1 Direct Aerobic Oxidation4.3.1.1 Aerobic Cometabolic Oxidation4.3.1.2 MTBE Degradation

4.4 In SituChemical Oxidation Systems

4.4.1 Advantages 4.4.2 Concerns4.4.3 Oxidation Chemistry4.4.3.1 Hydrogen Peroxide4.4.3.2 Potassium Permanganate 4.4.3.3 Ozone

4.4.4 Application4.4.4.1 Oxidation of 1,4-Dioxane by Ozone4.4.4.2 Biodegradation Enhanced by Chemical

Oxidation Pretreatment4.5 Nano-Scale Fe (0) Colloid Injection within an IRZ

4.5.1 Production of Nano-Scale Iron Particles4.5.2 Injection of Nano-Scale Particles in Permeable Sediments4.5.3 Organic Contaminants Treatable by Fe (0)

References

Oxidation-reduction process plays a major role in the mobility, transport, and fate of inorganic and organic contaminants in natural waters Hence, the manip- ulation of REDOX conditions to create an in situ reactive zone (IRZ) to meet the cleanup objectives was a predictable evolution …

is to design an engineered system for the systematic control of these reactions underthe naturally variable or heterogeneous conditions found in the field

The effectiveness of the reactive zone is determined largely by the relationshipbetween the kinetics of the target reactions and the rate at which the mass flux ofcontaminants passes through it with the moving groundwater Creation of a spatially

fixed reactive zone in an aquifer requires not only the proper selection of the reagents,but also the proper mixing of the injected reagents uniformly within the reactivezone Furthermore, such reagents must cause few side reactions and be relativelynontoxic in both its original and treated forms.

When dealing with dissolved inorganic contaminants such as heavy metals, theprocess sequence in a pump and treat system required to remove the dissolved heavymetals present in the groundwater becomes very complex, operation- and mainte-nance-intensive, and costly In addition, the disposal of the metallic sludge, in mostcases as a hazardous waste, is also very cost prohibitive Therefore, in situ treatmentmethods capable of achieving the same mass removal reactions for dissolved con-taminants in an in situ environment are evolving and gradually gaining prominence

in the remediation industry

The advantages of an in situ reactive zone to address the remediation of water contamination are as follows:

ground-• An in situ technology enables implementation of most ground treatment processes and eliminates the expensive infrastructure required for a pump and treat system with no disposal of water or wastes

Figure 4.1a Pictorial depiction of an in situ reactive zone (IRZ) formation.

Figure 4.1b Cross sectional view of the creation of an IRZ around an individual injection well

at a selected location.

Plan View

Source Area IRZ Grid

Contaminant Plume

Individual Reactive Zones Created by Individual Injection Points Providing a Collective In Situ Reactive Zone (IRZ) Curtain

Cross Sectional View

Contaminant Zone Reagent

• Inexpensive installation because primary capital expenditure for this technology

is the installation of injection wells at appropriate locations

• Inexpensive operation that allows inexpensive reagents to be injected at fairly low concentrations and, hence, should result in insignificant cost; only sampling required is for groundwater quality monitoring and performance monitoring parameters are usually done in the field; remediation of large volumes of contam- inated water without any pumping or disposal needs

• Can be used to remediate deep sites because cluster injection wells or in-well mixing systems can be installed to address deeper sites

• Unobtrusive because once the system is installed, site development and operations can continue with minimal obstructions

• In situ degradation of contaminants because organic contaminants and a few inorganics such as NH4+ , NO3, and CIO4 can be degraded by implementing the appropriate reactions

• Immobilization of contaminants because once the dissolved heavy metals are precipitated out, the capacity of the soils and sediments is utilized to adsorb, filter out, and retain inorganic contaminants

Manipulation of the reduction-oxidation (REDOX) potential of an aquifer is aviable approach for in situ remediation of REDOX-sensitive groundwater contami-nants In addition, various microbially induced or chemically induced reactions alsocan be achieved in an in situ environment As noted earlier, creation of spatiallyfixed reactive zones to achieve these reactions is very cost effective in comparison

to treating the entire plume as a reaction zone

Since the first IRZ for the precipitation and remediation of hexavalent chromium(Cr6+), was installed in 1993, this technology has advanced by leaps and bounds.1

Currently the application of this technology can be classified into three categoriesbased on the creation of specific bio-geo-chemical and REDOX environments: 1)engineered anaerobic systems, 2) engineered aerobic systems, and 3) in situchemicaloxidation

The engineered anaerobic systems can be further divided into enhanced reductivedechlorination (ERD) systems, in situ denitrification, in situ perchlorate transforma-tion, and in situ heavy metals precipitation The ERD application has been expanded

to many contaminants since the first trichloroethene (TCE) application site The IRZtechnology has been successfully applied to remediate the following chlorinatedcompounds:

• Chlorinated ethenes: tetrochloroethane (PCE), trichloroethane (TCE), hene (Cis 1,2 DCE, and 1,1 DCE), vinylchloride

dichloroet-• Chlorinated ethanes: 1,1,2,2 tetrachloroethane (1,1,2,2 PCA), 1,1,1 hane, (1,1,1 TCA), 1,1,2 trichloroethane (1,1,2 TCA), 1,1 and 1,2 dichloroethane (DCA), chloroethane (CA)

trichloroet-• Chlorinated phenols: pentachlorophenol (PCP), and tetrachlorophenol

• Chlorinated pesticides

• Perchlorate

In addition, the IRZ technology has been successfully applied to precipitate thefollowing dissolved metals at contaminated sites: Cr6+, Pb2+, Cd2+, Ni2+, Zn2+, Hg2+

4.2 ENGINEERED ANAEROBIC SYSTEMS

4.2.1 Enhanced Reductive Dechlorination (ERD) Systems

4.2.1.1 Early Evidence

The first microbially mediated reductive dechlorination of PCE and TCE wasobserved in the early 1980s, and this study2,3 reported the degradation of PCE tononchlorinated end products in an acetate-fed, continuous-flow methanogenic glassbead column It appeared that the first step in the degradation pathway was dechlori-nation to TCE Further anaerobic oxidation of TCE to carbon dioxide and hydrochloricacid was suggested In 19844, further evidence of dechlorination of PCE beyond TCEcame in an experiment where sediments from an aquifer recharge basin were incubatedwith PCE and methanol as the electron donor Significant concentrations of TCE, cis-1,2 DCE and VC were observed after three weeks, whereas in sterile controls nodechlorination had occurred Another study in the 1980s demonstrated that dechlori-nation of PCE to VC in a methanogenic column was achievable.5 Similar studies using

13C-TCE, showed that TCE was dechlorinated exclusively to cis-DCE in soil.6

In 1989, the first evidence of complete dechlorination of PCE to ethene undermethanogenic conditions with methanol as electron donor was demonstrated.7

Another study found PCE reduction via ethene to ethane with lactate as electrondonor in a flow-through column filled with a mixture of polluted sediment andanaerobic granular sludge.8 Meanwhile, numerous publications showed that micro-organisms capable of reductively dechlorinating chlorinated ethenes are abundant

in polluted anaerobic environments (An overview of the biological reductive rination pathway of chlorinated solvents is shown in Figure 4.2.) PCE and TCE aredechlorinated mainly to cis-DCE, although sometimes trans-DCE and 1,1-DCE havealso been found as products.9,10 However, the formation of the 1,1-DCE is believed

dechlo-to be a result of abiotic dechlorination in the presence of sulfide.10

Evidence from the earlier studies indicated that the dechlorination of PCE to

cis-DCE was found to be a relatively fast process, whereas, subsequent rates ofdechlorination of cis-DCE to VC and ethene were significantly slower or evenabsent.7,11 Dechlorination of 1,1-DCE and trans-DCE was less studied

In some of the earlier reports and studies the dechlorination of chlorinatedethenes was often found to be incomplete, both in the laboratory and in fieldexperiments, resulting in an accumulation of cis-DCE and VC It was not fullyunderstood at that time why dechlorination beyond these compounds was problem-atic, other than raising valid questions regarding the required microbial consortiafor complete dechlorination During that time (late 1980s and early 1990s) micro-organisms capable of dechlorinating DCE and VC had not been isolated yet, althoughseveral enrichment cultures existed Little was known about the substrate require-ments of these bacteria Later studies reported that PCE dechlorination in a contam-inated soil down to ethene was only achieved by adding a complex mixture of organicelectron donors Significant research was focused, during the early to mid 1990s,

on the microbial ecology that could perform complete dechlorination of PCE to

ethene and the biogeochemical conditions under which this biotransformation could

be achieved

The choice of a suitable electron donor for the stimulation of in situ nation is still a matter of discussion and may be dependent on local conditions; thiswill be discussed in detail in a later section When hydrogen is assumed to be themajor electron donor for dechlorination, its amendment can only be achieved byusing substrates yielding hydrogen after anaerobic degradation.12 Often, short-chainorganic acids are produced as intermediate products, which may lead to acidification

dechlori-of the groundwater and soil Additionally, electron donors that support dechlorinationare generally readily degraded by nondechlorinating microorganisms, leading tocompetition for the substrate and excessive bacterial growth in soil pores near theinjection well As a result, significantly more electron donor mass will be neededthan theoretically necessary to reduce all chlorinated ethenes present to ethene

4.2.1.1.1 Biostimulation vs Bioaugmentation

The first level of the treatment hierarchy for chlorinated ethenes is intrinsicbioremediation, or natural biodegradation, whereby indigenous microflora destroythe contaminant(s) of concern without any stimulation or enhancements The secondchoice in this hierarchy, biostimulation or enhanced biodegradation, involves stim-ulating the indigenous microbial populations and thus enhancing microbial activity

Figure 4.2 Biological and abiotic degradation pathways of the common chlorinated

com-pounds encountered at contaminated sites (adapted from McCarty and Semprini, 1994; after Vogel et al., 1987, and Wiedermeier et al., 1999).

CO2, H2O, CI-

1,1,1-TCE

1,1- DCA

CA

*

so that they destroy the target compounds at a rate that meets the cleanup objectives

at the site At almost every contaminated site a natural population of degradativemicroorganisms exists within the contaminated zone; however, specific nutrients,growth substrates inducers, electron donors, and electron acceptors may be required

to create optimal microbial activity.12 Thus, through the introduction of requiredadditional reagents, the native degradative microbial population can be stimulated

to grow, multiply, and destroy the target contaminants Most environments containmicroorganisms able to grow on and destroy a variety of chlorinated compounds;

at some sites, the persistence of these compounds, is not a consequence of theabsence of organisms but rather of the absence of the full set of conditions necessaryfor the indigenous species to function rapidly.12 In the past there was a significantdebate among remediation experts whether the microorganisms responsible forcometabolic degradation and dehalorespiration are ubiquitous Current belief is thatthese organisms are nearly ubiquitous

When intrinsic bioremediation or biostimulation is not feasible at a given sitedue to the absence of an appropriate microbial population, bioaugmentation may beutilized Bioaugmentation involves injection of selected exogenous microorganismswith the desired metabolic capabilities directly into the contaminated zones alongwith any required nutrients to effect the rapid biodegradation of target compounds.Two distinct bioaugmentation approaches have been developed for remediatingchlorinated ethenes In the first approach, degradative organisms are added to com-plement or replace the native microbial population The added microorganisms can

be selected for their ability to survive for extended periods or to occupy a specificniche within the contaminated environment If needed, stimulants or selective cosub-strates can be added to improve survival or enhance the activity of the addedorganism Thus, the goal of this approach is to achieve prolonged survival and growth

of the added organisms and degradation of the target contaminants

In the second bioaugmentation approach, large numbers of degradative bacteriaare added to a contaminated environment as biocatalysts which will degrade asignificant amount of the target contaminant before becoming inactive or perishing.12

Additional microbes can be added as needed to complete the remediation process.Attempts can be made to increase the production of the degradative enzymes or tomaximize catalytic efficiency or stability, but long-term survival, growth, and estab-lishment of an active microbial population are not the primary goals of this treatmentapproach

In the past, bioaugmentation has been implemented frequently and successfullyonly in bioreactors The conditions in these bioreactors are controlled and quitedifferent from those in nature, and prior to start-up, no microorganisms are presentanyway Hence, the addition of enriched cultures is essential Furthermore, bioreac-tors are engineered and controlled systems where conditions can be readily altered

or optimized for a particular process and can be designed to promote the cation and activity of the inoculated species — in contrast to contaminated field sites.The record of success of in situ bioaugmentation systems for chlorinated com-pounds has been rather spotty On the one hand, the initiation or enhancement ofdegradation has been reported (far more commonly in samples of the contaminatedenvironments in simulated laboratory experiments) following the addition of

multipli-enriched bacterial cultures that can metabolize and grow on chlorinated ethenes Onthe other hand, a number of failures in the field have been reported.

Such reports of failure of bioaugmentation came as no surprise to microbialecologists Without question, a species with a substrate uniquely available to it has

a distinct advantage, yet that advantage may not be sufficient to compensate formany other traits also necessary for survival, no less multiplication, in a naturalecosystem Possessing the requisite enzymes to metabolize a novel compound is anecessary attribute for the organism, but it is not sufficient for the organism tosucceed Populations of introduced microorganisms are subject to a variety of abioticand biotic stresses, and these must be overcome for these organisms to be able toexpress beneficial traits

The reasons for the frequent failures of bioaugmentation are many:12 limitingnutrients and growth factors in the uncontrolled natural environment, suppression

by predators and parasites, inability of the introduced bacteria to penetrate significantspace, metabolism of other nontarget organic compounds present, concentration ofthe target chlorinated compound too low to support multiplication, and other inhib-itory biogeochemical conditions such as pH, temperature, salinity, and toxins

In summary, the problems usually encountered in scaling up the bioaugmentationsuccesses achieved in laboratory experiments can be summarized as follows:12

• Contaminant rates established in controlled laboratory studies may differ tially from those in pilot-scale, full-scale, or even other laboratory studies.

substan-• Positive biotransformation results from small systems often are not reproduced in different systems.

• Instantaneous biotransformation rates vary widely and in an apparently stochastic manner, even in well-operated, steady-state systems.

4.2.1.2 Mechanisms of Reductive Dechlorination

Naturally occurring biological processes can degrade organic contaminants in situ or during transport in the subsurface under aerobic and/or anaerobic conditions.Microorganisms catalyze degradation reactions to obtain energy for growth, repro-duction, and cell maintenance Useable energy is recovered through a series ofREDOX reactions where the microorganisms act as “electron transport mediators”(Figure 4.3) Biologically mediated electron transfer couples the oxidation of anelectron donor (organic compound) with the reduction of an electron acceptor (inor-ganic or organic) and results in the production of useable energy for microbialconsortia.12,13,14 The bulk electron donor acts as a fuel source for the reactions andthe reactions proceed as long as there is a source of bioavailable electrons Fuelsources can be the target chlorinated compounds, native organic carbon, co-contam-inants such as fuel hydrocarbons, or organic compounds such as carbohydrates Inaerobic environments, the chlorinated compounds act as electron donors and underanaerobic conditions they act as electron acceptors

There are two primary mechanisms involved in the biodegradation of chlorinatedorganic contaminants (Table 4.1) First, biodegradation may be growth-linked andprovide carbon and energy to support growth when the compound is used as primary

substrate and directly utilized by the mediating organisms via the processes included

in Category 1 Some chlorinated solvents are used as electron donors and some areused as electron acceptors when serving as primary growth substrates When used

as an electron donor (under aerobic and anaerobic conditions) the contaminant isoxidized Conversely, when used as an electron acceptor, the contaminant is reducedvia the reductive dechlorination process called halorespiration.17

In addition to their use as a primary growth substrate, chlorinated solvents canalso be degraded via cometabolic pathways During cometabolism, microorganismsgain carbon and energy for growth from metabolism of a primary substrate, andchlorinated solvents are degraded fortuitously by enzymes present in the metabolic

Figure 4.3 Description of microorganisms acting as electron transport mediators (after

Schwarzenbach et al., 1993).

Table 4.1 Summary of the Categories of Degradation Pathways for Chlorinated Organic

Compounds (Adapted from Wiedemeier et al., 1999) 14

Halo-Direct Aerobic Oxidation

Direct Anaerobic Oxidation

Aerobic Cometabolism (co-oxidation)

Anaerobic Cometabolism (reductive dechlorination)

ne-(Contaminant)ox

(Contaminant)red+ ne-

-

ne-pathways Cometabolism is a process where the organism receives no direct benefitfrom the degradation of the organic compound.13,16 There are two types of cometa-bolic reactions: co-oxidation and reductive dechlorination, described as Category 2

in Table 4.1 Cometabolic reactions tend to be incomplete and can possibly lead to

an accumulation of more toxic daughter products To date, vinyl chloride (VC) anddichloroethene (cis/trans) are the only chlorinated solvents that can be degraded byall aerobic and anaerobic pathways.15

The predominant mechanism for the biodegradation of chlorinated solvents inanaerobic environments is reductive dechlorination, whether the organic compound

is a primary electron acceptor (halorespiration) or is cometabolized Before 1994,reductive dechlorination was thought to be strictly a cometabolic process becausethe organisms that cause these reactions are ubiquitous at most contaminatedsites.14,15 However, research has shown that combetabolic reductive dechlorination

is “sufficiently slow and frequently incomplete.”15,18 During reductive dechlorination,the chlorinated solvents act as an electron acceptor and a chlorine atom is replacedwith a hydrogen atom (Figure 4.4)

Cometabolic reduction of the chlorinated solvents is catalyzed by the reductivedehalogenase and reductase enzymes produced by microorganisms.14,20 Cometabolicdegradation occurs under iron reducing, manganese reducing, sulfate reducing, andmethanogenic environments.21 The enzymes of these reducing microorganisms areinduced to reduce abiotic forms of Fe (III) to Fe (II), Mn (IV) to Mn (II), sulphate

to sulfide or hydrogen sulfide, and carbon dioxide to methane Electrons are ferred to dissolved contaminants coincidentally during the reducing processes Thesedegradation reactions are often incomplete, resulting in an accumulation of toxicdaughter products

trans-Just as aerobic biodegradation systems utilize oxygen as a terminal electronacceptor to stimulate microbial activity, oxidative anaerobic systems require otherterminal electron acceptors, such as nitrate or ferric iron (Fe III), to stimulatebiodegradation Anaerobic oxidation occurs when anaerobic bacteria use the chlo-rinated contaminant as the electron donor and, in most instances, allow the micro-organism to derive useful amounts of energy from the reaction It has been shownthat vinyl chloride can be oxidized to carbon dioxide, water, and chloride ion via

Fe (III) reduction.22 Significant anaerobic mineralization of DCE, VC, and methylenechloride also have been reported in the literature

While in oxidative anaerobic systems the contaminant is used as an electrondonor, in reductive systems highly oxidized contaminants (such as PCE) are used

as electron acceptors The process begins by supplying excess reduced substrate(electron donor) to a microbial consortium, i.e., a cooperative community of micro-bial species (Figures 4.3 and 4.5) The presence of the substrate expedites theexhaustion of any naturally occurring electron acceptors As the natural electronacceptors are depleted, microorganisms capable of discharging electrons to otheravailable electron acceptors, such as oxidized contaminants, gain a selective advan-tage The intricacies of these microbial communities are complex, but recent researchhas provided some insight into methods for enhancing populations of contaminant-degrading microorganisms

The reductive dechlorination of PCE to ethene proceeds through a series ofhydrogenolysis reactions (Figure 4.4) Each reaction becomes progressively moredifficult to carry out; subsequently, the DCEs, particularly cis-DCE, and vinyl chlo-ride (VC), tend to accumulate in anaerobic environments under natural conditionsdue to the absence of sufficiently reducing conditions.

Figure 4.4 Hydrogenolysis reactions of PCE during reductive dechlorination with H 2 acting

as the electron donor and the chlorinated compounds acting as electron tors (adapted from Vogel et al., 1987, and Wiedermeier et al., 1999).

accep-+CI- H ion+

2 -

H

Electron Flow e

Ethane

h

H H

H

C C Ethene

CI

C C

H

C C

(Limited Biological Reaction) trans-1,2,-Dichloroethene

(Predominant Biological Reaction) cis-1,2,-Dichloroethene

CI

C C Trichloroethene

H H

C C

The oxidation-REDOX potential (ORP) affects the thermodynamics of reductive

dechlorination Microorganisms will facilitate only those oxidation-reduction

reactions that have a net yield of energy For reductive dechlorination to be

thermo-dynamically favorable the REDOX potential must be sufficiently low, thereby

excluding the presence of oxygen and nitrate as terminal electron acceptors

Fur-thermore, the presence of nitrate may have an inhibitory effect on PCE

dechlorina-tion.23 The REDOX potential range for reductive dechlorination is shown in Figure

4.6 It is important to note that the values of Eh ranges shown in Figure 4.6 and the

values of ORP measured in the field by remediation engineers are not the same

Both parameters have some correlation and do not represent the same conditions

Figure 4.5 summarizes the mechanisms and the required environmental condition

for the degradation of chlorinated solvents

4.2.1.3 Microbiology of Reductive Dechlorination

4.2.1.3.1 Cometabolic Dechlorination

A cometabolic process is defined here as a process in which the compound of

interest (e.g., PCE) is converted by a biological enzyme system or cofactor in which

the compound does not serve as a source of carbon or energy

Pure Microbial Cultures: Reductive dechlorination is the only biodegradative

conversion known for PCE This reaction can occur cometabolically or in a metabolic

energy-producing reaction In both cases, the cofactors of the enzymes involved are

metal-containing porphyrins Examples25 of acetogenic and methanogenic bacteria that

dechlorinate PCE cometabolically are listed in Table 4.2 In general, acetogenic bacteria

dechlorinate PCE at higher rates than methanogenic bacteria Metal-containing

cofac-tors have been found to catalyze the in vitro degradation of chlorinated ethenes.24,25

In general, reductive dechlorination rates decrease with a declining amount of

chlorine atoms in the molecule In vivo experiments with methanogenic and

aceto-genic bacteria indicate that dechlorination rates are low (0.5 to 235 nmol PCE

Figure 4.5 Pictorial description of the conditions which control reductive dechlorination.

Organic Compounds

Temperature pH

Anaerobic Conditions

Reductive Dechlorination

(mg protein)–1 day–1), compared with those of halorespiring bacteria25,26 (Table 4.3).

In vivo usually only one halogen atom is removed An exception is the reductive

dehalogenation of dibromoethene by Methanobacterium and Methanococcus that

yields acetylene as a product.27 However, in many studies the possible formation of

nonchlorinated products during dechlorination reactions was not included in the

carbon balance Complete dechlorination of PCE to ethene by pure cultures of

acetogenic or methanogenic bacteria has not been observed This is in contrast to

Figure 4.6 Optimal range for reductive dechlorination.

Table 4.2 Examples of Acetogenic and

Methanogenic Bacteria that Dechlorinate PCE

Cometabolic Dechlorination Cosubstrate

Methanosarcina sp Methanol

Methanosarcina mazei Methanol

Sporomusa ovata Methanol

Acetobacterium woodii Fructose

NO3- Reduction

REDOX Potential (Eh)

in millivolts (mV) at pH=7 and T=25˚C 1,000mV

findings with mixed anaerobic cultures in which more extensive dechlorination has

been observed.7,8,27-29 The latter may be due to the interactions between different

microorganisms Sometimes, however, it is difficult to distinguish between

comet-abolic and specific dechlorination in these mixed cultures, and often it is not even

clear which microorganisms are responsible for the dechlorination

The most often observed degradation pathway of PCE is via reductive

dechlo-rination to cis-DCE.10,25,30-32 Several dechlorination rates for chlorinated ethenes have

been reported in the literature, but it is difficult to compare the data because often

the numbers of bacteria involved were not known Nevertheless, it can be stated that

dechlorination rates in mixed cultures are generally higher than those found for

single acetogenic or methanogenic strains

There are a few reports on the degradation of PCE by granular methanogenic sludge

from upflow anaerobic sludge blanket reactors This sludge is enriched with acetogenic

and methanogenic bacteria and contains high concentrations of cofactors Such bacterial

consortia, therefore, are suitable as a source of cometabolic dechlorinating activity In

one of these reactors, fed with a mixture of sucrose, lactic acid, propionic acid, and

methanol as primary substrates, granular sludge showed a fast adaptation to high PCE

concentrations Influent concentrations of 360 to 420 mM PCE were completely

dechlo-rinated to ethene.26,33 Average removal rates of 7.6 mmol (g VSS)–1 day–1 were achieved,

with a maximum removal rate of 28.3 mmol (g VSS)–1 day–1 A bacterial consortium in

a similar reactor operated in batch mode converted PCE to TCE, cis- and trans-DCE

and traces of 1,1-DCE with ethanol as the primary substrate

4.2.1.3.2 Dechlorination by Halorespiring Microorganisms

Halorespiration is a type of anaerobic respiration in which a chlorinated

com-pound is used as a terminal electron acceptor In this reductive dechlorination

process, which enables the conservation of energy via electron transport

phospho-rylation, one or more chlorine atoms are removed and replaced by hydrogen

Exam-ples of halorespiring bacteria species are shown in Table 4.3

Halorespiration, also referred to as dehalorespiration, occurs when the organic

compound acts as an electron acceptor (primary growth substrate) during reductive

dechlorination During halorespiration, the chlorinated organic compounds are used

directly by microorganisms (termed halorespirators), such as an electron acceptor

while dissolved hydrogen serves as an electron donor:15,34

H2 + C – Cl Þ C – H + H+ + Cl– (4.1)

Table 4.3 Examples of Halorespiring Bacteria Halorespiration Electron Donor

Dehalobacter restrictus H 2

Dehalospirillum multivorans Pyruvate

Desulfitobacterium sp strain PCE1 Lactate

Desulfitobacterium sp strain TCE1 Lactate Strain MS-1 Yeast extract

where C – Cl represents the chlorine bond to the carbon in the chlorinated ethenemolecule Halorespiration occurs as a two-step process which results in the inter-species hydrogen transfer by two distinct strains of bacteria In the first step, bacteriaferment organic compounds to produce hydrogen During primary or secondaryfermentation, the organic compounds are transformed to compounds such as acetate,water, carbon dioxide, and dissolved hydrogen Fermentation substrates are eitherbiodegradable, nonchlorinated contaminants (i.e., BTEX — benzene, toluene, ethylbenzene, and xylenes — or sugar) or naturally occurring organic carbon In thesecond step, the nonfermenting microbial consortia utilize the hydrogen produced

by fermentation for halorespiration.35,36 Denitrifiers, iron reducers, sulfate reducers,methanogens, and halorespirators can all utilize hydrogen as an electron donor.14

Figure 4.7 shows which reducing environment is favored depending on the hydrogenconcentration Although compounds produced during fermentation and hydrogenhave been demonstrated to drive halorespiration,32 hydrogen appears to be the mostimportant electron donor for this process.36.37 Halorespiration has been found to belimited if available nutrients are not present Direct injection of H2 is able to serve

as an electron donor for reductive dechlorination of PCE to VC and eventually toethene in cultures provided with the proper nutritional supplements.34,38

Because reductive dechlorination of chlorinated ethenes is a reductive process,microorganisms may exist that can use chlorinated compounds as a terminal electronacceptor and possibly conserve the concomitant energy gain into ATP This hypoth-esis, developed in the early 1990s, proved to be true.25,26 The first evidence thatbacteria exist that can couple reductive dechlorination of PCE to growth (halores-piration) under strict anaerobic conditions was presented in the early 1990s.25,39 A

highly purified enrichment culture able to grow by the reduction of PCE to cis-DCE

using hydrogen as the electron donor was described The dechlorinating organism,

later designated Dehalobacter restrictus, uses only hydrogen as the electron donor and can couple growth to the reduction of PCE or TCE to cis-DCE A recent study25,40

described a new isolate, strain TEA, which is closely related to Dehalobacter restrictus Another strict anaerobic bacterium, Dehalospirillum multivorans, capable

of coupling dehalogenation of PCE to growth, was identified and describedrecently.25,41 This bacterium is less restricted concerning both electron donors andacceptors

Several dechlorinating strains belonging to the genus Desulfitobacterium were isolated from different sources The strictly anaerobic D dehalogenans, able to grow

by the reductive dechlorination of chlorinated phenolic compounds was isolated

recently Another Desulfitobacterium, strain PCE1, was isolated from polluted soil

and is reported to couple the reduction of both chlorinated phenols and chlorinatedethenes to growth.43 This strain dechlorinates PCE only to TCE, whereas other knownhalorespiring microorganisms dechlorinate PCE further The same authors also

described a Desulfitobacterium sp strain TCE1, which is able to use several electron donors for the reduction of PCE to cis-DCE.43,44 Another researcher has described

a Desulfitobacterium sp (strain PCE-S) that converts PCE to cis-DCE.45 All isolated

Desulfitobacterium strains are able to use a number of different electron donors and

acceptors for growth The nature and origin of the dechlorinating enzymes in theseorganisms are still unknown

A recent study described two facultative aerobic bacteria, strain MS-1 and the

closely related Enterobacter agglomerans biogroup 5, which can reductively logenate PCE to cis-DCE under anaerobic conditions.46 It is not clear yet whether

deha-strain MS-1 and E agglomerans biogroup 5 are actually halorespiring organisms Recently, an anaerobic bacterium, Desulfuromonas chloroethenica (strain TT4B), has been isolated which can not only reductively dechlorinate PCE to cis-

DCE with acetate as an electron donor, but also can reduce Fe (III) and polysulfide.These are unique features for PCE-dehalogenating organisms.47

All the above-mentioned organisms are only able to couple growth to the partial

reduction of PCE or TCE An exception is Dehalococcoides ethenogenes strain 195

that couples growth to rapid dehalogenation of PCE to VC, followed by a substantiallyslower reduction to ethene.37 Growth of this bacterium is restricted to the presence ofhydrogen, which is the only electron donor supporting the dechlorination reactions.Dechlorination was only sustained by using hydrogen, acetate, vitamin B12, anaerobicdigester sludge supernatant, and cell extracts from mixed cultures in the medium

Figure 4.7 Range of hydrogen concentrations for the different anaerobic metabolic pathways

(after Wiedermeier et al., 1999).

Chloroethene Reductive Dehalogenases: The biochemistry of PCE

dehalores-piration has been studied with enzymes that were purified from a reductively rinating pure culture or enrichment, which is able to couple dechlorination to energyconservation (halorespiration) PCE respiration has been studied most extensively

dechlo-in Dehalobacter restrictus42,49 and Dehalospirillum multivorans.26,41,45 In general, aPCE respiration chain should contain an electron-donating enzyme, electron carriers,

and a reductive dehalogenase as terminal reductase Studies with D multivorans and Desulfitobacterium strain PCE-S indicate that a proton gradient or a membrane

potential may also be essential for chloroethene respiration because several phores have been found to inhibit dechlorination in whole cell suspensions.45,50 The

iono-nature of the electron-donating enzyme depends on the electron donor In D tus, which uses hydrogen as electron donor for PCE respiration, hydrogenase activity

restric-has been localized on the membrane, facing the outside.49 D multivorans and Desulfitobacterium strain PCE-S are able to use several electron donors for dechlo-

rination, and different electron-donating activities have been found.45,50 The electronsthus generated are transported to the dehalogenase via electron carriers such asquinones and cytochromes It was demonstrated that menaquinone is involved as

electron carrier for PCE respiration in D restrictus,49 but not in D multivorans.45,50

Cytochrome b is present in both organisms, but its involvement in PCE respirationhas not been established

In contrast to the well studied PCE and TCE dechlorination, little is known aboutthe mechanism of DCE and VC dechlorination It was found that the enzymescatalyzing VC dechlorination in an enrichment culture are membrane bound and, incontrast to the known PCE reductase, cobalamin independent.51 It remains unclearwhether this enrichment is able to use VC as terminal electron acceptor Recently,

an enzyme has been obtained from an enrichment containing D ethenogenes that catalyzes the dechlorination of TCE to cis-DCE, VC, and ethane This cobalamin-

containing TCE-reductive dehalogenase is membrane bound and dechlorinates itssubstrates at similar rates, as have been reported for the PCE dehalogenases Moreresearch is needed to know what determines the difference in substrate specificity

of the cobalt-containing reductive dehalogenases

4.2.1.4 Electron Donors

The selection of an appropriate electron donor may be the most important designparameter for developing a healthy population of dechlorinating microorganismsduring implementation of an IRZ for enhanced reductive dechlorination Recentstudies have indicated a prominent role for molecular hydrogen (H2) in the reductivedechlorination of chloroethenes.34,39,48 Most known dechlorinators can use H2 as anelectron donor; some can use only H2 Because more complex electron donors arebroken down into metabolites and residual pools of H2 by other members of themicrobial community, they may also be used to support reductive dechlorination.From the small but growing pool of knowledge about dechlorinating organisms,

it thus appears that H2 may serve an important role in reductive dechlorination ofPCE in many environments The author recently has observed the quick or directtransformation of PCE or TCE to ethene under very reducing conditions leading to

Figure 4.8 Conceptual diagram of microbial activity to derive energy for growth and

multi-plication.

Figure 4.9 Distribution of electrons to generation and cell synthesis during the breakdown

of organic electron donors.

H electrons

First Intermediate Electron Donor Substrate

Target Electron Donor Substrate Carbon Substrate

Cell Synthesis Reactions

Electron Acceptor Substrate Energy Generation Reactions

fs electrons fe electrons

fs electrons fe electrons

fs electrons fe electrons

speculations of the probable effect of high H2 concentrations or reductive nation In natural systems, including contaminated aquifers, most H2 becomes avail-able to hydrogenotrophic microorganisms through the fermentation of more complexsubstrates by other members of the microbial consortium The dechlorinators mustthen compete with other organisms, such as methanogens and sulfate-reducingbacteria, for the evolved H2 (Figure 4.8) Figure 4.9 also describes the distribution

dechlori-of electrons during the microbial breakdown dechlori-of organic electron donor substrates.During studies in which ethanol or lactate was used to stimulate dechlorination inmixed anaerobic enrichment culture, both active dechlorination and methanogenesis

at high H2 levels was observed; however, when H2 levels fell, dechlorination tinued, albeit slowly, while methanogenesis ceased entirely It was speculated thatthe addition of electron donors fermented only under low H2 partial pressures mightgive selective advantage to dechlorinators over methanogens

con-One school of thought in the past was that the rate and quantity of H2 madeavailable to a degrading consortium must be carefully engineered to limit competi-tion for hydrogen from other microbial groups, such as methanogens and sulfate-reducers Competition for H2 by methanogens is a common cause of dechlorinationfailure in laboratory studies As the methanogen population increases, the portion

of reducing equivalents used for dechlorination quickly drops and methane tion increases.17,18,36 Speculation was that the use of slowly degrading nonmethano-genic substrates would help prevent this Recent thinking on this issue is evolving

produc-to be different and is discussed later

Many different compounds may serve as electron donors for the reductive rination of chlorinated solvents (Table 4.4) Several researchers suggest that themicrobial reductive dechlorination of chlorinated ethenes depends on the presence

dechlo-of molecular hydrogen as the actual electron donor, either directly available orproduced from other substrates by fermentation.32,34,55,56 Although this statementapplies to many studies, in several cases it does not hold Acetate, from which usually

no hydrogen is produced during anaerobic metabolism, has been shown to supportreductive dechlorination of chlorinated ethenes both in microcosms and environ-mental samples5,7,26,58 and in pure culture.47

Until recently, most research activities concerning the anaerobic degradation ofchlorinated compounds focused on methanogenic systems Such systems typicallyinvolve the introduction of a fermentable organic compound, such as acetate, lactate,hexoses (present in molasses) or even a co-contaminant such as toluene or phenol,which is fermented to produce hydrogen, among other things It is now clear thatthese systems probably contained at least two distinct strains of bacteria One strainfermented the organic carbon to produce hydrogen, and another utilized the hydrogen

as an electron donor for dehalorespiration.15 Only in the last two or three years haveresearchers finally recognized the role of hydrogen as the electron donor in thereductive dechlorination process

4.2.1.4.1 Production of H 2 by Fermentation

The production of H2 by different microorganisms is intimately linked with theirrespective energy metabolisms The production of H2 is one of the specific

mechanisms to dispose excess electrons through the activity of hydrogenase present

in H2 producing microorganisms.59 All hydrogen producing microorganisms can becategorized into four groups:60

• Hetertrophic facultative anaerobes that contain cytochromes and lyse formate to produce H2

• Desulfovibrio desulfuricans, which is the only strict anaerobe in this group with

a cytochrome system

• Photosynthetic bacteria with light-dependent evolution of H2 from reduced NADH

• Strict anaerobic heterotrophs that do not contain a cytochrome system (clostridia, micrococci, methanobacteria, etc.)

Production of H2 by obligate anaerobic microorganisms has optimum ometry (1:4, with glucose as substrate) compared with facultative anaerobes (1:2),although the latter process is comparatively simpler than the former.60

stoichi-Under natural conditions, fermentation is the process that generates the hydrogenused in reductive dechlorination In the absence of externally available electronacceptors, many organisms perform internally balanced (different portions of thesame substrate are oxidized and reduced) oxidation-reduction reactions of organic

compounds with the release of energy; this process is called fermentation Since

only partial oxidation of the carbon atoms of the organic compound occurs, tation yields substantially less energy per unit of substrate compared to oxidationreactions (Oxidation reactions are those in which external electron acceptors par-ticipate in the reaction) For instance, the fermentation of glucose to ethanol and

fermen-CO2 has a theoretical energy yield of –57 k cal/mole, enough to produce about

Table 4.4 Electron Donors That Have Been Used to Enhance

Reductive Dechlorination and Relative Costs per lb of PCE 51–53

Electron Donor

Bulk Price

$/lb

$/lb of PCE Soluble (Fast Release) Donors

6 ATP However, only 2 ATPs are produced, which implies that the organism operates

at considerably less than maximum efficiency.59

In any fermentation reaction, there must be a balance between oxidation andreduction In a number of these reactions, electron balance is maintained by theproduction of molecular hydrogen, H2 In H2 production, protons (H+) of the medium,derived from water, serve as electron acceptor The energetics of hydrogen produc-tion are actually somewhat unfavorable, so that most fermentative organisms onlyproduce a relatively small amount of hydrogen along with other fermentation prod-ucts Fermentation reactions that have pyruvate as an intermediate product have thepotential of producing more H2 Conversion of pyruvate to acetyl-CoA is an oxidationprocess and the excess electrons generated must either be used to make a morereduced end product, or can be used in the production of H2

Fermentation by bacteria can also be important in controlling the biogeochemicalenvironment of anaerobic aquifers Bacterial fermentation can be divided into twocategories:14,58

Primary fermentation is the fermentation of primary substrates such as sugars, amino

acids, and lipids to yield acetate, formate, CO2, and H2, but also yields ethanol, lactate, succinate, propionate, and butyrate While primary fermentation often yields

H2, production of H2 is not required for these reactions to occur.

Secondary fermentation or coupled fermentation is the fermentation of primary

fermentation products such as ethanol, lactate, succinate, propionate, and butyrate

to yield acetate, formate, H2, and CO2 Bacteria that carry out these reactions are

called obligate proton reducers because the reactions must produce hydrogen in

order to balance the oxidation of the carbon substrates These secondary tion reactions are energetically favorable only if hydrogen concentrations are very low (10 –2 to 10 –4 atm or 8000 to 80 nM dissolved hydrogen, depending on the fermentation substrate) Thus these fermentation reactions occur only when the produced hydrogen is utilized by other bacteria, such as methanogens that convert

fermenta-H2 and CO2 into CH4 and H2O The process by which hydrogen is produced by one

strain of bacteria and utilized by another is called interspecies hydrogen transfer.

It should be noted that the terminal products of anaerobic decomposition, CH4, and

CO2, respectively, are the most reduced and the most oxidized carbon compounds.There are a number of compounds besides the ones listed in Table 4.4 that can

be fermented to produce hydrogen (Figure 4.10) While anaerobic degradation ofBTEX compounds has been confirmed for a long time, there is still some controversy

as to whether aromatic compounds (without any oxygen in the molecule) such asthe BTEX compounds can be completely mineralized to CO2 without alternateelectron acceptors coupled solely by fermentation with methanogenesis

Based on a number of field observations of the presence of methane, it is wellknown that fermentation occurs at almost all sites where BTEX compounds are present

in groundwater.14,53 Since methane production requires fermentation products as anogenic substrates, the presence of methane is clear evidence that fermentation isoccurring Metabolism of BTEX compounds to produce hydrogen probably requiresthe involvement of several strains of bacteria One possible mechanism is a series ofreactions, in which other electron acceptors are used by nonfermenters to break downthe aromatics to simpler compounds that can be used by the fermenters

meth-4.2.1.4.2 Competition for H 2

In environments where hydrogen is the most important electron donor for rination of chlorinated solvents, competition for the uptake of hydrogen betweendifferent types of microorganisms, such as methanogenic, homoacetogenic, sulfi-dogenic, and dechlorinating bacteria, becomes important In several studies it has beenshown that dechlorinating organisms have a higher affinity for molecular hydrogenthan methanogens.27,35,55 This indicates that the dechlorinating organisms are able tosurvive at lower hydrogen levels, but will possibly be outcompeted by other microor-ganisms when elevated hydrogen levels are present These studies suggest that a moreeffective dechlorination may be achieved by using an electron donor that generateslow hydrogen concentrations during its fermentation, such as propionate or butyrate.The speculation is that this would then create more favorable conditions for dechlo-rinating bacteria than for hydrogen-consuming methanogens.27,35

dechlo-Reductive dechlorination of PCE requires the addition of two electrons for eachchlorine removed; for three of the seven recently identified dechlorinating organisms,

H2 is one of the substrates (and in some cases, the only one) that can serve as the

Figure 4.10 Steps in the process of biodegradation of PCE by reductive dechlorination As

shown, biodegradable organic matter is required as an electron donor to initiate the process Different types of microbes are involved at each stage The bottom step shows that PCE must compete for electrons with sulfate, iron, and carbon dioxide, meaning that a large amount of organic electron donors may be needed

to supply enough electrons Note: CDCE = cis-dichloroethene Source: after

McCarty, 1997.

direct electron donor Dehalobacter restrictus is another direct dechlorinator that

uses only H2 as an electron donor, but dechlorinates PCE only to

cis-1,2-dichloro-ethene (cisDCE).46,47 Dehalospirillum multivorans also dechlorinates PCE to

cis-DCE using H2, but has a much more widely varied biochemical repertoire: it isadditionally able to use various organic substrates such as pyruvate, lactate, ethanol,formate, and glycerol as electron donors.7,37,44 Other PCE-dechlorinating organismshave been isolated that do not use H2.34,61 It was later determined that the half-velocity constant with respect to H2 for this dechlorinator was one-tenth that of themethanogenic organisms in the culture The threshold H2 level for dechlorinationwas also correspondingly lower than values typically reported for methanogens.Though confirmed thus far with only this one dechlorinator, there are thermodynamicreasons (i.e., the relatively high free energy available from dechlorination) to suspectthat the threshold for H2 use by dechlorinators may generally be lower than that forhydrogenotrophic methanogens.15,27 This suggests a strategy for selective enhance-ment of dechlorination — managing H2 delivery so as to impart a competitiveadvantage to dechlorinators

Numerous microcosm and site studies have shown successful stimulation ofdechlorination with substrates such as methanol, ethanol, lactate, butyrate, andbenzoate.3,32,36,62,57 However, understanding the fate of the electron donors and that

of the H2 evolved from their degradation, as well as the extent to which their reducingequivalents are channeled to desirable dechlorination or competing H2 sinks, hasimportant implications for determining how best to effectively stimulate latent

dechlorinating activity for in situ enhanced reductive dechlorination in an IRZ.

Leading to a new school of thought, recent studies have suggested that the type

of substrates and the rate of fermentation may not have an impact on reductivedechlorination One study showed the ability of four fermentable substrates to sustainPCE dechlorination long-term (i.e., approximately four months).35 The choice oforganic substrates was based upon their rates of fermentation and the H2 partialpressures that could be developed and maintained Despite the difference in theresulting H2 partial pressures (ranging approximately 1 ¥ 10–5 to 3 ¥ 10–3 atm), nolong-term effect on dechlorination was observed This result may indicate either thatlow H2 partial pressures were not required to maintain a competitive dechlorinatingcommunity or that several isolated PCE respiring bacteria do not utilize H2 as anelectron donor.43,46 H2 was not the source of PCE-reducing equivalents in all systemstested Other laboratory and field studies have also suggested that the steady stateconcentration of hydrogen is controlled by the type of bacteria utilizing the hydrogenand is almost completely independent of the rate of hydrogen production

As discussed earlier, when hydrogen is produced by fermentative organisms, H2

is rapidly consumed by other bacteria This utilization of H2 by nonfermenters is

known as interspecies hydrogen transfer and is essential for fermentation reactions

to proceed.59 Note, for example, that a glucose fermenter is unable to utilize glucose

by itself so that both the glucose fermenter and the methanogen benefit from thissymbiotic relationship

Although H2 is a waste product of fermentation, it is a highly reduced molecule,which in turn makes it an excellent, high-energy electron donor In this symbioticrelationship, the hydrogen utilizing bacteria gain a high energy electron donor, while,

for the fermenters, the removal of hydrogen allows continuous fermentation to befavorable energetically.

In addition to methanogens, a wide variety of bacteria can utilize hydrogen as

an electron donor: denitrifiers, Fe (III) reducers, sulfate reducers and halorespirators

As discussed earlier, for dechlorination to take place, halorespirators must fully compete against all these hydrogen utilizers

success-It was suggested that the competition is mainly controlled by the Monod saturation constant Ks (H2) (the concentration at which a specific bacterial strain canutilize hydrogen at half the maximum utilization rate).14,27,56 The measured value of

half-Ks (H2) for halorespirators was 100 nM and for methanogens 1000 nM.14 This led

to the suggestion that halorespirators would compete successfully for H2 only at lowconcentrations

However, a more detailed analysis of halorespiration kinetics and competitionfor hydrogen based on the Monod kinetic model was performed recently.14,27 Usingthis model, the ability of hydrogen-utilizing bacteria to compete for hydrogen caneasily be predicted from substrate concentration and two properties of the bacteria,

mmax (maximum specific growth rate), and Ks Table 4.5 lists these parameters forthe various hydrogen-utilizing bacteria

Table 4.5 illustrates that, from the mmax term, halorespirators will outcompetemethanogens and sulfate reducers at any hydrogen concentration (since at highsubstrate concentration growth rate m ª mmax and at low substrate concentration m ª(mmax.S)/Ks) However, denitrifiers will probably outcompete halorespirators undermost conditions as their maximum specific growth rate is approximately three timesfaster than halorespirators’ Based on these detailed analyses and the synthesis ofwide ranging data from field observations, the following probable sequence takesplace at most sites undergoing halorespiration reactions:14,27

• Aerobic bacteria consume nonchlorinated organic substrates until the oxygen is depleted ; to implement enhanced reductive dechlorination, oxygen depletion is forced intentionally in an IRZ.

• Similarly, denitrifying bacteria will consume nonchlorinated organic substrates until the nitrate is exhausted ; nitrate depletion will be forced for enhanced reduc- tive dechlorination.

• Iron reducing bacteria consume nonchlorinated organic substrates until the able Fe (III) is expended.

avail-Table 4.5 Maximum SpeciÞc Growth Rate

(mmmmmax ) and Half Saturation CoefÞcient (K s ) for Various H 2 - Utilizing Bacteria (ModiÞed from Wiedermeier et al., 1999) Bacterial Strain mmmmmax (hr –1 ) K s (mg/L)

Halorespirator 0.019950 0.0002 DenitriÞer 0.058080 — Sulfate Reducer 0.003936 — Methanogen 0.003792 0.0019

• Fermentation processes consume nonchlorinated organic substrates and generate hydrogen; dechlorinating bacteria consume hydrogen for dechlorination, while sulfate-reducing bacteria consume nonchlorinated organic substrates and metha- nogens consume hydrogen to generate methane.

Recently researchers have found that steady state H2 concentrations in the fieldare controlled by the type of bacteria utilizing the hydrogen.14 For example, undernitrate reducing concentrations, steady state H2 concentrations were less than 0.05

nM, under Fe (III) reducing conditions they were less than 0.2 to 0.8 nM, undersulfate reducing conditions they were 1 to 4 nM, and under methanogenic conditionsthey were 5 to 14 nM (Figure 4.7) Thus it is clear that an increased rate of hydrogenproduction will result in increased halorespiration without affecting the competitionbetween various bacteria for the available hydrogen (Figure 4.11) Attempting tostimulate halorespiration with poor fermentation substrates, as has been suggested

in the past, may unnecessarily limit the amount of dechlorination taking place

It is clear from this this discussion that, during field scale enhanced reductive

dechlorination at contaminated sites, the oxidative poise contributed by dissolved

oxygen, nitrate, Fe (III), Mn (IV) and sulfate has to be depleted as quickly as possible

to achieve efficient steady state reductive dechlorination reactions.53 Thus it is dent to use the cheapest fermentable substrate available (see Table 4.4) to overcomethe oxidative poise (Figure 4.12)

pru-4.2.1.5 Mixture of Compounds on Kinetics

Because few studies have systematically investigated the effect of multiple taminants, and not all biochemical mediators of chlorinated aliphatic transformation

con-Figure 4.11 Effect of oxidative poise on H 2 concentrations during Þeld scale implementation

of enhanced reductive dechlorination systems, if methanogenic conditions can

be achieved and maintained (adapted from Weidermeier et al., 1999).

in methanogenic cultures are known, it is difficult to predict how any two chlorinatedaliphatics may influence each other’s transformation Several possible interactionscan be hypothesized wherein chlorinated aliphatic biotransformation rates mayincrease, decrease, or remain unaffected Increased rates may be observed throughinduction of transformation pathways or growth on one chlorinated aliphatic, such

as dichloromethane (DCM), which then supports transformation of other aliphaticspresent Decreased rates may result from competitive inhibition, competition forreducing equivalents, or synergistic toxicity effects There may be no observableeffect if transformation processes are independent, or concentrations of chlorinatedaliphatics are low

Mixtures of chlorinated aliphatics often result from reductive dechlorination of

a single parent compound; discerning the effect of mixtures on the transformation

of individual compounds is difficult The most frequently cited example is thesequential reductive dechlorination of polychlorinated ethenes (e.g., perchloroet-hene, trichloroethene) to ethene Rates of reductive dechlorination in this series tend

to decrease as the number of chlorine substituents decrease, so compounds such asvinyl chloride and dichloroethene often accumulate.64 Since all chlorinated ethenesare transformed by the same mechanism, it is conceivable that competitive interac-tions also influence the distribution of products, although it has not been systemat-ically investigated

The sequential reductive dechlorination of 3,5-dichlorobenzoate to zoate to benzoate has been reported.63 In this case, reduction of the 3-chlorobenzoatedid not proceed until the 3,5-dichlorobenzoate was completely transformed Thiscould not be explained by competing reaction rates, but was successfully described

3-chloroben-Figure 4.12 Pictorial depiction of oxidative poise to be overcome during implementation of

an IRZ for engineered anaerobic systems.

with a competitive inhibition model Similar behavior for the reduction of 3,5-dichlorobenzoate to 4-amino-3-chlorobenzoate also was observed.

4-amino-It is important to note that competition may be significant during degradation ofchlorinated methanes and ethanes as they can be transformed by reductive dechlo-rination and other mechanisms.65,66 Methylene chloride (DCM) can be oxidized to

CO2 or converted to acetate while serving as a growth substrate.7 Chloroform (CF)and carton tetrachloride (CT) can be hydrolyzed.66 1,1,1 Trichloroethane (TCA) can

be hydrolyzed or undergo dechlorination.19 Hydrolysis processes for these pounds are of particular interest since the products are reactive in some casesdecomposing to harmless end products or reacting with cell material — whereashydrolysis of CF and TCA yields strong nucleophiles (phosgene and acid halide),which may be toxic to the cell Subsequent hydrolysis of these intermediates yields

com-CO2 and acetate; both are subject to complete metabolism in anaerobic culture.The interaction of dechlorination amongst CM, CF, and TCA in methanogenicacetate-enrichment cultures was investigated in another study.67 Complex interac-tions occurred when mixtures of these chlorinated aliphatics were present: TCAtransformation rates were reduced by the presence of DCM or CF, DCM transfor-mation was enhanced by CF and TCA, and CF transformation rates increased ordecreased depending on the mixture Acetate utilization varied depending on themixture fed, complicating the interpretation of results Where acetate utilization wasinhibited, biomass concentrations decreased and steady-state conditions were notachieved during the study

In all cases where CF and TCA were fed together, the rate of their transformationswas lower than when they were fed individually The decrease in the rate of trans-formation increases with the concentration of either compound, CF being moreinhibitory than TCA on mg/L basis Results were described by a competitive inhi-bition model, which was more predictive for the effect of TCA on CF transformationthan the effect of CF on TCA transformation.67

Competitive interactions between substrates can introduce significant limitations

to bioremediation processes Studies investigating the cometabolic transformation

of chlorinated ethenes by methanotrophs,68,69 nitrifiers,70 and phenol-inducedaerobes71 have identified many problems at full scale that result from similar inter-actions The decrease of biodegradation rates that results from competitive inhibitionmay limit the applicability of bioremediation processes This is particularly true with

CF and TCA, whose growth and biotransformation rates decrease rapidly as theirconcentrations increase

The effect of multiple substrates on the kinetics of biotransformation reactionshas not been extensively studied The results reported in the literature demonstratethat a range of effects may be observed even with a mixture of compounds that arestructurally similar No unifying model can be constructed on the basis of theavailable information These studies also demonstrate the difficulty of assessing theinteractions that occur when the compounds of interest may have dissimilar degra-dation pathways

Unraveling the complex interactions of compounds such as those often tered in contaminated groundwaters will require further research It is clear fromthese studies that certain combinations of compounds will lead to decreased rates

encoun-of biotransformation This is certainly true for the combination encoun-of CF and TCA Forengineers hoping to implement anaerobic bioremediation, this is important informa-tion in the decision making and design processes If these interactions are notconsidered, any existing model will significantly underestimate the time requiredfor remediation Additional studies should attempt to enhance our fundamentalunderstanding of these interactions, and identify other mixtures commonly foundthat significantly affect biodegradation rates Such knowledge will minimize theapplication of bioremediation to sites where its efficacy would be limited.

4.2.1.6 Temperature Effects

Many studies have reported anaerobic reductive dechlorination of chlorinatedsolvents to occur within a mesophilic temperature range (20 to 37∞C) However,several authors have shown dechlorination of these compounds at more ambientgroundwater temperatures between 10 and 20∞C,8,26,55 indicating the applicability ofreductive dechlorination in many groundwater environments

Microbial dechlorination has also been demonstrated under thermophilic tions.26 An enrichment culture, obtained from polluted harbor sediment, rapidly dechlo-

condi-rinated PCE to cis-DCE at an optimum temperature of 65∞C Fumarate appeared to

be the best electron donor A large number of samples from high-temperature anaerobicenvironments has been investigated for the presence of dechlorinating microorganisms

as well, but no dechlorinating activity has been found

4.2.1.7 Anaerobic Oxidation

Microorganisms can anaerobically mineralize VC and DCE in the presence of

a complex, bioavailable electron acceptor such as Fe (III) - EDTA.72,73 Studies havefocused on the possibility of oxidation of VC and DCE when they are used as aprimary growth substrate under anaerobic environments.72,73,74 These results show

VC and DCE mineralization under methanogenic and iron reducing conditions inanaerobic streambed sediments without the accumulation of ethene or ethane andbuildup of carbon dioxide.14 Decreases of VC and DCE concentrations correspondedquantitatively to the production of carbon dioxide

4.2.1.8 Electron Acceptors and Nutrients

Nutrients: In addition to proper electron donor selection, nutrient availability

may be a critical factor in maintaining a healthy dechlorinating consortium In oneinstance, attempts to isolate a microbial species responsible for dechlorination led

to the discovery that nutritional factors probably had been supplied by other sortium members Highly enriched dechlorinating cultures required the addition ofvitamin B12 and sludge supernatant to sustain dechlorination.38 Speculation existsthat acetogens may supply the unknown nutritional factors required by the dechlo-rinating organism(s).34 Fortunately, in situ applications support a variety of microbial

con-species This microbial diversity, combined with the addition of nutritional ments, should support a healthy dechlorinating microbial community

supple-Alternative Electron Acceptors: Microbial dechlorination of chlorinated

ali-phatic hydrocarbons has been found to occur at low REDOX potentials, mainlyunder methanogenic conditions, although dechlorination under sulfate-reducing con-ditions has also been reported.26

A recent study found that, in reactions involving polychlorinated methanes andorganic reductants exhibiting mercapto groups, an alternative initial reaction stepmay be a halophilic dissociative two-electron transfer.75 The proposed reactionmechanisms(s) involving R-S-H or R-S-S-R groups in the complete dechlorination

of polychlorinated methanes may be helpful in the (re)interpretation of microbiallymediated dechlorination reactions of such compounds

Indirect microbial reductive dechlorination of PCE has also been observed underiron-reducing conditions due to magnetite formation by iron-reducing bacteria Mag-netite can chemically reduce PCE to lower chlorinated ethenes.76 Besides reduction

of chlorinated solvents under iron-reducing conditions, oxidation of cis-DCE and

VC has been reported to occur under conditions where Fe (III) is the final electronacceptor.73 The same authors also reported the oxidation of DCE and VC in meth-anogenic, organic compound-rich bed sediment, indicating that the oxidation of thesecompounds is coupled to the reduction of humic acid compounds.74

As discussed in the previous section, the successful application of enhancedreductive dechlorination depends upon the depletion of electron-accepting chemicalspecies The most environmentally relevant species include O2, NO3– , Mn (IV), Fe(III), and SO4–2 When evaluating a site for enhanced reductive dechlorination appli-cability, one must investigate the relative abundance of these compounds in both thegroundwater and the aquifer solids Although aqueous-phase acceptors such as O2and NO3– take primary consideration, it is imperative that aquifer solids be charac-terized because they can serve as a reservoir of relatively insoluble electron-acceptingspecies such as Fe (OH)3 or CaSO4 Once the electron-accepting species have beenquantified, the amount of electron donor required to deplete them can be estimated

by evaluating the stoichiometric relationship between the selected electron donorand each electron acceptor present on site (Figure 4.12) Higher levels of electronacceptor increase the oxidative poise and thus require more electron donor, thereforeraising treatment costs A series of generic reactions is given in Table 4.6 to illustratesome of the possible reactants and products

Once an electron donor has been selected and electron acceptors have beencharacterized, the stoichiometric relationship between them can be determined Anequation for each electron acceptor present at the site must be balanced using the

Table 4.6 Possible Reactants and Products of SpeciÞc Terminal

Electron-Accepting Processes

Electron donor + O 2 Æ CO 2 + H 2 O Aerobic respiration

Electron donor + NO 3 Æ CO 2 + H 2 O + N 2 DenitriÞcation

Electron donor + Mn 4+ Æ Mn 2+ + CO 2 + H 2 O Manganese reduction

Electron donor + Fe 3+ Æ Fe 2+ + CO 2 + H 2 O Iron reduction

Electron donor + SO 4–2 Æ H 2 S + CO 2 + H 2 O Sulfate reduction

selected electron donor Once balanced, the molar ratio of donor to acceptor can bedetermined from these equations.

These molar ratios represent an ideal case where the entire electron donor dosage

is used to reduce the electron acceptor present in the treatment zone When calculatingthe actual electron donor dosage, a safety factor must be incorporated to account foruncharacterized electron sinks and the advective transport of electron acceptors intothe treatment zone Site-specific conditions such as groundwater flow rate, surroundingelectron acceptor concentrations, depth to the water table, rainfall frequency, and level

of site characterization will influence the selection of the safety factor

Because treatment alternatives and budgetary constraints are different for each site,

no rule of thumb exists for screening sites based on electron acceptor concentrations.The required mass of electron donor should be estimated so its cost can be calculated.Afterwards, a site-specific, cost-benefit analysis must be undertaken to determine ifthe site is a good candidate for enhanced reductive dechlorination (ERD) application

4.2.1.9 Field Implementation of IRZ for Enhanced Reductive

an electron donor and supplemental nutrients to the contaminated groundwater zone

in order to provide the optimum biogeochemical environment conducive for tive dechlorination.1

reduc-The author’s wide experience in this technology is mostly based on the solubleelectron donor, such as molasses that must be added semicontinuously or in batchinjections1 in order to sustain the microbial activity for the fermentation reactions.Recently, cheese whey has been used as a slow release substrate in less permeablegeologic environments Preference of molasses and cheese whey is based purely oneconomics as illustrated in Figure 4.12 and Table 4.4.1

The geologic and hydrogeologic setting in which an IRZ system is installedgoverns its successful application IRZ systems rely on the delivery of dissolvedreagents, such as dilute molasses, throughout a contaminant plume; administeringdelivery of these amendments through both the vertical and horizontal extent ofcontaminant plumes sounds deceptively easy, but requires careful engineering and

a knowledge of the geologic parameters affecting groundwater flow and transport.Different configurations, in plan view and cross sections, used for IRZ systemdesigns are shown in Figures 4.13a and b, and 4.14a and b Creative engineeringconsiderations have to be taken into account to accommodate the requirements of asmaller plume vs a larger plume and a shallower plume vs a deeper plume Theultimate objective of the IRZ system design engineer should be to deliver the electrondonor as fast as possible and to create a uniformly mixed reactive zone in thesubsurface, as well as to maintain the optimum biogeochemical conditions forenhanced reductive dechlorination to occur

The total treatment time for an IRZ will encompass the time it takes to overcomethe oxidative poise (deplete available electron acceptors), acclimatize and stimulate

a healthy population of dechlorinating microorganisms, and allow the dechlorinationreactions to proceed to conclusion Site-specific conditions will obviously influencethe total time required for treatment; for instance, anaerobic and particularly meth-anogenic sites exhibiting a significant level of natural dechlorination will requireconsiderably less time than sites with aerobic groundwater and no evidence ofdechlorination Other factors incluencing the time required to treat a site includeaquifers with low hydraulic conductivities, which will require more time for delivery

of substrate throughout the subsurface, and the presence of DNAPL, which wouldrequire considerably longer treatment times due to the rate limitation imposed bydissolution of the contaminant

The time required for oxidative poise depletion depends on the electron donorsupply and utilization rate, on initial electron acceptor concentrations, and the rate

at which they are replenished by groundwater flow and recharge events The largenumber of variables affecting electron acceptor depletion makes it difficult to predictthe time lag; field data indicate that this lag could be anywhere from ten days toabout three months

When considering the time required to implement an enhanced reductive rination IRZ system, one should include a minimum of six months to perform a

dechlo-Figure 4.13a Staggered plume-wide injection grid for an IRZ system for remediation of a small

plume.

Figure 4.13b Source area staggered grid and containment curtains at mid-plume and

down-gradient locations for a large-size plume.

Injection Points

Flow Direction

Source Zone Grid

Containment Curtains Groundwater

Flow Direction

field pilot test to obtain the design parameters to design and scale up a full scalesystem This six month testing time frame assumes one to two months for creation

of a reducing environment after the depletion of the oxidative poise and three tofour months of evaluating treatment data The actual time required for a pilot testmay exceed six months and will depend on hydrogeologic conditions and whetherthe site was already reduced with partial declorination initially

The assessment of a particular site for IRZ application should include thedevelopment of a contaminant profile, a hydrogeological profile, and a bio-geochemical profile

An inventory of contaminants, their concentrations, and distribution throughoutthe plume will be the first step in assessing the feasibility of an IRZ The presence,relative concentration, and distribution of daughter products is particularly importantwhen assessing sites for enhanced dechlorination potential Co-contaminant impactsmay be either beneficial or detrimental, so it is important to assess before the onset

of a pilot study

The success of an IRZ application primarily depends upon the effective deliveryand distribution of the electron donor and nutrients throughout the contaminatedsubsurface Hence, the ability to control the movement of the injected reagents isimperative at sites with a hydraulic conductivity less than or equal to 10–5 cm/sec.These sites require the addition of a slow releasing electron donor Faster geologic

Figure 4.14a Injection well clusters for depths between 40 feet and 100 feet of saturated zone

containment.

Reactive Zones Dilute Molasses

settings require the addition of a soluble and fast releasing electron donor such asmolasses Figure 4.15 illustrates the need to inject a soluble electron donor at higherconcentrations to achieve a reasonable size reactive zone from each injection point

to achieve the scale up of the full scale system cost effectively

Biogeochemistry influences the potential for stimulating and maintaining bially catalyzed reductive dechlorination These microorganisms require highlyreducing conditions reflected by low REDOX potential measurements and the pro-duction of hydrogen sulfide and methane gas Additional biogeochemical parameterssuch as pH, alkalinity, temperature, and dissolved organic carbon can also affect thehealth and stability of dechlorinating microorganisms

Typically a pilot test or smaller-scale field test follows the initial screeningprocess The two primary issues to be addressed during the field testing phase arethe provision of properly placed observation wells and allowing for sufficient time

to demonstrate the success of ERD The amount of time it takes to see positive

Figure 4.14b In well mixing systems with submersible pumps for creating deeper IRZ systems.

results of the IRZ implementation is related to many factors, including groundwatervelocity, the time required for introduced reagents to overcome the ambient REDOXconditions in groundwater, and the locations of observation wells in relation to theinjection area It is prudent to evaluate geochemistry and achievable degradationrates from data collected from wells located at least several months of travel timeapart Consequently, the minimum duration of a typical pilot study is six monthswith the flexibility to extend the testing based on data collection and site-specificcosts Field tests that are shorter in duration, or are applied in too small an area,often do not provide information that is applicable to a successful or economicallarge-scale implementation This minimum period of time should be sufficient toovercome the initial aquifer REDOX conditions and allow for the degradation ofconstituents to a degree that will be observed in the pilot test Some observationwells should also be placed within one to two months’ groundwater travel time fromthe injection area This timing/placement should allow for early observations of theIRZ development, and allow for modification of the reagent injection program(strength and frequency) early enough in the planned test duration.

Reagent Delivery: The successful application of an IRZ to remediate chlorinated

solvents in groundwater first and foremost relies on the timely and consistent delivery

of the organic carbon reagent to the treatment zone

The author’s experience is primarily based on injecting a dissolvable sucrosesolution (molasses) as a reducing reagent This serves as a cost effective reagent(0.20– $0.30/pound) that can aggressively alter the REDOX state of groundwater(oxidative poise) in a short time period Other reagents, or electron donor substrates,such as edible oils and semisolid forms of lactate (such as Regenesis’ HRC®) willrely more on dissolution and diffusion for delivery On a unit cost basis, these donors

Figure 4.15 Effects of a soluble electron donor and a slow release electron donor in an IRZ

where the hydraulic conductivity is greater than 10 –5 cm/sec.

B - Distance of the Reactive Zone for the Soluble Electron Donor

Slow Releasing and Slower Degrading Electron Donor Such as Soybean Oil

Minimum Concentration Required for Adequate Production of H2

Distance From Injection Point

are more expensive However, the application of a slow diffusing reagent may bemore efficient in a highly reducing, slower groundwater velocity environment Theproper reagents should be selected based on the site hydrogeology and desiredtreatment time frame Cheese whey has been used by the author as the slow releasesubstrate in less permeable, slow moving groundwater environments.1

Based on the implementation of IRZs for the application of ERD to date, reagentdelivery becomes most complicated in low permeability geologic environments (10–5centimeters per second (cm/sec) or less hydraulic conductivity) or those with lowgroundwater flow velocities (less than 50 ft/yr) These settings can limit the area ofinfluence of individual reagent injection points due to the absence of sufficientreagent dispersion Poor donor delivery can also result in other potential complica-tions These complications can include:

• Uneven application of reagent and resulting treatment; not achieving treatment goals

• Lack of sufficient or timely demonstration of the technology during pilot phase

• Requirement of too many injection points for a full-scale application

In low permeability and/or low groundwater velocity environments, the reagentcan also accumulate in the vicinity of the injection point Careful monitoring of the

pH, ORP, and total organic carbon (TOC) levels in the groundwater near the injectionwell is necessary to avoid deleterious side effects These effects are related tofermentation and byproduct formation and are discussed later in this section

Natural Surfactant Effect: The injection of an abundant source of easily

degradable organic carbon during the application of ERD typically results in a rapidand large increase in the population of microorganisms in the treatment zone As inany microbiological system, this large population increase will also result in an

increase in production of natural biosurfactants and bioemulsifiers by the

microor-ganisms Natural biosurfactants result in desorption of the chlorinated contaminantsadsorbed to the aquifer media

To assimilate less soluble substrates, such as chlorinated solvents, isms require a large contact area between themselves and the contaminant Theyachieve this by emulsifying the adsorbed contaminants into an aqueous phase Mostmicrobes frequently synthesize and excrete chemicals that promote such emulsifi-cation These excreted chemicals fall into two main groups: biosurfactants andbioemulsifiers (Table 4.7)

microorgan-Table 4.7 Microbial Surfactants

Structural Type Producing Microorganism

Fatty acids — neutral lipids Pseudomonas, Acinetobacter,

Mycococcus, Micrococcus, Candida

Ornithine - lipids Pseudomonas, Thiobacillus,

Gluconobacter

Biosurfactants reduce the interfacial tension between water and the chlorinatedcontaminant so that the chlorinated contaminant (or less water soluble compoundssuch as PAHs) is easily micro-emulsified into the water phase These micro-emulsiondroplets are known to be smaller than microbial cells Some bacterial glycolipidsare extremely effective surfactants In addition to enhancing the “mobilization” ofthe contaminants by microemulsions, biosurfactants can also increase apparent sol-ubilities by the partitioning the contaminants into surfactant micelles.

This desorption, or natural surfactant effect, is observed in many biologicaltreatment processes as an increase in the constituent levels in the treatment zoneand, in some cases, downgradient of the treatment zone In some cases, the constit-uent concentrations in the treatment zone may remain unchanged, due to increasedsolubilization of the contaminants, for a short period even when biodegradation end-product data support the conclusion that sufficient mass is being degraded by theERD processes

The production of surfactants that facilitate the partitioning of contaminants fromthe DNAPL to the dissolved phase (thus resulting in enhanced biodegradation) hasreceived considerable attention recently.12,13 The success of this approach depends

on enhancing and maintaining biodegradation rates faster than the rate of masstransfer from NAPL to the dissolved phase

The magnitude and composition of soil organic carbon content combined withthe distinct differences in partitioning among the chlorinated alkenes have the poten-tial to develop additional mechanisms that cause temporary increases in constituentconcentrations during enhanced reductive dechlorination applications (Figure4.16a).76 A high TOC gradient present between the groundwater and the aquifer soilmatrix, resulting from the injection of molasses, will also result in desorption ofhydrophobic contaminants for the following reasons:76

Figure 4.16a Effect of K oc on Mass sorbed /Mass total

Suthersan, Suthan S “Chapter 4: In Situ Reactive Zones”

Natural and Enhanced Remediation Systems

Edited by Suthan S SuthersanBoca Raton: CRC Press LLC, 2001

• Successive dechlorination of alkene compounds is accompanied by successive decreases in organic carbon-water partition coefficients, Koc (the solubility of aliphatic compounds rapidly increases with decreasing molecular weight) If the degradation rate for daughter compounds is equal to or lower than the rate of parent compound degradation, temporary increases in aqueous-phase concentra- tions will be observed.

• Electron donors such as molasses, typically applied to enhance reductive rination, comprise soluble and colloidal carbon compounds, creating an aqueous- phase organic carbon pool that was essentially nonexistent prior to the creation

dechlo-of the IRZ Creation dechlo-of an aqueous-phase carbon pool is expected to result in a partial migration of chlorinated alkenes to aqueous-phase carbon sorption, result- ing in increased “apparent” concentrations Since the aqueous-phase carbon is mobile, chlorinated alkenes may also be transported from their point of origination.

As the soluble organic carbon is consumed by the microbial community, a portion

of the chlorinated alkenes may remain in dissolved phase for a while until eventual degradation within the IRZ.

Intuitively, the increased desorption of target constituents within the IRZ allowsfor greater access to the typically “untreatable” adsorbed and separate phase con-taminant mass present at source areas and DNAPL locations However, this microbialsurfactant effect must be anticipated and pilot or full-scale treatment should incor-porate provisions to evaluate and account for it For example, the potential for initialincreases of stable parent constituent trends can be of concern to both responsibleparties and regulatory bodies as the data would tend to indicate the technology isnot working and, in some cases, could be considered as actually making conditionsworse Therefore, during the full scale or pilot test planning stages the possibility

of this desorption effect must be evaluated in detail and anticipated in advance Also,the possibility for an increase of dissolved chlorinated solvent concentrations tooccur in areas downgradient of the treatment zone must be addressed Typically, an

“outside-in” approach is applied, whereby a steady state IRZ is established in adowngradient portion of the plume before applying ERD to the source area Desorbedconstituents would then move into an area already undergoing treatment and capable

of treating the increased level of mass flux

Fermentation and By-Product Formation: During application of ERD a highlyreducing biogeochemical environment is generally created throughout the treatmentzone This zone will also contain a large excess of organic carbon in the vicinity ofthe injection points, particularly if the geology is less permeable During the imple-mentation of an IRZ, at (10–5 cm/sec or less), these conditions can result in theformation of organic acids and alcohols in the groundwater as part of the degradationprocess If the formed acids and alcohols are not consumed quickly the zone aroundthe injection zone will mimic a fermenter where additional by-products can beformed

The formation of undesirable byproducts (including acetone and thiol pounds and 2-butanone) has been observed at sites where injection was initiatedwithout careful monitoring of altered groundwater conditions near the injectionwells The occurrences of these byproducts are generally limited in extent and oftensporadic in nature It is expected that these oxidized by-products will also be utilized

com-by microbes within the IRZ Therefore, the lessons learned regarding these potentialoccurrences are as follows:

• Careful and regular monitoring of groundwater within the treatment zone should

be provided in order to ensure that pH levels are not depressed below pH = 4.0–4.5, and TOC levels are not excessive (site specific, but generally within 2 to 3000 mg/L).

• The remediation plan for application of ERD should be flexible enough to allow for modification of both frequency of delivery and mass of organic carbon deliv- ered to prevent the build-up of organic carbon and creation of conditions amenable

to formation of these byproducts Modifications in reagent delivery should be tied

to the pH, ORP, and TOC monitoring in the treatment zone.

Overcoming Oxidizing Conditions/High Groundwater Velocity: As cussed, the implementation of an IRZ for the application of ERD relies on thecreation of a highly reducing biogeochemical environment through provision ofexcess organic carbon to the groundwater Achieving these conditions can be prob-lematic in groundwater flow systems in which the ambient conditions are veryoxidizing (due to shallow groundwater with abundant recharge) or the groundwatervelocity is very high (>1,000 ft/yr) In both situations, the amount of reagent needed

dis-to “overcome” the oxidative poise of the naturally oxidizing conditions will be costprohibitive In addition, the scale-up cost for the full-scale system will be uneco-nomical due to extremely narrow (cigar-shaped) zones of influence from each injec-tion point

In high groundwater velocity settings the limited transverse dispersion in water can limit the extent of the reactive zone created by an individual injectionpoint This is of particular importance in settings where drilling costs may be high(i.e., deep settings or complex geology) In such cases, these site-specific consider-ations need to be weighed against other treatment alternatives

ground-Biofilm Developments: When injecting an electron donor such as molasses (andelectron acceptors) into an aquifer via injection wells, biofilm development aroundthe injection wells should be anticipated Biofilms are large aggregations of bacteriaand other microorganisms bound together in a sticky mass of tangled polysaccharidefibers that connect cells together and tie them to a surface Aerobic and anaerobicbacteria not only can thrive side by side within biofilms when biogeochemicalconditions permit, but also actually seem to collaborate to make themselves morepowerful The polysaccharide coating acts like armor, giving the microorganismsprotection beyond their usual defense mechanisms

While the typical average diameter of a bacterium in established biofilms isabout 0.5–1 mm, biofilm bacteria rarely adhere directly to solid surfaces Instead, atdistances shorter than 1 nm, short-range forces such as hydrogen bonding and dipoleformation tend to be the dominant adhesion effects As bacteria are held in placeand fed by the organic and inorganic molecules trapped by these short-range forces,they form slime that anchors them to solid surfaces This slime becomes a home foradditional bacterial growth If the biofilm becomes too thick to permit adequateoxygen penetration, under aerobic conditions any additional biofilm growth mayactually decrease biofilm adherence due to shearing The thickness of the biofilm