Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins

Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins Biotreatment of industrial effluents CHAPTER 6 – chlorinated hydrocarbons and aromatics, and dioxins

C H A P T E R 6

Chlorinated

Hydrocarbons and

Aromatics, and Dioxins

Introduction

Organic pollutants are among the most ubiquitous in our environment They have accumulated because of a variety of anthropogenic causes and because

of their greater hydrophobicity (i.e., their lack of solubility in water)

Occurrence

Organics, which include polycylic aromatic hydrocarbons (PAHs), chlorinated aromatic hydrocarbons, chlorinated aliphatic hydrocarbons, halogenated hydrocarbons, biphenyls, phenols, aniline derivatives, phenol ethoxylates, and benzoic acid derivatives, are ubiquitous in our environ- ment Both anthropogenic and natural causes are known for their accumula- tion They are found in water, marine systems, soil, sewage, and air These are the most common pollutants and are known to persist in the environ- ment Some of them, such as the PAHs, are potent carcinogens All of them are reported to have adverse effects on human and animal health

Some of these, like the polyhalogenated aromatics, are chemically inert and therefore can only be degraded by biological means The degradation could be by either aerobic or anaerobic pathways A brief outline of both these pathways is necessary to be able to design suitable degradation pathways for

a given contaminant

Aerobic Degradation

A number of bacteria and fungi are known to adopt the aerobic pathway The enzymes involved in the fixing of oxygen (from air or water) into

65

organic molecules are called "oxygenases." Most of the oxygen required by the microorganisms is used for oxidative phosphorylation, which generates energy for cellular processes About 5 to 10% of the total oxygen require- ment is normally used by these "oxygenases." The ability of oxygenases to incorporate oxygen into organic compounds is important because many of the hydrophobic pollutants such as PAHs are high in carbon and hydrogen but low in oxygen Through the action of oxygenases, hydrophobic organic compounds become more water soluble and can be broken down by a large number of other microorganisms The end result of the oxygenase reaction on these hydrocarbons is hydroxyl or carbonyl compounds, which are normally more water soluble than the parent compounds

Two major classes of oxygenases are well known They are monooxyge- nase and dioxygenase Monooxygenases incorporate one atom of the oxygen molecule into the organic substrate while the second oxygen atom goes

to form water Dioxygenases incorporate both atoms of oxygen molecule into the substrates (Note: The division is not absolute.) These enzymes participate in the oxidative metabolism of a wide variety of chemicals of pharmaceutical, agricultural, and environmental significance

D i o x y g e n a s e s

Dioxygenases are very important in initiating the biodegradation of a variety

of chlorinated and nitrogenous aromatic compounds as well as nonsubsti- tuted PAHs There are two major types of dioxygenases One type requires

N A D H or NADPH, and these enzymes hydroxylate the substrates (Cerniglia, 1992) The other type has no specific requirement for NAD(P)H, and it cleaves the aromatic ring (Eltis et al., 1993) The overall mechanism of degradation can be summarized as shown in Fig 6-1 Dioxygenases are very important in initiating the biodegradation of a variety of chlorinated and nitrogenous aromatic compounds as well as nonsubstituted PAHs Their main substrates seem to be derived from crude oil and lignin, as these are the major sources of aromatic compounds in the environment Many of these compounds are first degraded to catechol or protocatechuate by oxygenases

M o n o o x y g e n a s e s

Monooxygenases are more abundant than the dioxygenases and are more commonly found in fungi and mammalian systems They can catalyze sev- eral different types of oxygen insertion reactions These classes of enzymes require two reductants (substrates) Since they oxidize two substrates, they are also called mixed function oxidases Since one of the main substrates becomes hydroxylated, they are also called hydroxylases (Gibson, 1993) The overall mechanism of degradation can be summarized as shown in Fig 6-2 The monooxygenases can initiate attack on aromatic compounds They are more abundant than dioxygenases

Chlorinated Hydrocarbons and Aromatics, and Dioxins 67

~ Benzene derivatives, PAHs,

biphenyl derivatives, &

fused ring a r o m a t i c /

eRases

.

I Cis-dihydrodiol derivatives [ Catechol derivatives i [' Intradiol cleavage ~ / ~ " ~ , , q Extradiol cleavage I

Muconic acid derivatives I I Muconic semialdehyde derivatives

Acetoacetate, pyruvate, etc., TCA cycle

FIGURE 6-1 Dioxygenase degradation mechanism

Anaerobic Degradation Pathways

The microbial mediated decomposition portion of the carbon cycle can

be coupled with oxygen or can occur with no external electron acceptor

With oxygen, respiratory metabolism occurs and results in higher energy

yields than fermentative metabolism (no external electron acceptor) does

Therefore, when oxygen is present, aerobic degradation predominates over anaerobic fermentation Nonetheless, anaerobic decomposition still plays a key role in the carbon cycle in the ecosphere because of ecological effects Since the late 1980s, an increasing number of novel microorganisms have been shown to utilize saturated and aromatic hydrocarbons as growth sub- strates under strictly anoxic conditions In the absence of oxygen, a wide variety of alternative electron acceptors are used by the anaerobic bacte- ria for oxidation of organic compounds Methanogenesis is predominant

in freshwater sediments, while sulfate reduction is a dominant process in

Phenol derivatives

I Trans-dihydrodiol derivatives I I Monohydroxylated derivatives

'

I Catecho/derivatives I

Dioxygenases !

I Ring fissi~ I FIGURE 6-2 Monooxygenase degradation mechanism

carbon metabolism in marine and estuarine sediments Denitrification can

be significant in regions of high nitrate input from agricultural runoff or sewage discharge Fe(III)reduction is important in several sediments and anoxic soils Halogenated aromatic compounds, phenols, benzoic acids, and PAHs are reported to have been degraded to carbon dioxide under a vari- ety of redox conditions, with nitrate, iron, sulfate, and carbon dioxide as alternative electron acceptors Oxidation of these substrates was coupled

to reduction of the respective electron acceptor Distinct anaerobic popu- lations are enriched and responsible for metabolic patterns under different redox conditions For the distinctive redox respiration to be effective, the anaerobic organisms grow in "syntrophic cocultures" with other anaerobes

or grow by anoxygenic photosynthesis The interactions and activities of diverse anaerobic communities need to be considered when evaluating the fate of anthropogenic contaminants in the environment and in developing bioremediation technologies The overall picture of the bioremediation of organics is summarized in Fig 6-3

Chlorinated Hydrocarbons and Aromatics, and Dioxins 69

\\

~ Chemotropic ~ ~ H20

Phototropic ~

anoxygenic ~ ~

" -

Cell mass < ~ , ( I'lY~176176176 )~Fe(lll) Cel ~lmass Fe(l,)

/ " " " ~ ~ ~ " " 4 Cell mass H2 s

v c o 2 Cell mass

FIGURE 6-3 Bioremediation of organics

There is no biochemical agent under anoxic conditions that exhibits the properties of the oxygen species involved in aerobic hydrocarbon activation; hence, the mechanisms of anaerobic hydrocarbon activation are completely different from oxygenase reactions Indeed, all of the anaerobic (degradation) activation reactions of hydrocarbons are mechanistically unprecedented in biochemistry (Rabus et al., 2001) The number of electrons released and the free energy of some of these reactions are shown in Fig 6-4

Thus in designing anaerobic degradation technologies, importance should be given to the following:

9 The type of inorganic substances present (nitrate, iron, sulfate, methane, etc.)

9 The different communities of the microorganisms in the medium

Polynuclear Aromatic Hydrocarbons

Polynuclear aromatic hydrocarbons (PAHs) constitute one class of toxic environmental pollutant that has accumulated in the environment because

of a variety of anthropogenic activities Incomplete combustion of organic materials, in particular fossil fuels, is considered to be the source of these PAHs Hence, domestic coal combustion, motor vehicle fuel combustion, and volatilization of the existing burden from contaminated soils are the primary sources Of these, 71 to 80% is due to traffic emissions (Guerin and

CH 4 + 3 H 2 0 ~ H C O 3 - + 9H + + 8 e -

SO42- + 9H + + 8 e - - - ~ H S - + 4 H 2 0

E in situ = - 0 2 4 8 V (E~ = - 0 2 1 7 V)

C16H34 + 1 9 6 N O 3- + 3.6H +

16HCO 3- + 9.8 N 2 + 10.8 H 2 0

A G' = - 9 8 3 KJ / mole of N 2 formed

C16H34 + 12.25 SO42-

16HCO 3 - + 12.25 H S - + 3.75 H + + H 2 0

A G' = - 6 1 KJ / mole of H S - formed

C16H34 + 11.25 H 2 0

12.25 CH 4 + 3.75 HCO 3- + 3.75 H +

A G' = - 3 3 KJ / mole of CH 4 formed

FIGURE 6-4 Anaerobic degradation of hydrocarbons

Jones, 1988) Several PAHs have been shown to be acutely toxic The most potent carcinogens of the PAH group in addition to benzo[a]pyrene include: the benzofluoranthenes, benzo[a]anthracene, dibenzo[ah]anthracene, and indenol[1,2,3-cd]pyrene (Fig 6-5) Most of the PAHs are recognized by regulatory agencies such as the European Community (EC) and the U.S Envi- ronmental Protection Agency as priority pollutants Some of them are also classified as persistent organic pollutants (POPs)

General Aspects of PAH Degradation

The persistence of PAHs in the environment depends on the physical and chemical characteristics of the PAHs The greater their lipophilic character (and corresponding hydrophobic character), the greater is their persistence PAHs are degraded by photooxidation, chemical oxidation, and biological transformation Microbial-mediated biological transformation is probably the most prevailing route of PAH cleanup (Mueller et al., 1989) The basic pathway of degradation is via the cis dihydrodiol formation as shown in Fig 6-6 (Juhasz and Naidu, 2000) Although most bacteria possess the enzymes for the catabolism of PAHs, degradation of these compounds may not occur because these compounds are unable to pass through the bacterial

Chlorinated Hydrocarbons and Aromatics, and Dioxins 71

Chrysene

Benzo [a] anthracene

Benzo [a] pyrene

Indeno [1,2,3,c-d] pyrene

Benzo [b] fluranthene

Benzo [k] fluranthene

Dibenzo [a,h] anthracene

Benzo [g,h] peryiene

CH 3

1 -methylphenanthrene 2-methylphenanthrene

CH 3 9-methylphenanthrene

F I G U R E 6-5 S t r u c t u r e s of s o m e PAHs

cell walls On the other hand, the ability of the fungi to produce extracellu- lar enzymes such as lignin peroxidases (LIP)overcomes this problem (Duran

complete degradation of PAHs

Halogenated Organic Compounds

Halogenated compounds constitute one of the largest groups of environ- mental pollutants Contamination of marine and freshwater sediments by anthropogenic halogenated organic compounds, such as solvents (tetra- chloro ethylene[PCE], trichloro ethylene [TCE], dichloro ethylene [DCE], chloroethylene [CE], trichloro methane[TCM], and dichloro methane

Benzo [a] pyrene

Hll

" l;', H'OH HO - ~

/-

HOOC

HOOC

F I G U R E 6-6 B i o d e g r a d a t i o n of PAHs

[DCM]), pesticides (DDT, TCE), polychloro dibenzofurans (PCDFs), and diox- ins, is a matter of increasing concern The majority of these compounds are chlorinated, but brominated and fluorinated aromatic compounds are also in use Microbial processes based on the metabolic activities of anaerobic bacte- ria are very effective in the degradation of these compounds They are known

to play important roles in nature by preparing many of these compounds for subsequent biodegradation, predominantly by the aerobic means A critical step in degradation of organohalides is the cleavage of the carbon-halogen bond (Leisinger, 1996); two main strategies can be differentiated:

9 The halogen substituent is removed as an initial step in degradation via

reductive, hydrolytic, or oxygenolytic mechanisms, as shown in Fig 6-7

9 Dehalogenation occurs after cleavage of the aromatic ring This generally happens with lower chlorinated aromatics, wherein microorganisms may open the ring with dioxygenases before removal of chlorines, as shown in Fig 6-8 This reaction is very similar to those acting on nonhalogenated substrates

Chlorinated Hydrocarbons and Aromatics, and Dioxins 73

OH

CI ~ CI

OH

OH +GSH= C I ~ C I -HCI CI f y NSG

O

GS SC-CH2-enzyme +

+ - SC-CH2-enzyme

I

OH

c I ~ C I

CI f y ~ ' H

OH Reductive dehalogenation

Cl ~ ~ COOH

3-chloroacrylate

+H20

Dehalogenase

OH

CI ~ COOH

~ -HCI

O

H ~ COOH

Dehalogenation by hydration

+ 02 + NADH + H + ~ ~ C ) H

% -

CI

Oxidative dehalogenation

C/ OH H

1

CH~ ,-COOH

OH

OH

FIGURE 6-7 Critical step in the degradation of organohalide

I Dioxygenase 02

CI

O

i

o

Dioxetan

CI

OH

OH Catechol

CI

OH

OH

Cis-dihydrodiol

S ~ P H + H +

NADP +

FIGURE 6-8 Degradation of organohalides by dioxygenases

Reductive dehalogenation under aerobic conditions is brought about

by conjugation with glutathione A good example is the reaction catalyzed

by tetrachlorohydroquinone reductive dehalogenase from Sphingomonas chlorophenolicus This reaction requires 2 mol of reduced glutathione (GSH)

per reaction In the first part of the reaction, one molecule of GSH is oxidized and becomes attached to the substrate at the site of dechlorination In the second part, another molecule of GSH extracts the first glutathione to form glutathione disulfide (GSSG)while replacing it with a hydrogen on the ring (Fig 6-7) Under anaerobic conditions, reductive dehalogenation can yield energy for the microorganism through the process of halorespiration, where reductive dehalogenation is coupled to energy metabolism (Mohn and Tiedje, 1992) Here a halogenated compound like tetrachloroethene (PCE) serves as

a terminal electron acceptor during oxidation of an electron-rich compound such as hydrogen, benzene, toluene, and similar organic substrates (Fig 6-9) However, this process is often partly inhibited by other electron acceptors such as sulfate or nitrate Several studies show that alternate electron accep- tors, such as sulfate, iron (III), or nitrate, can support anaerobic degradation

of halogenated phenols and benzoates Mineralization of these compounds

to CO2 may be coupled to sulfate, iron(III), or nitrate reduction, as shown in Fig 6-10

In an interesting study on bioremediation of hazardous wastes, aro- matic hydrocarbons such as benzene, toluene, ethyl benzene, xylenes, phenols, and cresols were used as electron donors to biologically reduce halo- genated hydrocarbons (electron acceptors) such as tetrachloroethylene (PCE) and trichloroethylene (TCE), thereby achieving the degradation of both (U.S Patent No 5922204)