Caister Academic Press

Microbial Biodegradation, Bioremediation and Biotransformation

Edited by: Eduardo Díaz
Biodegradation of aromatics, molecular detection, predictive modelling, regulatory networks, bioavailability, chemotaxis, transport, functional genomics, and much more.

Microbial Biodegradation, Bioremediation and Biotransformation

Interest in the microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to cleanup contaminated environments. These bioremediation and biotransformation methods endeavour to harness the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons (e.g. oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclides and metals. Major methodological breakthroughs in recent years have enabled detailed genomic, metagenomic, proteomic, bioinformatic and other high-throughput analyses of environmentally relevant microorganisms providing unprecedented insights into key biodegradative pathways and the ability of organisms to adapt to changing environmental conditions.

The elimination of a wide range of pollutants and wastes from the environment is an absolute requirement to promote a sustainable development of our society with low environmental impact. Biological processes play a major role in the removal of contaminants and they take advantage of the astonishing catabolic versatility of microorganisms to degrade/convert such compounds. New methodological breakthroughs in sequencing, genomics, proteomics, bioinformatics and imaging are producing vast amounts of information. In the field of Environmental Microbiology, genome-based global studies open a new era providing unprecedented in silico views of metabolic and regulatory networks, as well as clues to the evolution of degradation pathways and to the molecular adaptation strategies to changing environmental conditions. Functional genomic and metagenomic approaches are increasing our understanding of the relative importance of different pathways and regulatory networks to carbon flux in particular environments and for particular compounds and they will certainly accelerate the development of bioremediation technologies and biotransformation processes.

Aerobic Biodegradation of Organic Pollutants
The burgeoning amount of bacterial genomic data provides unparalleled opportunities for understanding the genetic and molecular bases of the degradation of organic pollutants. Aromatic compounds are among the most recalcitrant of these pollutants and lessons can be learned from the recent genomic studies of Burkholderia xenovorans LB400 and Rhodococcus sp. strain RHA1, two of the largest bacterial genomes completely sequenced to date. These studies have helped expand our understanding of bacterial catabolism, non-catabolic physiological adaptation to organic compounds, and the evolution of large bacterial genomes. First, the metabolic pathways from phylogenetically diverse isolates are very similar with respect to overall organization. Thus, as originally noted in pseudomonads, a large number of "peripheral aromatic" pathways funnel a range of natural and xenobiotic compounds into a restricted number of "central aromatic" pathways. Nevertheless, these pathways are genetically organized in genus-specific fashions, as exemplified by the b-ketoadipate and Paa pathways. Comparative genomic studies further reveal that some pathways are more widespread than initially thought. Thus, the Box and Paa pathways illustrate the prevalence of non-oxygenolytic ring-cleavage strategies in aerobic aromatic degradation processes. Functional genomic studies have been useful in establishing that even organisms harboring high numbers of homologous enzymes seem to contain few examples of true redundancy. For example, the multiplicity of ring-cleaving dioxygenases in certain rhodococcal isolates may be attributed to the cryptic aromatic catabolism of different terpenoids and steroids. Finally, analyses have indicated that recent genetic flux appears to have played a more significant role in the evolution of some large genomes, such as LB400's, than others. However, the emerging trend is that the large gene repertoires of potent pollutant degraders such as LB400 and RHA1 have evolved principally through more ancient processes. That this is true in such phylogenetically diverse species is remarkable and further suggests the ancient origin of this catabolic capacity.

Anaerobic Biodegradation of Organic Pollutants
Anaerobic microbial mineralization of recalcitrant organic pollutants is of great environmental significance and involves intriguing novel biochemical reactions. In particular, hydrocarbons and halogenated compounds have long been doubted to be degradable in the absence of oxygen, but the isolation of hitherto unknown anaerobic hydrocarbon-degrading and reductively dehalogenating bacteria during the last decades provided ultimate proof for these processes in nature. Many novel biochemical reactions were discovered enabling the respective metabolic pathways, but progress in the molecular understanding of these bacteria was rather slow, since genetic systems are not readily applicable for most of them. However, with the increasing application of genomics in the field of environmental microbiology, a new and promising perspective is now at hand to obtain molecular insights into these new metabolic properties. Several complete genome sequences were determined during the last few years from bacteria capable of anaerobic organic pollutant degradation. The ~4.7 Mb genome of the facultative denitrifying "Aromatoleum aromaticum" strain EbN1 was the first to be determined for an anaerobic hydrocarbon degrader (using toluene or ethylbenzene as substrates). The genome sequence revealed about two dozen gene clusters (including several paralogs) coding for a complex catabolic network for anaerobic and aerobic degradation of aromatic compounds. The genome sequence forms the basis for current detailed studies on regulation of pathways and enzyme structures. Further genomes of anaerobic hydrocarbon degrading bacteria were recently completed for the iron-reducing species Geobacter metallireducens (accession nr. NC_007517) and the perchlorate-reducing "Dechloromonas aromatica" (accession nr. NC_007298), but these are not yet evaluated in formal publications. Complete genomes were also determined for bacteria capable of anaerobic degradation of halogenated hydrocarbons by halorespiration: the ~1.4 Mb genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1 and the ~5.7 Mb genome of Desulfitobacterium hafniense strain Y51. Characteristic for all these bacteria is the presence of multiple paralogous genes for reductive dehalogenases, implicating a wider dehalogenating spectrum of the organisms than previously known. Moreover, genome sequences provided unprecedented insights into the evolution of reductive dehalogenation and differing strategies for niche adaptation.

Detecting Bacteria Involved in Biodegradation
Traditional molecular analyses have led to great understanding of the microbial diversity in natural systems. These approaches can tell the presence of a particular group of bacteria, but does not address the activity. Molecular methods, including microautoradiography, mRNA analysis, growth assays, and incorporation of stable isotopes, can be used to determine which bacteria are involved in biodegradation of chemical pollutants. This information leads to a greater understanding of the role of microbial community structure and function with respect to bioremediation.

Bioremediation with Extracellular Electron Transfer
Geobacter species are often the predominant organisms when extracellular electron transfer is an important bioremediation process in subsurface environments. Therefore, a systems biology approach to understanding and optimizing bioremediation with Geobacter species has been initiated with the ultimate goal of developing in silico models that can predict the growth and metabolism of Geobacter species under a diversity of subsurface conditions. To date, these studies have included sequencing the genomes of multiple Geobacter species and detailed functional genomic/physiological studies on one species, Geobacter sulfurreducens. Genome-based models of several Geobacter species that are able to predict physiological responses under different environmental conditions are now available. Quantitative analysis of gene transcript levels during in situ uranium bioremediation has demonstrated that it is possible to track in situ rates of metabolism and the in situ metabolic state of Geobacter in the subsurface. Initial attempts to link in silico Geobacter models with existing subsurface hydrological and geochemical models are underway. It is expected that this systems approach to bioremediation with Geobacter will provide the opportunity to evaluate multiple Geobacter-catalyzed bioremediation strategies in silico prior to field implementation, thus providing substantial savings when initiating large-scale in situ bioremediation projects for groundwater polluted with uranium and/or organic contaminants.

Signalling Networks and Pollutant Biosensors
The two elements needed for an efficient utilization of aromatic compounds by bacteria are the enzymes responsible for their degradation and the regulatory elements that control the expression of the catabolic operons to ensure the more efficient output depending on the presence/absence of the aromatic compounds or alternative environmental signals. Transcriptional regulation seems to be the more common and/or most studied mechanism of expression of catabolic clusters, although post-transcriptional control also plays an important role. Transcription is dependent on specific regulators that channel the information between specific signals and the target gene(s). A more complex network of signals connects the metabolic and the energetic status of the cell to the expression of particular catabolic clusters, overimposing the specific transcriptional regulatory control. In general, the regulatory networks that control the operons involved in the catabolism of aromatic compounds are endowed with an extraordinary degree of plasticity and adaptability. Elucidating such regulatory networks pave the way for a better understanding of the regulatory intricacies that control microbial biodegradation of aromatic compounds, which are key issues for the rational design of more efficient recombinant biodegraders, bacterial biosensors, and biocatalysts.

Bioavailability, Chemotaxis, and Transport of Organic Pollutants
Bacterial pathways for the degradation of organic pollutants have been the subject of intense study for decades. However, important physiological events that precede the catabolism of these compounds have recently been receiving significant scientific attention. Bioavailability, or the amount of a substance that is physiochemically accessible to microorganisms is a key factor in the efficient biodegradation of pollutants. Chemotaxis, or the directed movement of motile organisms towards or away from chemicals in the environment is an important physiological response that may contribute to effective catabolism of molecules in the environment. In addition, mechanisms for the intracellular accumulation of aromatic molecules via various transport mechanisms are also important.

Solvent Tolerance and Pumps That Extrude Toxic Chemicals
Organic solvents are toxic for microorganisms because they dissolve in the cytoplasmic membranes, a process that alters the membrane's physical structure and renders the cell unable to synthesize ATP. The degree of toxicity varies depending on the chemical and the strain involved, and chemical toxicity correlates with the partition coefficient of the compound in a mixture of octanol and water. Microbial tolerance to solvents can be mediated by physical and biochemical barriers. Physical barriers are usually based on increased membrane rigidity through the alteration of the cis/trans unsaturated fatty acid ratio, the increase in the saturated:unsaturated fatty acid ratio, or alteration in the phopholipd head groups. Although these barriers counteract the effect of initial toxicity, long-term resistance is based on the active extrusion of solvents, which is mainly mediated by extrusion pumps. Genomic analyses in Gram-negative bacteria have revealed that the resistance-nodulation-cell division (RND) family of efflux pumps is the main group involved in the removal of solvents from the cell. These pumps are made up of three components that span the membranes and extrude solvents from the inner membrane or cytoplasm to the outer medium. The level of expression of these extrusion pumps is finely modulated by transcriptional regulators belonging to different families, but which act in a similar fashion. Some regulators act as repressors that prevent access of the RNA polymerase to the promoter region of the cognate operon. These regulators recognize multiple drugs through a series of overlapping binding pockets, and upon the binding of an effector, transmit a signal to the DNA binding region so that the regulator is released and the genes encoding the efflux pumps are transcribed.

Evolution of Catabolic Pathways and Bacterial Adaptation to Xenobiotic Compounds
Bacteria adapt and become quite rapidly selected to xenobiotic compounds introduced into the environment, mainly via the usage of the compound as carbon, energy or nitrogen source. Important examples include chlorobenzenes, the herbicide 2,4-dichlorophenoxyacetic acid, chloroalkanes, lindane, atrazine and nitroaromatic compounds. At the genomic level, such bacteria show evidence for genetic rearrangements mediated by transposable elements or general recombination, the result being most often an expansion of existing catabolic properties with additional gene modules from outside sources. DNA from outside sources appears to have been trapped and mobilized via conjugative plasmids and genomic islands. Genomic evidence shows that most bacterial genomes contain considerable numbers of insertion elements, integrases, prophages and/or plasmids, which can contribute to their adaptation capacities.

Oil Biodegradation in Marine Systems
Petroleum oil is toxic for most life forms and episodic and chronic pollution of the environment by oil causes major ecological perturbations. Marine environments are especially vulnerable since oil spills of coastal regions and the open sea are poorly containable and mitigation is difficult. In addition to pollution through human activities, millions of tons of petroleum enter the marine environment every year from natural seepages. Despite its toxicity, a considerable fraction of petroleum oil entering marine systems is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB). Alcanivorax borkumensis, a paradigm of HCB and probably the most important global oil degrader, was the first to be subjected to a functional genomic analysis. This analysis has yielded important new insights into its capacity for (i) n-alkane degradation including metabolism, biosurfactant production and biofilm formation, (ii) scavenging of nutrients and cofactors in the oligotrophic marine environment, as well as (iii) coping with various habitat-specific stresses. The understanding thereby gained constitutes a significant advance in efforts towards the design of new knowledge-based strategies for the mitigation of ecological damage caused by oil pollution of marine habitats. HCB also have potential biotechnological applications in the areas of bioplastics and biocatalysis.

Emerging Technologies to Analyze Natural Attenuation and Bioremediation
Natural attenuation is one of several cost-saving options for the treatment of polluted environment, in which microorganisms contribute to pollutant degradation. For risk assessments and endpoint forecasting, natural attenuation sites should be carefully monitored (monitored natural attenuation). When site assessments require rapid removal of pollutants, bioremediation, categorized into biostimulation (introduction of nutrients and chemicals to stimulate indigenous microorganisms) and bioaugmentation (inoculation with exogenous microorganisms), can be applied. In such a case, special attention should be paid to its influences on indigenous biota and the dispersal and outbreak of inoculated organisms. Recent advances in microbial ecology have provided molecular technologies, e.g., detection of degradative genes, community fingerprinting and metagenomics, which are applicable to the analysis and monitoring of indigenous and inoculated microorganisms in polluted sites. Scientists have started to use some of these technologies for the assessment of natural attenuation and bioremediation in order to increase their effectiveness and reliability.

Analysis of Waste Biotreatment in Confined Environments
Sustainable development requires the promotion of environmental management and a constant search for new technologies to treat vast quantities of wastes generated by increasing anthropogenic activities. Biotreatment, the processing of wastes using living organisms, is an environmentally friendly, relatively simple and cost-effective alternative to physico-chemical clean-up options. Confined environments, such as bioreactors, have been engineered to overcome the physical, chemical and biological limiting factors of biotreatment processes in highly controlled systems. The great versatility in the design of confined environments allows the treatment of a wide range of wastes under optimized conditions. To perform a correct assessment, it is necessary to consider various microorganisms having a variety of genomes and expressed transcripts and proteins. A great number of analyses are often required. Using traditional genomic techniques, such assessments are limited and time-consuming. However, several high-throughput techniques originally developed for medical studies can be applied to assess biotreatment in confined environments.

Metabolic Engineering and Biocatalytic Applications of the Pollutant Degradation Machinery
The study of the fate of persistent organic chemicals in the environment has revealed a large reservoir of enzymatic reactions with a large potential in preparative organic synthesis, which has already been exploited for a number of oxygenases on pilot and even on industrial scale. Novel catalysts can be obtained from metagenomic libraries and DNA-sequence based approaches. Our increasing capabilities in adapting the catalysts to specific reactions and process requirements by rational and random mutagenesis broadens the scope for application in the fine chemical industry, but also in the field of biodegradation. In many cases, these catalysts need to be exploited in whole cell bioconversions or in fermentations, calling for system-wide approaches to understanding strain physiology and metabolism and rational approaches to the engineering of whole cells as they are increasingly put forward in the area of systems biotechnology and synthetic biology.

Further reading