Análisis de puntos fuertes y débiles del producto

Culture-independent approaches used in microbial ecology are mainly applied to conduct microbial community profiling and are a preliminary steps before the more advanced (e.g. metagenomic, metatrascriptomic) analysis is performed. Metagenomics and metatranscriptomics are advanced methods since they allow to study genetic material recovered directly from environmental samples, while traditional microbiology and genomics rely upon cultivated microbial cultures (Desai

et al., 2010). High-throughput technologies from the genomics, metagenomics, proteomics and metabolomics are the most promising tools to understand the biodegradation processes in depth (Trigo et al., 2009). Application of “omics” gives not only the opportunity to confirm the processes/mechanisms occurring in the environment, but also to answer questions arising during the environmental studies (e.g. explanation of test failures, detection of new degraders, characterization of functional genes and possible biodegradation pathways). A variety of emerging high- throughput technologies have proved to be useful for studying biodegradation processes in the environment since they link microbial phylogeny to its ecological function. The potential application of these techniques (Table 1.5) for development of new bioremediation strategies of contaminated sites was overviewed by Stenuit et al. (2008). Advanced high throughput techniques like 454 sequencing enable fast and in depth screening of environmental samples for specific degraders and functional genes involved in biodegradation pathways of chemicals. In depth analysis of microbial consortia present in environmental compartments is useful for studying their function, community structure and how they are affected by different factors. Information obtained through application of such techniques is essential for better understanding of microbial networks present in natural ecosystems since it allows specific functions to be assigned to certain groups of microbes. ‘Omics’ enable transition between the environment and laboratory tests, and vice versa, which is crucial for extrapolating results into the environment, and therefore, for better biodegradation assessment under environmentally realistic conditions.

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Table 1.5: Application and potential of high-throughput technologies in biodegradation studies.

Source: Stenuit et al., 2008

Technique Typical target molecule Application Potential Fingerprinting techniques, phylogenetic oligonucleotide arrays (PAOs), community genome arrays (CGAs), real-time Polymerase Chain Reaction (QPCR) DNA RNA  Community structure and dynamics Relate microbial community analysis to the metabolic function of specific groups of bacteria Metatranscriptomics Metaproteomics Metabolomics RNA Proteins Metabolites  Community function Identification of the biodegradation potential and the function of specific microbial communities Biosensors Proteins Metabolites  Presence of undefined chemicals  Nature and concentration of the chemicals  Toxicity of the

chemicals for living organisms  Decrease in chemical concentration, and/or transformation Monitoring “real-time” the performance of a bioremediation process Cultivation (+ genome sequencing), Metagenomics

DNA  Use of exogenous

biocatalysts

 Search for new

catabolic activities

New metabolic functions of microbial communities

41 1.8.1. Metagenomics

Recent advances in metagenomics in which collective microbial genomes are sequenced and screened for functional genes and phylogenetic markers provide an opportunity for linking microbial diversity with function (Malik et al., 2008). The recent development of ultra-high throughput sequencing technologies do not require cloning or PCR amplification, and can produce huge number of DNA reads at an affordable cost. The analysis of such datasets aims to determine and compare the biological diversity and the functional activity of different microbial communities (Huson et al., 2009). For example Mackelprang et al. (2011) reported that metagenomics allowed examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic matrix. Their permafrost metagenome analyses revealed changes in many microbial, phylogenetic and functional gene abundances and pathways they studied which were caused by thaw of permafrost. Development of metagenomic arrays for uncultured microorganisms from contaminated environments can greatly improve our understanding of microbial interaction and metabolism to facilitate the development of suitable bioremediation strategies for environment clean up (Malik et al., 2008).

1.8.2. Metatranscriptomics

Another approach for studying bacterial communities is metatranscriptomics, which refers to the analysis of the collective transcriptomes of a given habitat. It is based on direct retrieval and analysis of microbial transcripts from environmental samples, where environmental mRNA obtained from total RNA is reverse transcribed, amplified and used for analysis (Stenuit et al., 2008). For example, transcriptomic analysis of a marine bacterial community enriched with dimethylsulfoniopropionate (DMSP) was reported by Vila-Costa et al. (2010). They observed increased abundance of transcripts for Gammaproteobacteria and Bacteroidetes as well as overexpression of genes involved in the biodegradation of C3 compounds after DMSP addition.

42 1.8.3. Metaproteomics

Metaproteomics is the study of the entire protein content of a given habitat, and it has greater potential than genomics for the functional analysis of microbial communities. It is known that mRNA expression levels (the transcriptome) may be unreliable indicators of the abundance of the corresponding proteins. In metaproteomics, complex mixtures of proteins from an environmental sample are separated and fractions of interest are analyzed by high-throughput mass spectrometry-based analytical platforms (Stenuit et al., 2008). Exploring the differential expression of a wide variety of proteins and screening of the entire genome for proteins that interact with particular mineralization regulatory factors is helpful to get insights into biodegradation (Vieites et al., 2009). Proteomics also plays an essential role in determining the physiological changes of microorganisms under specific environmental influences, while functional proteomics would predict metabolism of contaminants by degrading organisms (Chauhan and Jain, 2010).

1.8.4. Systems biology approach to biodegradation

Microbial interactions are essential for biodegradation processes in natural environments. General knowledge about the processes operating within microbial community is not sufficient enough to determine the role of individual members and their interactions within the community. For biodegradation studies it is crucial to understand how those relationships could be affected by the environment and emerging chemicals, and vice versa (Zengler and Palsson, 2012). Also, the biodegradation processes are framed in a complex web of metabolic and regulatory interactions which are extremely difficult to approach with the traditional molecular methods. For a long time experimental procedures only allowed the analysis of a few enzymes at a time. Therefore, new techniques and in particular large-scale approaches were developed. They use the system, which is divided, into parts to study them individually but also it is possible to look into interactions between the parts and how they influence each other. The recent accumulation of knowledge about the biochemistry and genetics of the biodegradation process, and creation of

43 structured databases, has opened the door to systems biology. It has been set up to examine complex biological interactions and processes, but it also allows gathering of more information about the biodegradation process in the real environment where it takes place (Trigo etal., 2009).

1.8.5. Advantages and disadvantages of high throughput methods

Emergence of specialized techniques for studying the microbial genome, transcriptome, proteome, metabolome enabled development and successful execution of bioremediation processes (Desai et al., 2010). Recent molecular and “omics” approaches enable exploration and better understanding of microbial biodegradation processes (Figure 1.4), and therefore, improvement of chemical risk assessment in environmental compartments. This will lead to development and application of new, more effective strategies to sustain a cleaner environment.

Although, high throughput techniques are emerging tools in biodegradation studies they are still relatively new techniques. One of the biggest disadvantages of “omics” is their blind application. While there is a place for discovery science using high- throughput techniques progress is usually achieved in studies that are applied with a clear hypothesis in mind. The major concerns arising around “omics” are also linked with constant development of technologies and urge to bring new improvements and applications by companies such as Roche or Illumina. This requires constant development and education of scientists in order to be on top of those approaches. Another issue is the interpretation and management of massive amount of data, which is generated by high throughput technologies. Therefore, development of efficient statistical algorithms, pipelines and other bioinformatic tools is crucial to obtain the good quality data and to perform further analysis. Moreover, the costs of generating high throughput data and data analysis are still high and not always affordable. Hopefully, in the near future, decrease in the costs of such analysis will increase the availability of these techniques and their applicability for biodegradation studies (Shendure and Ji, 2008; Desai et al., 2010).

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Figure 1.4: Schematic of an approach to study effects of environmental realism on biodegradation.