## 3.7.2. Biodegradation

Author: John Parsons

Reviewers: Steven Droge, Russell Davenport

Learning objectives:

You should be able to:

- the contribution of biochemical reactions in removing chemicals from the environment
- explain the differences between biotransformation, primary biodegradation and mineralization
- describe the most important biodegradation reactions under aerobic and anaerobic conditions

Keywords: Primary biodegradation, mineralisation, readily biodegradable chemicals, persistent chemicals, oxygenation reactions, reductions reactions

Introduction:

Biodegradation and biotransformation both refer to degradation reactions that are catalyzed by enzymes. In general, biodegradation is usually used to describe the degradation carried out by microorganisms and biotransformation often refers to reactions that follow the uptake of chemicals by higher organisms. This distinction is important and arises from the role that bacteria and other microorganisms play in natural biogeochemical cycles. As a result, microorganisms have the capacity to degrade most (perhaps all) naturally occurring organic chemicals in organic matter and convert them to inorganic end products. These reactions supply the microorganisms with the nutrients and energy they need to grow. This broad degradative capacity means that they are able to degrade many anthropogenic chemicals and potentially convert them to inorganic end products, a process referred to as mineralisation.

Although higher organisms are also able to degrade (metabolise) many anthropogenic chemicals, these chemicals are not taken up as source of nutrients and energy. Many anthropogenic chemicals can disturb cell functioning processes, and the biotransformation process has been proposed as a detoxification mechanism. Undesirable chemicals that may accumulate to potentially harmful levels are converted to products that are more rapidly excreted. In most cases, a polar and/or ionizable unit is attached to the chemical in one or two steps, making the compound more soluble in blood and more readily removed via the kidneys to the urine. This also renders most hazardous chemicals less toxic than the original chemical. Such biotransformation steps always costs energy (ATP, or through the use of e.g. NADH or NADPH in the enzymatic reactions) from the organism. Biotransformation is sometimes also used to describe degradation by microorganisms when this is limited to a conversion of a chemical into a new product.

Biodegradation is for many organic contaminants the major process that removes them from the environment. Measuring the rates of biodegradation therefore is a prominent aspect of chemical risk assessment. Internationally recognized standardised protocols have been developed to measure biodegradation rates of chemicals. Well know examples of these are the OCED Guidelines. These guidelines include screening tests designed to identify chemicals can be regarded as readily (i.e. rapidly) biodegradable as well as more complex tests to measure biodegradation rates of chemicals that degrade slowly in a variety of simulated environments. For more complex mechanistic studies, microorganisms able to degrade specific chemicals are isolated from environmental samples and cultivated in laboratory systems.

In principle, biodegradation of a chemical can be determined by either following the concentration of the chemical during the test or by following the conversion to end products (in most cases by either measuring oxygen consumption or CO2 production). Although measuring the concentration gives the most directly relevant information on a chemical, it requires the availability or development of analytical methods which is not always within the capability of routine testing laboratories. Measuring the conversion to CO2 is comparatively straightforward but the production of CO2 from other chemicals present in the test system (such as soil or dissolved organic matter) should be accounted for. This can be done by using 14C-labelled chemicals in the tests but not all laboratories have facilities for this. The main advantage of this approach is that demonstration of quantitative conversion of a chemical to CO2 etc. means that there is no concern about the accumulation of potentially toxic metabolites.

Since it is an enzymatically catalysed process, the rates of biodegradation should be modelled using the Michaelis Menten kinetics, or Monod kinetics if growth of the microorganisms is taken into account. In practice, however, first order kinetics are often used to model biodegradation in the absence of significant growth of the degrading microorganisms. This is more convenient that using Michaelis Menten kinetics but there is some justification for this simplification since the concentrations of chemicals in the environment are in general much lower than the half saturation concentrations of the degrading enzymes.

Table 1. Influence of molecular structure on the biodegradability of chemicals in the aerobic environment.

Type of compounds or substituents | More biodegradable | Less biodegradable
hydrocarbons | linear alkanes < C12 | linear alkanes > C12
| alkanes with not too high molecular weight | high molecular weight alkanes
| linear chain | branched chain
| -C-C-C- | -C-O-C-
| aliphatic | aromatic
aliphatic chlorine | Cl more than 6 carbons from terminal C | Cl at less than 6 carbons from terminal C
Substituents to an aromatic ring | -OH | -F
| -CO2H | -Cl
| -NH2 | -NO2
| -OCH3 | -CF3

Whether expressed as terms of first order kinetics or Michaelis Menten parameters, rates of biodegradation vary widely for different chemicals showing that chemical structure has a large impact on biodegradation. Large variations in biodegradation rates are however often observed for the same chemical in different experimental systems. This shows that environmental properties and conditions also play a key role in determining removal by biodegradation and it is often almost impossible to distinguish the effects of chemical properties from those of environmental properties. In other words, there is no such thing as an intrinsic biodegradation rate of a chemical. Nevertheless, we can derive some generic relationships between the structure and biodegradability of chemicals, as listed in Table 1. Examples are that branched hydrocarbon structures are degraded more slowly than linear hydrocarbon structures, and cyclic and in particular aromatic chemicals are degraded more slowly than aliphatic (non-aromatic) chemicals. Substituents and functional groups also have a major impact on biodegradability with halogens and other electron withdrawing substituents having strongly negative effects. It is therefore no surprise than the list of persistent organic pollutants is dominated by organohalogen compounds and in particular those with aromatic or alicyclic structures.

It should be recognized that biodegradation rates have often been observed to change over time. Long term exposure of microbial communities to new chemicals has often been observed to lead to increasing biodegradation rates. This phenomenon is called adaptation or acclimation and is often the case following repeated application of a pesticide at the same location. An example is shown for atrazine in Figure 2 where degradation rates increase following longer exposure to the pesticide.

Figure 1. Effect of chlorination on (aerobic) biodegradation rates. Adapted from Janssen et al. (2005) by Steven Droge.

Figure 2. Comparison of the atrazine removal rates with (days 43 and 105) or without the addition of carbon and nitrogen sources (day 274). Redrawn from Zhou et al. (2017) by Wilma IJzerman.

Another recent example is the differences in biodegradation rates of the builder L-GLDA (tetrasodium glutamate diacetate) by activated sludge from different waste water treatment plants in the USA. Sludge from regions where L-GLDA was not or only recently on the market required long lag time before degradation started whereas sludge from regions where L-GLDA –containing products had been available for several months required shorted lag phases.

Figure 3. Biodegradation as a function of time following initial shipment of L-GLDA-containing products. Redrawn from Itrich et al. (2015) by Wilma Ijzerman.

Adaptation can results from i) shifts in composition or abundances of species in a bacterial community, ii) mutations within single populations, iii) horizontal transfer of DNA or iv) genetic recombination events, or combinations of these.

Biodegradation reactions and pathways

Biodegradation of chemicals that we regard as pollutants takes place when these chemicals are incorporated into the metabolism of microorganisms. The reactions involved in biodegradation are therefore similar to those involved in common metabolic reactions, such as hydrolyses, oxidations and reductions. Since the conversion of an organic chemical to CO2 is an overall oxidation reaction, oxidation reactions involving molecular oxygen are probably the most important reactions. These reactions with oxygen are often the first but essential step in degradation and can be regarded as activation step converting relatively stable molecules to more reactive intermediates. This is particularly important for aromatic chemicals since oxygenation is required to make aromatic rings susceptible to ring cleavage and further degradation. These reactions are catalysed by enzymes called oxygenases of which there are broadly speaking two classes. Monoxygenases are enzymes catalysing reactions in which one oxygen atom of O2 reacts with an organic molecule to produce a hydroxylated product. Examples of such enzymes are the cytochrome P450 family and are present in all organisms. These enzymes are for example involved in the oxidation of alkanes to carboxylic acids as part of the “beta-oxidation” pathway, which shortens linear alkanoic acids in steps of C2-units, as shown in Figure 4.

Figure 4. Typical oxidation steps of an alkane to an alkanoic acid, and subsequent beta-oxidation pathway from dodecanoic acid to decanoic acid, involving Coenzym-A. Redrawn from Schwarzenbach et al. (2003) by Steven Droge.

Dioxygenases are enzymes catalysing reactions in which both oxygen atoms of O2 react with organic chemicals and appear to be unique to microorganisms such as bacteria. Examples of these reactions are shown for benzene in Figure 5. Similar reactions are involved in the degradation of more complex aromatic chemicals such as PAHs and halogenated aromatics.

Figure 5. Examples of bacterial dioxygenation reactions and ring cleavage of toluene and benzene. Redrawn from Van Leeuwen and Vermeire (2007) by Steven Droge.

The absence of oxygen in anaerobic environments (sediments and groundwater) does not preclude oxidation of organic chemicals. Other oxidants present (nitrate, sulphate, Fe(III) etc) may be present in sufficiently high concentrations to act as oxidants and terminal electron acceptors supporting microbial growth. In the absence of oxygen, activation relies on other reactions, the most important reactions seem to be carboxylation or addition of fumarate. Figure 6 shows an example of the degradation of naphthalene to CO2 in sediment microcosms under sulphate-reducing conditions.

Figure 6. Proposed anaerobic oxidation pathway of naphthalene (redrawn from Kleeman and Merckenstock 2017). This involves two initial reaction mechanisms: carboxylation to naphthoic acid, methylation to 2-methylnaphthalene. Addition of fumarate (process b) could follow methylation and produce another way of transforming to 2-naphthoic acid. In both cases, 2-naphthoic acid is oxidized to CO2.

Other important reactions in anaerobic ecosystems (sediments and groundwater plumes) are reductions. This affects functional groups, for example reduction of acids to aldehydes to alcohols, nitro groups to amino groups and, particularly important, substitution of halogens by hydrogen. The latter reactions can contribute to the conversion of highly chlorinated chemicals, that are resistant to oxidative biodegradation, to less chlorinated products which are more amenable to aerobic biodegradation. Many examples of these reductive dehalogenation reactions have been shown to occur in, for example, tetrachloroethene-contaminated groundwater (e.g. from dry-cleaning processes) and PCB-contaminated sediment. These reactions are exothermic under anaerobic conditions and some microorganisms are able to harvest this energy to support their growth. This can be considered to be a form of respiration based on dechlorination and is sometimes referred to as chlororespiration.

As is the case for abiotic degradation, hydrolyses are also important reactions in biodegradation pathways, particularly for chemicals that are derivatives of organic acids, such as carbamate, ester and organophosphate pesticides where hydrolyses are often the first step in their biodegradation. These reactions are similar to those described in the section on Chemical degradation.

References

Itrich, N.R., McDonough, K.M., van Ginkel, C.G., Bisinger, E.C., LePage, J.N., Schaefer, E.C., Menzies, J.Z., Casteel, K.D., Federle, T.W. (2015). Widespread microbial adaptation to L-glutamate-N,N,-diacetate (L-GLDA) following its market introduction in a consumer cleaning product. Environmental Science & Technology 49, 13314-13321.

Janssen, D. B., Dinkla, I. J. T., Poelarends, G. J., Terpstra, P. (2005). Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities, Environmental Microbiology 7, 1868-1882.

Kleemann, R., Meckenstock, R.U. (2017). Anaerobic naphthalene degradation by Gram-positive, iron-reducing bacteria. FEMS Microbial Ecology 78, 488-496.

Schwarzenbach, R.P., Gschwend, P.M., Imboden, D.M. (2017). Environmental Organic Chemistry, Third Edition, Wiley, ISBN 978-1-118-76723-8

Van Leeuwen, C., Vermeire, T.G. (2007). Risk Assessment of Chemicals: An Introduction (2nd ed.), Springer, ISBN 978-1-4020-6101-1

Zhou, Q., Chen, L. C., Wang, Z., Wang, J., Ni, S., Qiu, J., Liu, X., Zhang, X., Chen, X. (2017). Fast atrazine degradation by the mixed cultures enriched from activated sludge and analysis of their microbial community succession. Environmental Science & Pollution Research 24, 22152-22157.